HLA class II-restricted T cell receptor for RAS with the G12R mutation

Abstract

Disclosed is an isolated or purified T cell receptor (TCR) having antigen specificity for a mutant human RAS amino acid sequence in which glycine at position 12 is substituted with arginine. Related polypeptides and proteins, as well as related nucleic acids, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions, are also provided. Also disclosed are methods for detecting the presence of cancer in a mammal and for treating or preventing cancer in a mammal.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of U.S. Provisional Patent Application No. 62 / 795,203, filed Jan. 22, 2019, which is incorporated by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support awarded by the National Cancer Institute, National Institutes of Health, under Project No. ZIABC010984. The Government has certain rights in this invention.

[0003] Incorporation by Reference of Electronically Submitted Documents The computer readable nucleotide / amino acid sequence listing, which was submitted contemporaneously herewith and is identified as follows, is incorporated by reference in its entirety into this specification: a 74,093 byte ASCII (text) file entitled "746666_ST25.txt", dated January 21, 2020. [Background technology]

[0004] Some cancers may have very limited treatment options, especially when the cancer becomes metastatic and unresectable.For example, despite advances in treatment such as surgery, chemotherapy, and radiation therapy, many cancers, such as pancreatic, colorectal, lung, endometrial, ovarian, and prostate cancers, may have poor prognosis.Therefore, there is an unmet need for additional cancer treatments. Summary of the Invention

[0005] An embodiment of the present invention provides an isolated or purified T cell receptor (TCR), the TCR having antigen specificity for a mutant human RAS amino acid sequence in which glycine at position 12 is substituted with arginine, the mutant human RAS amino acid sequence being the amino acid sequence of a mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS), a mutant human Harvey rat sarcoma viral oncogene homolog (HRAS), or a mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS), and wherein position 12 is defined by reference to the wild-type (WT) human KRAS, WT human HRAS, or WT human NRAS protein, respectively.

[0006] Another embodiment of the present invention provides an isolated or purified polypeptide comprising a functional portion of a TCR of the present invention, wherein the functional portion comprises the amino acid sequence of (a) all of SEQ ID NOs: 1 to 3, (b) all of SEQ ID NOs: 4 to 6, (c) all of SEQ ID NOs: 7 to 9, (d) all of SEQ ID NOs: 10 to 12, (e) all of SEQ ID NOs: 1 to 6, or (f) all of SEQ ID NOs: 7 to 12.

[0007] Yet another embodiment of the present invention provides an isolated or purified protein comprising at least one of the polypeptides of the present invention.

[0008] Embodiments of the invention further provide nucleic acids, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions related to the TCRs, polypeptides, and proteins of the invention.

[0009] An embodiment of the invention provides an isolated or purified nucleic acid comprising, in a 5' to 3' direction, a first nucleic acid sequence and a second nucleotide sequence, wherein the first and second nucleotide sequences encode the amino acid sequences of SEQ ID NOs: 13 and 14; 14 and 13; 15 and 16; 16 and 15; 30 and 31; 31 and 30; 32 and 33; 33 and 32; 34 and 35; 35 and 34; 36 and 37; 37 and 36; 41 and 42; 42 and 41; 43 and 44; 44 and 43; 45 and 46; 46 and 45; 47 and 48; 48 and 47; 49 and 50; 50 and 49; 51 and 52; or 52 and 51, respectively.

[0010] Further provided by embodiments of the invention are methods of detecting the presence of cancer in a mammal, methods of treating or preventing cancer in a mammal, methods of inducing an immune response against cancer in a mammal, methods of generating host cells expressing a TCR with antigenic specificity for the peptide of SEQ ID NO:39, and methods of generating the TCRs, polypeptides, and proteins of the invention. [Brief description of the drawings]

[0011] [Figure 1] Figure 1 shows the number of interferon gamma (IFNγ) positive spots detected per 2x104 (2E4) cells upon co-culture of 4270 TCR-transduced cells with autologous DCs. Prior to co-culture, autologous DCs were pulsed with serial dilutions (ng / mL) of WT RAS peptide (squares) or G12R RAS peptide (circles). [Diagram 2] 1 is a graph showing the concentration (pg / mL) of IFNγ detected when 4270 TCR-transduced cells were co-cultured with COS7 cells. Prior to co-culture, COS7 cells were transfected with one of the indicated HLA molecules and then pulsed with G12R RAS peptide. HLA-DRA01:01 is the second member of the heterodimer for each of DRB1*11:01, DRB1*15:01, DRB3*02:02, DRB4*01:01, and DRB5*01. [Diagram 3]Figure 1 shows the number of IFNγ positive spots per 2E4 cells detected when 4268 TCR-transduced cells were co-cultured with autologous DCs. Prior to co-culture, autologous DCs were pulsed with serial dilutions (ng / mL) of WT RAS peptide (squares) or G12R RAS peptide (circles). [Figure 4] 1 is a graph showing the concentration (pg / mL) of IFNγ detected when 4268 TCR-transduced cells were co-cultured with COS7 cells. Prior to co-culture, COS7 cells were transfected with one of the indicated HLA molecules and then pulsed with G12R RAS peptide. HLA-DRA01:01 is the second member of the heterodimer for each of DRB1*11:01, DRB1*12:01, DRB3*02, DRB4*01, and DRB5*01. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] RAS family proteins belong to a large family of small GTPases. Without being bound to a particular theory or mechanism, it is believed that when mutated, RAS proteins may be involved in signal transduction in the early stages of carcinogenesis in many human cancers. A single amino acid substitution can activate the protein. Mutant RAS protein products can be constitutively activated. Mutant RAS proteins can be expressed in any of a variety of human cancers, such as, for example, pancreatic cancer (e.g., pancreatic cancer), colorectal cancer, lung cancer (e.g., lung adenocarcinoma), endometrial cancer, ovarian cancer (e.g., epithelial ovarian cancer), and prostate cancer. Human RAS family proteins include KRAS, HRAS, and NRAS.

[0013] KRAS is also referred to as GTPase KRas, V-Ki-Ras2 Kirsten rat sarcoma viral oncogene, or KRAS2. There are two transcript variants of KRAS: KRAS variant A and KRAS variant B. WT KRAS variant A has the amino acid sequence of SEQ ID NO: 17. WT KRAS variant B has the amino acid sequence of SEQ ID NO: 18. Hereinafter, reference to "KRAS" (mutated or non-mutated (WT)) refers to both variant A and variant B, unless otherwise specified. When activated, mutant KRAS binds guanosine-5'-triphosphate (GTP) and converts GTP to guanosine 5'-diphosphate (GDP).

[0014] HRAS is another member of the RAS protein family. HRAS is also called Harvey rat sarcoma virus oncoprotein, V-Ha-Ras Harvey rat sarcoma virus oncogene homolog, or Ras family small GTP-binding protein H-Ras. WT HRAS has the amino acid sequence of SEQ ID NO: 19.

[0015] NRAS is yet another member of the RAS protein family. NRAS is also called GTPase NRas, V-Ras neuroblastoma RAS viral oncogene homolog, or NRAS1. WT NRAS has the amino acid sequence of SEQ ID NO:20.

[0016] An embodiment of the invention provides an isolated or purified TCR, the TCR having antigen specificity for a mutant human RAS amino acid sequence in which glycine at position 12 is substituted with arginine, the mutant human RAS amino acid sequence being the amino acid sequence of mutant human KRAS, mutant human HRAS, or mutant human NRAS, where position 12 is defined by reference to the WT human KRAS, WT human HRAS, or WT human NRAS protein, respectively. References hereinafter to "TCR" also refer to functional parts and functional variants of the TCR, unless otherwise specified.

[0017] The mutant human RAS amino acid sequence can be a mutant human KRAS amino acid sequence, a mutant human HRAS amino acid sequence, or a mutant human NRAS amino acid sequence. The amino acid sequences of the WT human KRAS, NRAS, and HRAS proteins are 188 or 189 amino acid residues long, respectively, and have a high degree of identity to each other. For example, the amino acid sequence of the WT human NRAS protein is 86.8% identical to the amino acid sequence of the WT human KRAS protein. Amino acid residues 1-86 of the WT human NRAS protein and the WT human KRAS protein are 100% identical. The amino acid sequence of the WT human HRAS protein is 86.3% identical to the amino acid sequence of the WT human KRAS protein. Amino acid residues 1-94 of the WT human HRAS protein and the WT human KRAS protein are 100% identical. Hereinafter, references to "RAS" (mutated or non-mutated (WT)) refer collectively to KRAS, HRAS, and NRAS, unless otherwise specified.

[0018] In an embodiment of the invention, the mutant human RAS amino acid sequence comprises a human RAS amino acid sequence in which glycine at position 12 is replaced with arginine, where position 12 is defined by reference to the corresponding WT RAS protein. The WT RAS protein may be any one of the WT KRAS protein (SEQ ID NO: 17 or 18), the WT HRAS protein (SEQ ID NO: 19), or the WT NRAS protein (SEQ ID NO: 20), because, as explained above, amino acid residues 1-86 of the WT human NRAS protein and the WT human KRAS protein are 100% identical, and amino acid residues 1-94 of the WT human HRAS protein and the WT human KRAS protein are also 100% identical. Thus, the amino acid residue at position 12 of each of the WT KRAS, WT HRAS, and WT NRAS proteins is the same, i.e., glycine.

[0019] The mutant human RAS amino acid sequence has an arginine substitution for glycine at position 12. In this regard, embodiments of the present invention provide a TCR with antigen specificity for any human RAS protein, polypeptide, or peptide amino acid sequence having a G12R mutation.

[0020] RAS mutations and substitutions are defined herein by reference to the amino acid sequence of the corresponding WT RAS protein. Accordingly, RAS mutations and substitutions are described herein by reference to the amino acid residue present at a particular position (i.e., position 12) of a WT RAS protein, followed by the position number, followed by the amino acid residue that replaces said residue in the particular mutation or substitution under consideration. A RAS amino acid sequence (e.g., a RAS peptide) may contain fewer than all of the amino acid residues of a full-length WT RAS protein. Accordingly, position 12 is defined herein by reference to a WT full-length RAS protein (i.e., any one of SEQ ID NOs: 17-20), with the understanding that the actual positions of the corresponding residues may differ in specific examples of RAS amino acid sequences. When a position is as defined by any one of SEQ ID NOs: 17-20, the term "G12" refers to the glycine normally present at position 12 of any one of SEQ ID NOs: 17-20, and "G12R" indicates that the glycine normally present at position 12 of any one of SEQ ID NOs: 17-20 has been replaced with an arginine. For example, a specific example of a RAS amino acid sequence is TEYKLVVVGA. G GVGKSALTIQLI (SEQ ID NO:25), an exemplary WT KRAS peptide corresponding to consecutive amino acid residues 2-24 of SEQ ID NO:17, "G12R" refers to the substitution of the underlined glycine in SEQ ID NO:25 with a valine, even though the actual position of the underlined glycine in SEQ ID NO:25 is 11. Hereinafter, a human RAS amino acid sequence having a G12R mutation is referred to as "G12R RAS."

[0021] Examples of full-length RAS proteins having the G12R mutation are listed in Table 1 below.

[0022] [Table 1]

[0023] In an embodiment of the present invention, the TCR has antigen specificity for a RAS peptide having the G12R mutation, and the G12R RAS peptide has any length. In an embodiment of the present invention, the G12R RAS peptide has any length suitable for binding to any of the HLA class II molecules described herein. For example, the TCR may have antigen specificity for a RAS peptide having a G12R mutation, the RAS peptide having a length of about 11 to about 30 amino acid residues, about 12 to about 24 amino acid residues, or about 18 to about 20 amino acid residues. The G12R RAS peptide may include any consecutive amino acid residues of a mutant RAS protein containing a G12R mutation. In an embodiment of the present invention, the TCR may have antigen specificity for a mutant RAS peptide having a G12R mutation, the mutant RAS peptide having a length of about 30 amino acid residues, about 29 amino acid residues, about 28 amino acid residues, about 27 amino acid residues, about 26 amino acid residues, about 25 amino acid residues, about 24 amino acid residues, about 23 amino acid residues, about 22 amino acid residues, about 21 amino acid residues, about 20 amino acid residues, about 19 amino acid residues, about 18 amino acid residues, about 17 amino acid residues, about 16 amino acid residues, about 15 amino acid residues, about 14 amino acid residues, about 13 amino acid residues, about 12 amino acid residues, about 11 amino acid residues, or a range of any two of the above values. An example of a specific peptide having a G12R mutation that can be recognized by the TCR of the present invention is MTEYKLVVVGA. R GVGKSALTIQLI (SEQ ID NO: 39). In an embodiment of the invention, the TCR has antigen specificity for the mutant human RAS amino acid sequence of SEQ ID NO: 39. In an embodiment of the invention, the TCR does not have antigen specificity for the wild-type human RAS amino acid sequence of SEQ ID NO: 40.

[0024] In an embodiment of the present invention, the TCR of the present invention can recognize the G12R RAS presented by HLA class II molecules. In this regard, the TCR can induce an immune response when it binds to the G12R RAS in the frame of the HLA class II molecule. The TCR of the present invention can recognize the G12R RAS presented by the HLA class II molecule, and can bind to the HLA class II molecule in addition to the G12R RAS.

[0025] In an embodiment of the present invention, the HLA class II molecule is an HLA-DR heterodimer. The HLA-DR heterodimer is a cell surface receptor that includes an alpha chain and a beta chain. The alpha chain of HLA-DR is encoded by the HLA-DRA gene. The beta chain of HLA-DR is encoded by the HLA-DRB1 gene, the HLA-DRB3 gene, the HLA-DRB4 gene, or the HLA-DRB5 gene. Examples of molecules encoded by HLA-DRB1 include, but are not limited to, HLA-DR1, HLA-DR2, HLA-DR3, HLA-DR4, HLA-DR5, HLA-DR6, HLA-DR7, HLA-DR8, HLA-DR9, HLA-DR10, HLA-DR11, HLA-DR12, HLA-DR13, HLA-DR14, HLA-DR15, HLA-DR16, and HLA-DR17. The HLA-DRB3 gene encodes HLA-DR52. The HLA-DRB4 gene encodes HLA-DR53. The HLA-DRB5 gene encodes HLA-DR51. In an embodiment of the invention, the HLA class II molecule is an HLA-DRB5:HLA-DRA heterodimer. In a particularly preferred embodiment, the HLA class II molecule is HLA-DRB5:HLA-DRA heterodimer. * 01:HLA-DRA * 01:01 allele (i.e., HLA-DRB5 * 01:HLA-DRA * 01:01 heterodimer).

[0026] In another embodiment of the present invention, the HLA class II molecule is an HLA-DQ heterodimer. The HLA-DQ heterodimer is a cell surface receptor that includes an α chain and a β chain. The α chain of HLA-DQ is encoded by the HLA-DQA1 gene. The HLA-DQA1 allele includes DQA1 * 01:01, DQA1 * 01:02, DQA1 * 01:03, DQA1 * 01:04, DQA1 * 02:01, DQA1 * 03:01, DQA1 * 03:02, DQA1 * 03:03, DQA1 * 04:01, DQA1 * 05:01, DQA1 * 05:05 and DQA1 * The HLA-DQ beta chain is encoded by the HLA-DQB1 gene. The HLA-DQB1 allele is * 02:01, HLA-DQB1 * 02:02, HLA-DQB1 * 02:03, HLA-DQB1 * 03:01, HLA-DQB1 * 03:02, HLA-DQB1 * 03:03, HLA-DQB1 * 03:04, HLA-DQB1 * 03:05, HLA-DQB1 * 04:01, HLA-DQB1 * 04:02, HLA-DQB1 * 05:01, HLA-DQB1 * 05:02, HLA-DQB1 * 05:03, HLA-DQB1 * 05:04, HLA-DQB1 * 06:01, HLA-DQB1 * 06:02, HLA-DQB1 * 06:03, HLA-DQB1 * 06:04, HLA-DQB1 * 06:05, and HLA-DQB1 *06:09. In an embodiment of the invention, the HLA class II molecule is an HLA-DQA1:HLA-DQB1 heterodimer. In a particularly preferred embodiment, the HLA class II molecule is an HLA-DQA1:HLA-DQB1 heterodimer. * 05:05:HLA-DQB1 * 03:01 allele (i.e., HLA-DQA1 * 05:05:HLA-DQB1 * 03:01 heterodimer).

[0027] The TCRs of the present invention may provide any one or more of a variety of advantages, including when expressed by cells used for adoptive cell transfer. G12R RAS is expressed in cancer cells and not in normal non-cancer cells. Without being bound to a particular theory or mechanism, it is believed that the TCRs of the present invention advantageously target the destruction of cancer cells while minimizing or eliminating, and thereby reducing, the destruction of normal non-cancer cells, e.g., by minimizing or eliminating toxicity. Furthermore, since the G12R mutation is likely to occur in the early stages of tumorigenesis, the G12R RAS mutation may be expressed in substantially all of the patient's cancer cells. The TCRs of the present invention may advantageously successfully treat or prevent G12R RAS-positive cancers that do not respond to other types of treatment, such as chemotherapy, surgery, or radiation. Additionally, the TCRs of the present invention may be used to successfully treat or prevent G12R RAS-positive cancers that are not responsive to other types of treatment, such as chemotherapy, surgery, or radiation. Additionally, the TCRs of the present invention may be used to treat tumors that are not engineered (e.g., tumors that have not been treated with interferon (IFN)-γ, and that express G12R RAS and HLA-DRB5). * 01:HLA-DRA * 01:01, G12R RAS and / or HLA-DQA1 * 05:05:HLA-DQB1 * 03:01, 03:02, 03:03, 03:04, 03:05, 03:06, 03:07, 03:08, 03:09, 03:10, 03:11, 03:12, 03:13, 03:14, 03:15, 03:16, 03:17, 03:18, 03:19, 03:20, 03:21, 03:22, 03:23, 03:24, 03:25, 03:26, 03:27, 03:28, 03:29, 03:30, 03:31, 03:32, 03:33, 03:34, 03:35, 03:36, 03:37, 03:38, 03:39, 03:39, 03:31, 03:32, 03:34, 03:35, 03:36, 03:37, 03:38, 03:39 ...9, 03:39, 03:39, 03:39, 03:39, 03:39, 03:39, 03:39, 03:39, 03:39, 03:39, 03:39, 03:39, *The 01 allele is expressed at a frequency of approximately 18% in the human population and is associated with HLA-DQA1 * 05:05:HLA-DQB1 * 03:01 is expressed in approximately 11% of humans of Caucasian ethnicity in the United States. Thus, the TCRs of the present invention may not be eligible for immunotherapy using TCRs that recognize G12R RAS presented by other MHC molecules. * 01:HLA-DRA * 01:01 Allele and HLA-DQA1 * 05:05:HLA-DQB1 * The number of immunotherapy-eligible cancer patients can be expanded to include those who express one or both 03:01 alleles. HLA-DRB5 in various cancers in the United States * 01:HLA-DRA * 01:01 and HLA-DQA1 * 05:05:HLA-DQB1 * Based on the estimated frequency of the 03:01 allele and G12R RAS expression, it is estimated that more than about 1000 pancreatic cancer patients may be eligible for treatment with one of the TCRs of the present invention each year. For example, the KRAS G12R mutation is expressed in more than about 8% of pancreatic cancer patients.

[0028] The phrase "antigen specificity" as used herein means that the TCR is capable of specifically binding to and immunologically recognizing G12R RAS with high avidity. For example, the TCR is capable of expressing about 1×10 IgG1-expressing G12R RAS when co-cultured with (a) antigen-negative HLA class II molecule-positive target cells pulsed with a low concentration of G12R RAS peptide (e.g., about 0.05 ng / mL to about 10 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 8 ng / mL, 10 ng / mL, or a range defined by any two of the above values) or (b) antigen-negative HLA class II molecule-positive target cells into which a nucleotide sequence encoding G12R RAS has been introduced such that the target cells express G12R RAS. 4 ~Approx. 1×10 5T cells may be considered to have "antigen specificity" for G12R RAS if they secrete at least about 200 pg / mL or more (e.g., 200 pg / mL or more, 300 pg / mL or more, 400 pg / mL or more, 500 pg / mL or more, 600 pg / mL or more, 700 pg / mL or more, 1000 pg / mL or more, 5,000 pg / mL or more, 7,000 pg / mL or more, 10,000 pg / mL or more, 20,000 pg / mL or more, or a range defined by any two of the above values). Cells expressing the TCR of the present invention can also secrete IFN-γ when co-cultured with antigen-negative HLA class II molecule-positive target cells pulsed with a higher concentration of G12R RAS peptide. The HLA class II molecule may be any of the HLA class II molecules described herein (e.g., HLA-DRB5, HLA-DRB6, HLA-DRB7, HLA-DRB8, HLA-DRB9, HLA-DRB10, HLA-DRB11, HLA-DRB12, HLA-DRB13, HLA-DRB14, HLA-DRB15, HLA-DRB16, HLA-DRB17, HLA-DRB18, HLA-DRB20, HLA-DRB21, HLA-DRB22, HLA-DRB30, HLA-DRB40, HLA-DRB50, HLA-DRB51, HLA-DRB52, HLA-DRB53, HLA-DRB54, HLA-DRB55, HLA-DRB61, HLA-DRB62, HLA-DRB73, HLA-DRB84, HLA-DRB95, HLA-DRB155, H * 01:HLA-DRA * 01:01 Heterodimer or HLA-DQA1 * 05:05:HLA-DQB1 * 03:01 heterodimer).

[0029] Alternatively, or in addition, a TCR may be considered to have "antigen specificity" for G12R RAS if T cells expressing the TCR secrete at least two-fold more IFN-γ when co-cultured with (a) antigen-negative HLA class II molecule-positive target cells pulsed with a low concentration of G12R RAS peptide or (b) antigen-negative HLA class II molecule-positive target cells into which a nucleotide sequence encoding G12R RAS has been introduced such that the target cells express G12R RAS, compared to the amount of IFN-γ expressed by a negative control. The negative control may be, for example, (i) T cells expressing the TCR co-cultured with (a) antigen-negative HLA class II molecule-positive target cells pulsed with the same concentration of an irrelevant peptide (e.g., some other peptide having a sequence different from the G12R RAS peptide) or (b) antigen-negative HLA class II molecule-positive target cells into which a nucleotide sequence encoding the irrelevant peptide has been introduced such that the target cells express the irrelevant peptide, or (ii) non-transduced TCR cells (e.g., derived from PBMCs not expressing the TCR) co-cultured with (a) antigen-negative HLA class II molecule-positive target cells pulsed with the same concentration of G12R RAS peptide or (b) antigen-negative HLA class II molecule-positive target cells into which a nucleotide sequence encoding G12R RAS has been introduced such that the target cells express G12R RAS. The HLA class II molecule expressed by the negative control target cells is the same HLA class II molecule expressed by the target cells co-cultured with the T cells to be tested. The HLA class II molecule may be any of the HLA class II molecules described herein (e.g., HLA-DRB5 * 01:HLA-DRA * 01:01 Heterodimer or HLA-DQA1 * 05:05:HLA-DQB1 * 03:01 heterodimer). IFN-γ secretion can be measured by methods known in the art, for example, enzyme-linked immunosorbent assay (ELISA).

[0030] Alternatively, or in addition, a TCR may be considered to have "antigen specificity" for G12R RAS if at least two times more T cells expressing the TCR secrete IFN-γ when co-cultured with (a) antigen-negative HLA class II molecule-positive target cells pulsed with a low concentration of G12R RAS peptide or (b) antigen-negative HLA class II molecule-positive target cells into which a nucleotide sequence encoding G12R RAS has been introduced such that the target cells express G12R RAS, compared to the number of negative control T cells secreting IFN-γ. The HLA class II molecule, concentration of peptide, and negative control may be as described herein for other aspects of the invention. The number of cells secreting IFN-γ can be measured by methods known in the art, for example, by ELISPOT.

[0031] Alternatively, or additionally, a TCR may be considered to have "antigen specificity" for G12R RAS if T cells expressing the TCR upregulate expression of one or more T cell activation markers, as measured, for example, by flow cytometry, following stimulation with a target cell expressing G12R RAS. Examples of T cell activation markers include 4-1BB, OX40, CD107a, CD69, and cytokines that are upregulated upon antigenic stimulation (e.g., tumor necrosis factor (TNF), interleukin (IL)-2, etc.).

[0032] Embodiments of the present invention provide a TCR comprising two polypeptides (i.e., polypeptide chains), such as, for example, a TCR alpha (α) chain, a TCR beta (β) chain, a TCR gamma (γ) chain, a TCR delta (δ) chain, or a combination thereof. The polypeptides of the TCRs of the present invention may comprise any amino acid sequence, so long as the TCR has antigen specificity for the G12R RAS. In some embodiments, the TCR is not naturally occurring.

[0033] In an embodiment of the invention, the TCR comprises two polypeptide chains, each comprising a variable region comprising TCR complementarity determining region (CDR) 1, CDR2, and CDR3. In an embodiment of the invention, the TCR comprises a first polypeptide chain comprising CDR1 (CDR1 of the alpha chain of 4268 TCR) comprising the amino acid sequence of SEQ ID NO: 1, CDR2 (CDR2 of the alpha chain of 4268 TCR) comprising the amino acid sequence of SEQ ID NO: 2, and CDR3 (CDR3 of the alpha chain of 4268 TCR) comprising the amino acid sequence of SEQ ID NO: 3, and a second polypeptide chain comprising CDR1 (CDR1 of the beta chain of 4268 TCR) comprising the amino acid sequence of SEQ ID NO: 4, CDR2 (CDR2 of the beta chain of 4268 TCR) comprising the amino acid sequence of SEQ ID NO: 5, and CDR3 (CDR3 of the beta chain of 4268 TCR) comprising the amino acid sequence of SEQ ID NO: 6.

[0034] In another embodiment of the invention, the TCR comprises a first polypeptide chain comprising a CDR1 (CDR1 of the alpha chain of 4270 TCR) comprising the amino acid sequence of SEQ ID NO:7, a CDR2 (CDR2 of the alpha chain of 4270 TCR) comprising the amino acid sequence of SEQ ID NO:8, and a CDR3 (CDR3 of the alpha chain of 4270 TCR) comprising the amino acid sequence of SEQ ID NO:9, and a second polypeptide chain comprising a CDR1 (CDR1 of the beta chain of 4270 TCR) comprising the amino acid sequence of SEQ ID NO:10, a CDR2 (CDR2 of the beta chain of 4270 TCR) comprising the amino acid sequence of SEQ ID NO:11, and a CDR3 (CDR3 of the beta chain of 4270 TCR) comprising the amino acid sequence of SEQ ID NO:12.

[0035] In this regard, the TCR of the present invention may comprise any one or more of the amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 12. In an embodiment of the present invention, the TCR comprises the amino acid sequences of (a) all of SEQ ID NOs: 1 to 3, (b) all of SEQ ID NOs: 4 to 6, (c) all of SEQ ID NOs: 7 to 9, (d) all of SEQ ID NOs: 10 to 12, (e) all of SEQ ID NOs: 1 to 6, or (f) all of SEQ ID NOs: 7 to 12. In a particularly preferred embodiment, the TCR comprises the amino acid sequences of (i) all of SEQ ID NOs: 1 to 6 or (ii) all of SEQ ID NOs: 7 to 12.

[0036] In an embodiment of the invention, the TCR comprises an amino acid sequence of a variable region of a TCR comprising the above CDRs. In this regard, the TCR may comprise the amino acid sequence of SEQ ID NO: 13 (variable region of the alpha chain of 4268 TCR with N-terminal signal peptide); SEQ ID NO: 14 (variable region of the beta chain of 4268 TCR with N-terminal signal peptide); SEQ ID NO: 15 (variable region of the alpha chain of 4270 TCR with N-terminal signal peptide); SEQ ID NO: 16 (variable region of the beta chain of 4270 TCR with N-terminal signal peptide); SEQ ID NO: 43 (variable region of the alpha chain of 4268 TCR without N-terminal signal peptide); SEQ ID NO: 44 (variable region of the beta chain of 4268 TCR without N-terminal signal peptide); SEQ ID NO: 41 (variable region of the alpha chain of 4270 TCR without N-terminal signal peptide); SEQ ID NO: 42 (variable region of the beta chain of 4270 TCR without N-terminal signal peptide); both SEQ ID NOs: 13 and 14; both SEQ ID NOs: 15 and 16; both SEQ ID NOs: 41 and 42; or both SEQ ID NOs: 43 and 44. Preferably, the TCR comprises the amino acid sequences of: (i) both SEQ ID NOs: 13 and 14; (ii) both SEQ ID NOs: 15 and 16; (iii) both SEQ ID NOs: 41 and 42; or (iv) both SEQ ID NOs: 43 and 44.

[0037] The TCRs of the invention may further comprise an alpha chain constant region and a beta chain constant region. The constant regions may be from any suitable species, such as, for example, human or mouse. In an embodiment of the invention, the TCR further comprises a mouse alpha and beta chain constant region or a human alpha and beta chain constant region. As used herein, the terms "mouse" or "human" when referring to a TCR or any component of a TCR described herein (e.g., CDRs, variable regions, constant regions, alpha chains, and / or beta chains) refer to a TCR (or component thereof) of mouse or human origin, respectively, i.e., a TCR (or component thereof) that originates from or was once expressed by a mouse T cell or a human T cell, respectively.

[0038] An embodiment of the present invention provides a chimeric TCR comprising a human variable region and a mouse constant region, the TCR having antigen specificity for a mutated human RAS amino acid sequence in which glycine at position 12 is replaced with arginine. The mouse constant region may provide any one or more advantages. For example, the mouse constant region may reduce mispairing of the TCR of the present invention with the endogenous TCR of the host cell into which the TCR of the present invention is introduced. Alternatively, or additionally, the mouse constant region may increase expression of the TCR of the present invention compared to the same TCR with a human constant region. The chimeric TCR may comprise the amino acid sequence of SEQ ID NO: 28 (WT mouse α chain constant region), SEQ ID NO: 29 (WT mouse β chain constant region), or both SEQ ID NO: 28 and 29. Preferably, the TCR of the present invention comprises the amino acid sequence of both SEQ ID NO: 28 and 29. The chimeric TCR may comprise any of the mouse constant regions described herein in combination with any of the CDR regions described herein with respect to other aspects of the invention. In this regard, the TCR may comprise the amino acid sequence of (a) all of SEQ ID NOs: 1-3 and 28; (b) all of SEQ ID NOs: 4-6 and 29; (c) all of SEQ ID NOs: 7-9 and 28; (d) all of SEQ ID NOs: 10-12 and 29; (e) all of SEQ ID NOs: 1-6 and 28-29; or (f) all of SEQ ID NOs: 7-12 and 28-29. In another embodiment of the invention, the chimeric TCR may comprise any of the murine constant regions described herein in combination with any of the variable regions described herein with respect to other aspects of the invention. In this regard, the TCR may comprise the amino acid sequence of (i) both of SEQ ID NOs: 13 and 28; (ii) both of SEQ ID NOs: 14 and 29; (iii) both of SEQ ID NOs: 15 and 28; (iv) both of SEQ ID NOs: 16 and 29; (v) all of SEQ ID NOs: 13-14 and 28-29; or (vi) all of SEQ ID NOs: 15-16 and 28-29.

[0039] In another embodiment of the invention, the TCR is selected from SEQ ID NO:34 (4268 TCR alpha chain with WT mouse constant region and N-terminal signal peptide), SEQ ID NO:35 (4268 TCR beta chain with WT mouse constant region and N-terminal signal peptide), SEQ ID NO:36 (4270 TCR alpha chain with WT mouse constant region and N-terminal signal peptide), SEQ ID NO:37 (beta chain of 4270 TCR with WT mouse constant region and N-terminal signal peptide), SEQ ID NO:49 (alpha chain of 4268 TCR with WT mouse constant region and no N-terminal signal peptide), SEQ ID NO:50 (beta chain of 4268 TCR with WT mouse constant region and no N-terminal signal peptide), SEQ ID NO:51 (alpha chain of 4270 TCR with WT mouse constant region and no N-terminal signal peptide), SEQ ID NO:52 (4270 TCR with WT mouse constant region and no N-terminal signal peptide). TCR β chain), both of the amino acid sequences of SEQ ID NOs: 34 to 35, both of SEQ ID NOs: 36 to 37, both of SEQ ID NOs: 49 to 50, or both of SEQ ID NOs: 51 to 52 (multiple sequences are possible).

[0040] In an embodiment of the invention, the TCR comprises an alpha chain comprising a variable region and a constant region, and a beta chain comprising a variable region and a constant region. In this regard, the TCR may comprise: (a) an α chain comprising the amino acid sequence of SEQ ID NO: 30, wherein (i) X at position 180 of SEQ ID NO: 30 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 30 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 246 of SEQ ID NO: 30 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 247 of SEQ ID NO: 30 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp (α chain of 4268 TCR with N-terminal signal peptide); (b) a β chain comprising the amino acid sequence of SEQ ID NO: 31, wherein X at position 198 of SEQ ID NO: 31 is Ser or Cys (α chain of 4268 TCR with N-terminal signal peptide). (c) an α chain comprising the amino acid sequence of SEQ ID NO: 32, in which (i) X at position 188 of SEQ ID NO: 32 is Thr or Cys; (ii) X at position 252 of SEQ ID NO: 32 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 254 of SEQ ID NO: 32 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 255 of SEQ ID NO: 32 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp (a 4270 TCR α chain with an N-terminal signal peptide); (d) a β chain comprising the amino acid sequence of SEQ ID NO: 33, in which X at position 191 of SEQ ID NO: 33 is Ser or Cys (a 4270 TCR α chain with an N-terminal signal peptide). (e) both (a) and (b); (f) both (c) and (d); (g) (i) X at position 161 of SEQ ID NO:45 is Thr or Cys; (ii) X at position 225 of SEQ ID NO:45 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 227 of SEQ ID NO:45 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp;and (iv) an α chain comprising the amino acid sequence of SEQ ID NO: 45, in which X at position 228 of SEQ ID NO: 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp (the α chain of a 4268 TCR without an N-terminal signal peptide); (h) a β chain comprising the amino acid sequence of SEQ ID NO: 46, in which X at position 178 of SEQ ID NO: 46 is Ser or Cys (the α chain of a 4268 TCR without an N-terminal signal peptide). (i) an α chain comprising the amino acid sequence of SEQ ID NO: 47, in which X at position 168 of SEQ ID NO: 47 is Thr or Cys; (ii) X at position 232 of SEQ ID NO: 47 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 234 of SEQ ID NO: 47 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 235 of SEQ ID NO: 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp (a 4270 TCR α chain without an N-terminal signal peptide); (j) a β chain comprising the amino acid sequence of SEQ ID NO: 48, in which X at position 171 of SEQ ID NO: 48 is Ser or Cys (a 4270 TCR α chain without an N-terminal signal peptide). (k) both (g) and (h); or (l) both (i) and (j);

[0041] In an embodiment of the invention, the TCR comprises a substituted constant region. In this regard, the TCR may comprise the amino acid sequence of any of the TCRs described herein, with one, two, three, or four amino acid substitution(s) in one or both of the α and β chain constant regions. Preferably, the TCR comprises a murine constant region with one, two, three, or four amino acid substitution(s) in one or both of the α and β chain murine constant regions. In a particularly preferred embodiment, the TCR comprises a murine constant region with one, two, three, or four amino acid substitution(s) in the murine constant region of the α chain and one amino acid substitution in the murine constant region of the β chain. In some embodiments, the TCR comprising the substituted constant region advantageously has a higher G12R RAS constant region than a parent TCR comprising an unsubstituted (wild type) constant region. +and / or increased target recognition, increased expression by host cells, decreased mispairing with endogenous TCR, and increased anti-tumor activity. In general, the substituted amino acid sequences of the murine constant regions of the α and β chains of the TCR, SEQ ID NOs:26 and 27, respectively, correspond to all or a portion of the unsubstituted murine constant region amino acid sequences, SEQ ID NOs:28 and 29, respectively, with SEQ ID NO:26 having one, two, three, or four amino acid substitution(s) compared to SEQ ID NO:28, and SEQ ID NO:27 having one amino acid substitution compared to SEQ ID NO:29. In this regard, embodiments of the invention provide a TCR comprising the amino acid sequence of (a) SEQ ID NO: 26 (constant region of the α chain) in which (i) X at position 48 is Thr or Cys; (ii) X at position 112 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 114 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 115 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) SEQ ID NO: 27 (constant region of the β chain) in which X at position 57 is Ser or Cys; or (c) both SEQ ID NOs: 26 and 27. In an embodiment of the invention, a TCR comprising SEQ ID NO: 26 does not comprise SEQ ID NO: 28 (unsubstituted mouse constant region of the α chain). In an embodiment of the invention, a TCR comprising SEQ ID NO: 27 does not comprise SEQ ID NO: 29 (unsubstituted murine constant region of the β chain).

[0042] In an embodiment of the invention, the substituted constant region comprises a cysteine ​​substitution in one or both of the constant regions of the α and β chains to provide a cysteine-substituted TCR. Opposing cysteines in the α and β chains provide a disulfide bond that links the α and β chain constant regions of the substituted TCR together and is not present in a TCR comprising an unsubstituted mouse constant region. In this regard, the TCR may be a cysteine-substituted TCR in which one or both of the native Thr at position 48 of SEQ ID NO:28 (Thr48) and the native Ser at position 57 of SEQ ID NO:29 (Ser57) may be substituted with Cys. Preferably, both the native Thr48 of SEQ ID NO:28 and the native Ser57 of SEQ ID NO:29 are substituted with Cys. Exemplary constant region sequences for cysteine-substituted TCRs are set forth in Table 2. In embodiments of the invention, the cysteine ​​replaced TCR comprises (i) SEQ ID NO: 26, (ii) SEQ ID NO: 27, or (iii) both SEQ ID NOs: 26 and 27, both of which are as defined in Table 2. The cysteine ​​replaced TCRs of the invention may comprise a replaced constant region in addition to any of the CDRs or variable regions described herein.

[0043] In an embodiment of the invention, the cysteine-substituted chimeric TCR comprises a full-length alpha chain and a full-length beta chain. Exemplary sequences of the alpha and beta chains of cysteine-substituted chimeric TCRs are set forth in Table 2. In an embodiment of the invention, the TCR comprises (i) SEQ ID NO: 30, (ii) SEQ ID NO: 31, (iii) SEQ ID NO: 32, (iv) SEQ ID NO: 33, (v) both SEQ ID NOs: 30 and 31, (vi) both SEQ ID NOs: 32 and 33, (vii) SEQ ID NO: 45, (viii) SEQ ID NO: 46, (ix) SEQ ID NO: 47, (x) SEQ ID NO: 48, (xi) both SEQ ID NOs: 45 and 46, or (xii) both SEQ ID NOs: 47 and 48, where SEQ ID NOs: 30-33 and 45-48 are all as defined in Table 2.

[0044] [Table 2]

[0045] In an embodiment of the invention, the replacement amino acid sequence comprises the replacement of one, two or three amino acids in the transmembrane (TM) domain of one or both of the α and β chain constant regions with hydrophobic amino acids to provide a hydrophobic amino acid replaced TCR (also referred to herein as "LVL modified TCR"). The hydrophobic amino acid replacement(s) in the TM domain of the TCR may increase the hydrophobicity of the TM domain of the TCR compared to a TCR that does not have the hydrophobic amino acid replacement(s) in the TM domain. In this regard, the TCR is an LVL modified TCR in which one, two or three of the native Ser112, Met114 and Gly115 of SEQ ID NO:28 may be independently replaced with Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; preferably Leu, Ile or Val. Preferably, all three of the native Ser112, Met114, and Gly115 of SEQ ID NO:28 may be independently substituted with Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; preferably Leu, Ile, or Val. In embodiments of the invention, the LVL-modified TCR comprises (i) SEQ ID NO:26, (ii) SEQ ID NO:27, or (iii) both SEQ ID NOs:26 and 27, both of which are as defined in Table 3. The LVL-modified TCRs of the invention may comprise a substituted constant region in addition to any of the CDRs or variable regions described herein.

[0046] In an embodiment of the invention, the LVL-modified TCR comprises a full-length alpha chain and a full-length beta chain. Exemplary sequences of the alpha and beta chains of the LVL-modified TCR are set forth in Table 3. In an embodiment of the invention, the LVL-modified TCR comprises (i) SEQ ID NO: 30, (ii) SEQ ID NO: 31, (iii) SEQ ID NO: 32, (iv) SEQ ID NO: 33, (v) both SEQ ID NOs: 30 and 31, (vi) both SEQ ID NOs: 32 and 33, (vii) SEQ ID NO: 45, (viii) SEQ ID NO: 46, (ix) SEQ ID NO: 47, (x) SEQ ID NO: 48, (xi) both SEQ ID NOs: 45 and 46, or (xii) both SEQ ID NOs: 47 and 48, where SEQ ID NOs: 30-33 and 45-48 are all as defined in Table 3.

[0047] [Table 3-1]

[0048] [Table 3-2]

[0049] In an embodiment of the invention, the replaced amino acid sequence comprises a cysteine ​​substitution in one or both of the constant regions of the α and β chains in combination with a hydrophobic amino acid substitution(s) of one, two or three amino acids in the transmembrane (TM) domains of one or both of the constant regions of the α and β chains (also referred to herein as "cysteine-substituted LVL-modified TCRs"). In this regard, the TCR is a cysteine-substituted LVL-modified chimeric TCR in which native Thr48 of SEQ ID NO:28 is replaced with Cys; one, two or three of native Ser112, Met114 and Gly115 of SEQ ID NO:28 are replaced independently with Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; preferably Leu, Ile or Val; and native Ser57 of SEQ ID NO:29 is replaced with Cys. Preferably, all three of the native Ser112, Met114, and Gly115 of SEQ ID NO:28 may be independently substituted with Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; preferably Leu, Ile, or Val. In an embodiment of the invention, the cysteine-substituted LVL-modified TCR comprises (i) SEQ ID NO:26, (ii) SEQ ID NO:27, or (iii) both SEQ ID NOs:26 and 27, both of which are as defined in Table 4. The cysteine-substituted LVL-modified TCR of the invention may comprise a substituted constant region in addition to any of the CDRs or variable regions described herein.

[0050] In an embodiment, the cysteine ​​substituted LVL modified TCR comprises a full length alpha chain and a full length beta chain. In an embodiment of the invention, the cysteine ​​substituted LVL modified TCR comprises (i) SEQ ID NO: 30, (ii) SEQ ID NO: 31, (iii) SEQ ID NO: 32, (iv) SEQ ID NO: 33, (v) both SEQ ID NOs: 30 and 31, (vi) both SEQ ID NOs: 32 and 33, (vii) SEQ ID NO: 45, (viii) SEQ ID NO: 46, (ix) SEQ ID NO: 47, (x) SEQ ID NO: 48, (xi) both SEQ ID NOs: 45 and 46, or (xii) both SEQ ID NOs: 47 and 48, where SEQ ID NOs: 30-33 and 45-48 are all as defined in Table 4.

[0051] [Table 4-1]

[0052] [Table 4-2]

[0053] Also provided by the invention are polypeptides comprising a functional portion of any of the TCRs described herein. The term "polypeptide" as used herein includes oligopeptides and refers to a single chain of amino acids linked by one or more peptide bonds.

[0054] In relation to the polypeptides of the invention, a functional portion may be any portion comprising consecutive amino acids of the TCR of which it is a part, so long as it specifically binds to G12R RAS. The term "functional portion", when used in relation to a TCR, refers to any portion or fragment of a TCR of the invention that retains the biological activity of the TCR of which it is a part (the parent TCR). A functional portion may, for example, retain the biological activity of the parent TCR to a similar extent, the same extent, or to a greater extent (e.g., HLA-DRB5 * 01:HLA-DRA * 01:01 Heterodimer or HLA-DQA1 * 05:05:HLA-DQB1 *03:01 heterodimer) or that retains the ability to detect, treat, or prevent cancer. With respect to the parent TCR, the functional portion may constitute, for example, about 10%, about 25%, about 30%, about 50%, about 68%, about 80%, about 90%, about 95%, or more of the parent TCR.

[0055] A functional portion may comprise additional amino acids at the amino or carboxy terminus of the portion, or at both termini, that are not found in the amino acid sequence of the parent TCR. Desirably, the additional amino acids do not interfere with the biological function of the functional portion, e.g., specifically binding to G12R RAS and / or having the ability to detect, treat or prevent cancer. More desirably, the additional amino acids enhance the biological activity compared to the biological activity of the parent TCR.

[0056] The polypeptide may comprise a functional portion of either or both of the alpha and beta chains of the TCR of the invention, for example a functional portion comprising one or more of the CDR1, CDR2 and CDR3 of the variable region(s) of the alpha and / or beta chain of the TCR of the invention. In an embodiment of the invention, the polypeptide may comprise the amino acid sequence of SEQ ID NO: 1 (alpha chain CDR1), SEQ ID NO: 2 (alpha chain CDR2), SEQ ID NO: 3 (alpha chain CDR3), SEQ ID NO: 4 (beta chain CDR1), SEQ ID NO: 5 (beta chain CDR2), SEQ ID NO: 6 (beta chain CDR3), or a combination thereof. In another embodiment of the invention, the polypeptide may comprise the amino acid sequence of SEQ ID NO: 7 (alpha chain CDR1), SEQ ID NO: 8 (alpha chain CDR2), SEQ ID NO: 9 (alpha chain CDR3), SEQ ID NO: 10 (beta chain CDR1), SEQ ID NO: 11 (beta chain CDR2), SEQ ID NO: 12 (beta chain CDR3), or a combination thereof.

[0057] In this regard, the polypeptide of the present invention may comprise any one or more of the amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 12. In an embodiment of the present invention, the TCR comprises the amino acid sequences of (a) all of SEQ ID NOs: 1 to 3, (b) all of SEQ ID NOs: 4 to 6, (c) all of SEQ ID NOs: 7 to 9, (d) all of SEQ ID NOs: 10 to 12, (e) all of SEQ ID NOs: 1 to 6, or (f) all of SEQ ID NOs: 7 to 12. In a preferred embodiment, the polypeptide comprises the amino acid sequences of (i) all of SEQ ID NOs: 1 to 6 or (ii) all of SEQ ID NOs: 7 to 12.

[0058] In an embodiment of the invention, the polypeptide of the invention may comprise a variable region of a TCR of the invention, for example comprising a combination of the above CDR regions. In this regard, the polypeptide may comprise the amino acid sequence of (i) SEQ ID NO: 13 (variable region of the α chain), (ii) SEQ ID NO: 14 (variable region of the β chain), (iii) both SEQ ID NOs: 13 and 14, (iv) SEQ ID NO: 15 (variable region of the α chain), (v) SEQ ID NO: 16 (variable region of the β chain), (vi) both SEQ ID NOs: 15 and 16, (vii) SEQ ID NO: 41 (variable region of the α chain), (viii) SEQ ID NO: 42 (variable region of the β chain), (ix) both SEQ ID NOs: 41 and 42, (x) SEQ ID NO: 43 (variable region of the α chain), (xi) SEQ ID NO: 44 (variable region of the β chain), or (xii) both SEQ ID NOs: 43 and 44. Preferably, the polypeptide comprises the amino acid sequences of (i) both SEQ ID NOs:13 and 14, (ii) both SEQ ID NOs:15 and 16, (iii) both SEQ ID NOs:41 and 42, or (iv) both SEQ ID NOs:43 and 44.

[0059] In an embodiment of the invention, the polypeptide of the invention may further comprise a constant region of a TCR of the invention as described above. In this regard, the polypeptide may further comprise the amino acid sequence of SEQ ID NO: 28 (WT mouse constant region of the α chain), SEQ ID NO: 29 (WT mouse constant region of the β chain), SEQ ID NO: 26 (substituted mouse constant region of the α chain), SEQ ID NO: 27 (substituted mouse constant region of the β chain), both SEQ ID NO: 28 and 29, or both SEQ ID NO: 26 and 27. Preferably, the polypeptide further comprises the amino acid sequence of both SEQ ID NO: 26 and 27, or both SEQ ID NO: 28 and 29, in combination with any of the CDR regions or variable regions described herein with respect to other aspects of the invention. In an embodiment of the invention, one or both of SEQ ID NO: 26 and 27 of the polypeptide are as defined in any one of Tables 2 to 4.

[0060] In an embodiment of the invention, the polypeptide of the invention may comprise the full length of the α-chain or β-chain of a TCR as described herein. In this regard, the polypeptide of the invention may comprise the amino acid sequence of SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, both SEQ ID NOs:30-31, both SEQ ID NOs:32-33, both SEQ ID NOs:34-35, both SEQ ID NOs:36-37, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, both SEQ ID NOs:45 and 46, both SEQ ID NOs:47 and 48, both SEQ ID NOs:49 and 50, or both SEQ ID NOs:51 and 52. Alternatively, the polypeptide of the invention may comprise both chains of a TCR as described herein.

[0061] For example, the polypeptide of the present invention may comprise: (a) an amino acid sequence of SEQ ID NO:30, in which: (i) X at position 180 of SEQ ID NO:30 is Thr or Cys; (ii) X at position 244 of SEQ ID NO:30 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 246 of SEQ ID NO:30 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 247 of SEQ ID NO:30 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp. (b) the amino acid sequence of SEQ ID NO:31, in which X at position 198 of SEQ ID NO:31 is Ser or Cys; (c) (i) X at position 188 of SEQ ID NO:32 is Thr or Cys; (ii) X at position 252 of SEQ ID NO:32 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 254 of SEQ ID NO:32 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 255 of SEQ ID NO:32 is Gly, Ala, Val, Leu, Ile, Pro, Phe, or Trp. (d) the amino acid sequence of SEQ ID NO: 33, wherein X at position 191 of SEQ ID NO: 33 is Ser or Cys; (e) both (a) and (b); (f) both (c) and (d); (g) (i) X at position 161 of SEQ ID NO: 45 is Thr or Cys; (ii) X at position 225 of SEQ ID NO: 45 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 227 of SEQ ID NO: 45 is Met, Ala, Val, Le and (iv) the amino acid sequence of SEQ ID NO: 45, wherein X at position 228 of SEQ ID NO: 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (h) the amino acid sequence of SEQ ID NO: 46, wherein X at position 178 of SEQ ID NO: 46 is Ser or Cys; (i) (i) the X at position 168 of SEQ ID NO: 47 is Thr or Cys; (ii) the X at position 232 of SEQ ID NO: 47 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp;(iii) the amino acid sequence of SEQ ID NO: 47, in which X at position 234 of SEQ ID NO: 47 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) the amino acid sequence of SEQ ID NO: 47, in which X at position 235 of SEQ ID NO: 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (j) the amino acid sequence of SEQ ID NO: 48, in which X at position 171 of SEQ ID NO: 48 is Ser or Cys; (k) both (g) and (h); or (l) both (i) and (j). In an embodiment of the invention, any one or more of SEQ ID NOs: 30 to 33 and 45 to 48 of the polypeptide are as defined in any one of Tables 2 to 4.

[0062] The present invention further provides a protein comprising at least one of the polypeptides described herein.By "protein" is meant a molecule comprising one or more polypeptide chains.

[0063] In an embodiment, the protein of the present invention may comprise: (a) a first polypeptide chain comprising an amino acid sequence of any one of SEQ ID NOs: 1 to 3 and a second polypeptide chain comprising an amino acid sequence of any one of SEQ ID NOs: 4 to 6; or (b) a first polypeptide chain comprising an amino acid sequence of any one of SEQ ID NOs: 7 to 9 and a second polypeptide chain comprising an amino acid sequence of any one of SEQ ID NOs: 10 to 12.

[0064] In another embodiment of the invention, the protein may comprise: (i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:13 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:14; (ii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:15 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:16; (iii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:41 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:42; or (iv) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:43 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:44.

[0065] The proteins of the invention may further comprise any of the constant regions described herein with respect to other aspects of the invention. In this regard, in embodiments of the invention, (i) the first polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 26 and the second polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 27; or (ii) the first polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 28 and the second polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 29. In embodiments of the invention, one or both of SEQ ID NOs: 26 and 27 of the protein are as defined in any one of Tables 2-4.

[0066] Alternatively, or in addition, the protein of the present invention has the following structure: (a) (i) X at position 180 of SEQ ID NO: 30 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 30 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 246 of SEQ ID NO: 30 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 247 of SEQ ID NO: 30 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Tr. (b) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 31, wherein X at position 198 of SEQ ID NO: 31 is Ser or Cys; (c) (i) X at position 188 of SEQ ID NO: 32 is Thr or Cys; (ii) X at position 252 of SEQ ID NO: 32 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 254 of SEQ ID NO: 32 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp. and (iv) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 32, wherein X at position 255 of SEQ ID NO: 32 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (d) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 33, wherein X at position 191 of SEQ ID NO: 33 is Ser or Cys; (e) both (a) and (b); (f) both (c) and (d); (g) (i) X at position 161 of SEQ ID NO: 45 is Thr or Cys; (ii) X at position 225 of SEQ ID NO: 45 is Ser, Ala, (iii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 45, wherein X at position 227 of SEQ ID NO: 45 is Met, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; and (iv) X at position 228 of SEQ ID NO: 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (h) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 46, wherein X at position 178 of SEQ ID NO: 46 is Ser or Cys;(i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 47, wherein (i) X at position 168 of SEQ ID NO: 47 is Thr or Cys; (ii) X at position 232 of SEQ ID NO: 47 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 234 of SEQ ID NO: 47 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 235 of SEQ ID NO: 47 is GIy, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (j) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 48, wherein X at position 171 of SEQ ID NO: 48 is Ser or Cys; (k) both (g) and (h); or (l) both (i) and (j). In an embodiment of the invention, one or more of SEQ ID NOs: 30-33 and 45-48 are as defined in any one of Tables 2-4;

[0067] The protein of the invention may be a TCR. Alternatively, the protein of the invention may be a fusion protein, for example, when the protein comprises one polypeptide chain comprising the amino acid sequences of both SEQ ID NOs: 30 and 31, both SEQ ID NOs: 32 and 33, or when the first and / or second polypeptide chain(s) of the protein further comprises another amino acid sequence, for example an amino acid sequence encoding an immunoglobulin or a portion thereof. In this regard, the invention also provides fusion proteins comprising at least one of the polypeptides of the invention described herein, together with at least one other polypeptide. The other polypeptide may be present as a separate protein of a fusion protein, or as a polypeptide expressed in frame (in tandem) with one of the polypeptides of the invention described herein. The other polypeptide may encode any peptidic or proteinaceous molecule, or a portion thereof, including, but not limited to, an immunoglobulin, CD3, CD4, CD8, an MHC molecule, a CD1 molecule, e.g., CD1a, CD1b, CD1c, CD1d, etc.

[0068] A fusion protein may contain one or more copies of a polypeptide of the invention and / or one or more copies of another polypeptide. For example, a fusion protein may contain 1, 2, 3, 4, 5, or more copies of a polypeptide of the invention and / or another polypeptide. Suitable methods for making fusion proteins are known in the art and include, for example, recombinant methods.

[0069] In some embodiments of the invention, the TCRs, polypeptides, and proteins of the invention may be expressed as a single protein comprising a linker peptide linking the alpha and beta chains. In this regard, the TCRs, polypeptides, and proteins of the invention may further comprise a linker peptide. The linker peptide may advantageously facilitate expression of the recombinant TCRs, polypeptides, and / or proteins in a host cell. The linker peptide may comprise any suitable amino acid sequence. The linker peptide may be a cleavable linker peptide. For example, the linker peptide may be a furin-SGSG-P2A linker comprising the amino acid sequence of SEQ ID NO: 38. Once the construct comprising the linker peptide is expressed by the host cell, the linker peptide may be cleaved, resulting in separated alpha and beta chains. In embodiments of the invention, the TCRs, polypeptides, or proteins may comprise an amino acid sequence comprising a full-length alpha chain, a full-length beta chain, and a linker peptide located between the alpha and beta chains.

[0070] The protein of the invention may be a recombinant antibody or antigen-binding portion thereof comprising at least one of the polypeptides of the invention described herein. As used herein, "recombinant antibody" refers to a recombinant (e.g., genetically engineered) protein comprising at least one of the polypeptides of the invention and an antibody polypeptide chain, or an antigen-binding portion thereof. The antibody polypeptide or antigen-binding portion thereof may be the antibody heavy chain, light chain, heavy or light chain variable or constant region, single chain variable region (scFv), or Fc, Fab, or F(ab)2' fragment, etc. The antibody polypeptide chain or antigen-binding portion thereof may be present as a separate polypeptide of the recombinant antibody. Alternatively, the antibody polypeptide chain or antigen-binding portion thereof may be present as a polypeptide expressed in frame (tandem) with a polypeptide of the invention. The antibody polypeptide or antigen-binding portion thereof may be any antibody or any antibody fragment polypeptide, including any of the antibodies and antibody fragments described herein.

[0071] Functional variants of the TCRs, polypeptides, or proteins of the present invention described herein are included within the scope of the present invention. The term "functional variant" as used herein refers to a TCR, polypeptide, or protein that has substantial or significant sequence identity or similarity to a parent TCR, polypeptide, or protein, and the functional variant retains the biological activity of the TCR, polypeptide, or protein of which it is a variant. Functional variants include, for example, variants of the TCRs, polypeptides, or proteins described herein (parent TCRs, polypeptides, or proteins) that retain to a similar, identical, or greater extent than the parent TCR, polypeptide, or protein the antigen specificity of the parent TCR or the ability to specifically bind to G12R RAS to which the parent polypeptide or protein specifically binds. With respect to a parent TCR, polypeptide, or protein, a functional variant may, for example, be at least about 30%, about 50%, about 75%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or more identical in amino acid sequence to the parent TCR, polypeptide, or protein, respectively.

[0072] A functional variant may, for example, comprise an amino acid sequence of a parent TCR, polypeptide, or protein with at least one conservative amino acid substitution. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid with particular physical and / or chemical properties is exchanged for another amino acid with the same chemical or physical properties. For example, a conservative amino acid substitution may be the substitution of an acidic amino acid with another acidic amino acid (e.g., Asp or Glu), an amino acid having a non-polar side chain with another amino acid having a non-polar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid with another basic amino acid (Lys, Arg, etc.), an amino acid having a polar side chain with another amino acid having a polar side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), etc.

[0073] Alternatively, or in addition, the functional variant may comprise the amino acid sequence of the parent TCR, polypeptide or protein with at least one non-conservative amino acid substitution, where the non-conservative amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant, preferably the non-conservative amino acid substitution is capable of enhancing the biological activity of the functional variant, such that the biological activity of the functional variant is increased compared to the parent TCR, polypeptide or protein.

[0074] A TCR, polypeptide, or protein may consist essentially of the specified amino acid sequence(s) described herein, such that other components of the TCR, polypeptide, or protein, e.g., other amino acids, do not substantially alter the biological activity of the TCR, polypeptide, or protein. In this regard, a TCR, polypeptide, or protein of the invention may consist essentially of the amino acid sequence of, for example, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, both SEQ ID NOs:30-31, both SEQ ID NOs:32-33, both SEQ ID NOs:34-35, or both SEQ ID NOs:36-37. Also, for example, a TCR, polypeptide, or protein of the invention may consist essentially of the amino acid sequence(s) of (i) SEQ ID NO:13, (ii) SEQ ID NO:14, (iii) SEQ ID NO:15, (iv) SEQ ID NO:16, (v) both SEQ ID NOs:13 and 14, or (vi) both SEQ ID NOs:15 and 16. Furthermore, the TCR, polypeptide, or protein of the present invention may essentially consist of the amino acid sequence of: (a) any one or more of SEQ ID NOs: 1 to 12; (b) all of SEQ ID NOs: 1 to 3; (c) all of SEQ ID NOs: 4 to 6; (d) all of SEQ ID NOs: 7 to 9; (e) all of SEQ ID NOs: 10 to 12; (f) all of SEQ ID NOs: 1 to 6; or (g) all of SEQ ID NOs: 7 to 12.

[0075] The TCRs, polypeptides, and proteins of the invention may be of any length, i.e., may contain any number of amino acids, so long as the TCR, polypeptide, or protein retains its biological activity, such as the ability to specifically bind to G12R RAS; detect cancer in a mammal; or treat or prevent cancer in a mammal. For example, a polypeptide may range from about 50 to about 5000 amino acids in length, such as about 50, about 70, about 75, about 100, about 125, about 150, about 175, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000 or more amino acids in length. In this regard, the polypeptides of the invention also include oligopeptides.

[0076] The TCRs, polypeptides and proteins of the invention may contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline, lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0077] The TCRs, polypeptides or proteins of the invention may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, e.g. via disulfide bridges, or converted into acid addition salts, and / or optionally dimerized or multimerized, or conjugated.

[0078] The TCRs, polypeptides, and / or proteins of the invention can be obtained by methods known in the art, such as de novo synthesis. Polypeptides and proteins can also be produced recombinantly using standard recombinant techniques and the nucleic acids described herein. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual ,4 th ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Alternatively, companies such as, for example, Synpep (Dublin, CA), Peptide Technologies Corp. (Gaithersburg, MD), and Multiple Peptide Systems (San Diego, CA) can commercially synthesize the TCRs, polypeptides, and / or proteins described herein. In this regard, the TCRs, polypeptides, and proteins of the invention may be synthetic, recombinant, isolated, and / or purified. An embodiment of the invention provides an isolated or purified TCR, polypeptide, or protein encoded by any of the nucleic acids or vectors described herein with respect to other aspects of the invention. Another embodiment of the invention provides an isolated or purified TCR, polypeptide, or protein resulting from the intracellular expression of any of the nucleic acids or vectors described herein with respect to other aspects of the invention. Yet another embodiment of the invention provides a method of producing any of the TCRs, polypeptides, or proteins described herein, comprising culturing any of the host cells or populations of host cells described herein such that the TCRs, polypeptides, or proteins are produced.

[0079] Also included within the scope of the invention are conjugates, e.g., bioconjugates, comprising any of the TCRs, polypeptides, or proteins of the invention (including any functional portion or variant thereof), nucleic acids, recombinant expression vectors, host cells, populations of host cells, or antibodies or antigen-binding portions thereof. Conjugates, and generally methods for synthesizing conjugates, are known in the art.

[0080] An embodiment of the present invention provides a nucleic acid comprising a nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein. As used herein, "nucleic acid" includes "polynucleotides," "oligonucleotides," and "nucleic acid molecules," and generally refers to a polymer of DNA or RNA, which may be single-stranded or double-stranded, may contain natural, non-natural, or modified nucleotides, and may contain natural, non-natural, or modified internucleotide linkages, such as phosphoramidate or phosphorothioate linkages, in place of the phosphodiesters found between nucleotides in unmodified oligonucleotides. In an embodiment, a nucleic acid comprises a complementary DNA (cDNA). It is generally preferred that a nucleic acid does not contain any insertions, deletions, inversions, and / or substitutions. However, in some instances, it may be preferred that a nucleic acid contains one or more insertions, deletions, inversions, and / or substitutions, as discussed herein.

[0081] Preferably, the nucleic acid of the present invention is recombinant. As used herein, the term "recombinant" refers to (i) a molecule constructed outside a living cell by linking a natural or synthetic nucleic acid segment to a nucleic acid molecule capable of replicating in the living cell, or (ii) a molecule obtained by replicating what is described in (i) above. For purposes herein, the replication may be in vitro or in vivo.

[0082] Nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, e.g., Green and Sambrook, supra. For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed upon hybridization. Examples of modified nucleotides that can be used to generate nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N 6 -Isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6 -Substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6 -isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxocine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the present invention can be purchased from companies such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0083] The nucleic acid may comprise any nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein. In an embodiment of the invention, the nucleic acid comprises a codon-optimized nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein. Without being bound to any particular theory or mechanism, it is believed that codon optimization of a nucleotide sequence increases the translation efficiency of an mRNA transcript. Codon optimization of a nucleotide sequence may involve replacing a native codon with another codon that encodes the same amino acid but that can be translated by a more readily available tRNA in the cell, thereby increasing translation efficiency. Optimization of a nucleotide sequence may also reduce secondary structures in the mRNA that interfere with translation, thereby increasing translation efficiency.

[0084] The present invention also provides a nucleic acid comprising a nucleotide sequence that is complementary to the nucleotide sequence of any of the nucleic acids described herein, or that hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.

[0085] Nucleotide sequences that hybridize under stringent conditions preferably hybridize under highly stringent conditions. By "highly stringent conditions" is meant that a nucleotide sequence specifically hybridizes to a target sequence (any nucleotide sequence of a nucleic acid described herein) in an amount detectably greater than non-specific hybridization. Highly stringent conditions include conditions that distinguish polynucleotides with exact complementary sequences or those that contain only a few scattered mismatches from random sequences that happen to have a few small regions (e.g., 3-10 bases) of nucleotide sequence matching. Such small regions of complementarity melt more easily than full-length complements of 14-17 bases or more, making them easily distinguishable by highly stringent hybridization. Relatively high stringency conditions include low salt and / or high temperature conditions, such as those provided by about 0.02-0.1 M NaCl or equivalent, at temperatures of about 50-70°C. Such highly stringent conditions tolerate little, if any, mismatch between the nucleotide sequence and the template or target strand and are particularly suitable for detecting expression of any of the TCRs of the present invention. It will generally be appreciated that conditions can be made more stringent by adding increasing amounts of formamide.

[0086] Also, embodiments of the invention provide a nucleic acid comprising a nucleotide sequence that is at least about 70% or more identical to any of the nucleic acids described herein, e.g., about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any of the nucleic acids described herein. In this regard, the nucleic acid may consist essentially of any of the nucleotide sequences described herein.

[0087] An embodiment of the invention provides an isolated or purified nucleic acid comprising, in a 5' to 3' direction, a first nucleic acid sequence and a second nucleotide sequence, wherein the first and second nucleotide sequences encode the amino acid sequences of SEQ ID NOs: 13 and 14; 14 and 13; 15 and 16; 16 and 15; 30 and 31; 31 and 30; 32 and 33; 33 and 32; 34 and 35; 35 and 34; 36 and 37; 37 and 36; 41 and 42; 42 and 41; 43 and 44; 44 and 43; 45 and 46; 46 and 45; 47 and 48; 48 and 47; 49 and 50; 50 and 49; 51 and 52; or 52 and 51, respectively.

[0088] In an embodiment of the invention, the isolated or purified nucleic acid further comprises a third nucleotide sequence interposed between the first and second nucleotide sequences, the third nucleotide sequence encoding a cleavable linker peptide. In an embodiment of the invention, the cleavable linker peptide may comprise the amino acid sequence of SEQ ID NO:38.

[0089] The nucleic acids of the invention can be incorporated into a recombinant expression vector. In this regard, the invention provides a recombinant expression vector comprising any of the nucleic acids of the invention. In an embodiment of the invention, the recombinant expression vector comprises a nucleotide sequence encoding an α chain, a β chain, and a linker peptide.

[0090] For purposes herein, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct, in which the construct comprises a nucleotide sequence encoding an mRNA, a protein, a polypeptide, or a peptide, and the cell is capable of expressing the mRNA, protein, polypeptide, or peptide when the vector is contacted with the host cell under conditions sufficient to express the mRNA, protein, polypeptide, or peptide in the host cell. The vector of the present invention is not naturally occurring in its entirety. However, a portion of the vector may be naturally occurring. The recombinant expression vector of the present invention may comprise any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and may contain natural, non-natural, or modified nucleotides. The recombinant expression vector may comprise naturally occurring internucleotide bonds, non-naturally occurring internucleotide bonds, or both types of bonds. Preferably, the non-naturally occurring or modified nucleotides or internucleotide bonds do not interfere with the transcription or replication of the vector.

[0091] The recombinant expression vector of the present invention may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and propagation, or for expression, or both, such as plasmids and viruses. The vector may be selected from the group consisting of the pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 may also be used. Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). Preferably, the recombinant expression vector is a viral vector, such as a retroviral vector. In a particularly preferred embodiment, the recombinant expression vector is a MSGV1 vector. In an embodiment of the invention, the recombinant expression vector is a transposon or lentiviral vector.

[0092] The recombinant expression vectors of the invention can be prepared using standard recombinant DNA techniques, for example, as described in Green and Sambrook et al., supra. Expression vector constructs, which may be circular or linear, can be prepared to contain a replication system that functions in prokaryotic or eukaryotic host cells. Replication systems can be derived, for example, from ColEl, 2μ plasmid, lambda, SV40, bovine papilloma virus, etc.

[0093] Desirably, the recombinant expression vector includes regulatory sequences, e.g., transcription and translation initiation and termination codons that are specific for the type of host cell (e.g., bacterial, fungal, plant, or animal) into which the vector will be introduced, as appropriate, and considering whether the vector is DNA or RNA based.

[0094] The recombinant expression vector may contain one or more marker genes to allow for the selection of transformed or transfected host cells. Marker genes include biocide resistance, resistance to e.g., antibiotics, heavy metals, etc., complementation in auxotrophic hosts to confer prototrophy, etc. Suitable marker genes for the expression vectors of the present invention include, for example, the neomycin / G418 resistance gene, the hygromycin resistance gene, the histidinol resistance gene, the tetracycline resistance gene, and the ampicillin resistance gene.

[0095] The recombinant expression vector may include a native or non-native promoter operably linked to a nucleotide sequence encoding a TCR, polypeptide, or protein, or to a nucleotide sequence that is complementary or hybridizes to a nucleotide sequence encoding a TCR, polypeptide, or protein. For example, the selection of strong, weak, inducible, tissue-specific, and developmental stage-specific promoters is within the skill of one of ordinary skill in the art. Similarly, the combination of a nucleotide sequence and a promoter is within the skill of one of ordinary skill in the art. The promoter may be a non-viral promoter or a viral promoter, such as the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, and the promoter found in the long terminal repeat of the murine stem cell virus.

[0096] The recombinant expression vectors of the invention can be designed for transient expression, stable expression, or both. Additionally, the recombinant expression vectors can be made for constitutive or inducible expression.

[0097] Additionally, the recombinant expression vector may be made to contain a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the death of a cell expressing the suicide gene. A suicide gene may be a gene that confers sensitivity to an agent, such as a drug, on the cell in which it is expressed, and causes the cell to die when the cell comes into contact with or is exposed to the agent. Suicide genes are known in the art and include, for example, herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, nitroreductase, and the inducible caspase 9 gene system.

[0098] Another embodiment of the present invention further provides a host cell comprising any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that may contain a recombinant expression vector of the present invention. The host cell may be a eukaryotic cell, such as a plant, animal, fungus, or algae, or a prokaryotic cell, such as a bacterium or a protozoan. The host cell may be a cultured or primary cell, i.e., a cell directly isolated from an organism, such as a human. The host cell may be an adherent or suspension cell, i.e., a cell that grows in suspension. Suitable host cells are known in the art and include, for example, DH5α E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For purposes of amplifying or replicating a recombinant expression vector, the host cell is preferably a prokaryotic cell, such as a DH5α cell. For purposes of producing a recombinant TCR, polypeptide, or protein, the host cell is preferably a mammalian cell. Most preferably, the host cell is a human cell. The host cell may be any cell type, originate from any type of tissue, and may be at any stage of development, but the host cell is preferably a peripheral blood lymphocyte (PBL) or peripheral blood mononuclear cell (PBMC). More preferably, the host cell is a T cell. In an embodiment of the invention, the host cell is a human lymphocyte. In another embodiment of the invention, the host cell is selected from the group consisting of a T cell, a natural killer T (NKT) cell, an invariant natural killer T (iNKT) cell, and a natural killer (NK) cell. Yet another embodiment of the invention provides a method of generating a host cell expressing a TCR having antigen specificity for a peptide of SEQ ID NO: 39, comprising contacting the cell with any of the vectors described herein under conditions that allow the introduction of the vector into the cell.

[0099] For purposes herein, a T cell may be any T cell, such as a cultured T cell, such as a primary T cell, or a T cell from a cultured T cell line, such as Jurkat, SupT1, etc., or a T cell obtained from a mammal. If obtained from a mammal, the T cells may be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The T cells may also be enriched or purified. Preferably, the T cells are human T cells. The T cells may be any type of T cell and may be at any stage of development, such as CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells, e.g., Th1 and Th2 cells, CD4 + T cells, CD8 + These include, but are not limited to, T cells (e.g., cytotoxic T cells), tumor infiltrating lymphocytes (TIL), memory T cells (e.g., central memory T cells and effector memory T cells), naive T cells, and the like.

[0100] Also provided by the present invention is a population of cells comprising at least one host cell as described herein. The population of cells may be a heterogeneous population comprising host cells comprising any of the described recombinant expression vectors in addition to at least one other cell, e.g., a host cell (e.g., a T cell), that does not comprise any of the recombinant expression vectors, or a cell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, a muscle cell, a brain cell, etc. Alternatively, the population of cells may be a substantially homogenous population, comprising primarily host cells comprising (e.g., consisting essentially of) a recombinant expression vector. The population may also be a clonal population of cells, where all cells of the population are clones of one host cell comprising a recombinant expression vector, such that all cells of the population comprise a recombinant expression vector. In one embodiment of the present invention, the population of cells is a clonal population comprising host cells comprising a recombinant expression vector as described herein.

[0101] In embodiments of the invention, the number of cells in a population can be rapidly expanded. Expansion of T cell numbers can be achieved by any of a number of methods known in the art, for example, as described in U.S. Patent Nos. 8,034,334, 8,383,099, U.S. Patent Application Publication No. 2012 / 0244133, Dudley et al., J. Immunother., 26:332-42 (2003), and Riddell et al., J. Immunol. Methods, 128:189-201 (1990). In embodiments, expansion of T cell numbers is performed by culturing the T cells with OKT3 antibody, IL-2, and feeder PBMCs (e.g., irradiated allogeneic PBMCs).

[0102] The TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, and host cells (including populations thereof) of the present invention may be isolated and / or purified. The term "isolated" as used herein means removed from its natural environment. The term "purified" as used herein means increased purity, and "purity" is a relative term and is not necessarily to be construed as absolute purity. For example, the purity may be at least about 50%, may be greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 95%, or may be about 100%.

[0103] The TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, and host cells (including populations thereof) of the invention (all of which are hereinafter collectively referred to as the "TCR materials of the invention") may be formulated into compositions, such as pharmaceutical compositions. In this regard, the invention provides pharmaceutical compositions comprising any of the TCRs, polypeptides, proteins, nucleic acids, expression vectors, and host cells (including populations thereof) described herein and a pharma- ceutically acceptable carrier. Pharmaceutical compositions of the invention containing any of the TCR materials of the invention may include more than one TCR material of the invention, e.g., a polypeptide and a nucleic acid, or two or more different TCRs. Alternatively, a pharmaceutical composition may include the TCR materials of the invention in combination with another pharma- ceutical active agent(s) or drug(s), e.g., a chemotherapeutic agent, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, and the like.

[0104] Preferably, the carrier is a pharma- ceutically acceptable carrier. For pharmaceutical compositions, the carrier may be any of those conventionally used for the particular TCR material of the present invention under consideration. Methods for preparing administrable compositions are known or apparent to those skilled in the art, and are described, for example, in Remington: The Science and Practice of Pharmacy, 22 nd Ed., Pharmaceutical Press (2012). Preferably, a pharma- ceutically acceptable carrier is one that has no detrimental side effects or toxicity under the conditions of use.

[0105] The choice of carrier is determined in part by the specific TCR material of the present invention, as well as by the specific method used to administer the TCR material of the present invention.Therefore, there are various suitable formulations of the pharmaceutical composition of the present invention.Suitable formulations may include those for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, intratumoral, or intraperitoneal administration.More than one route may be used to administer the TCR material of the present invention, and in certain instances, a particular route may provide a more immediate and effective response than another route.

[0106] Preferably, the TCR material of the invention is administered, for example, by injection intravenously. When the TCR material of the invention is a host cell (or population thereof) expressing a TCR of the invention, the pharmaceutically acceptable carrier for the cells for injection may include any isotonic carrier, for example, normal saline (about 0.90% w / v NaCl in water, about 300 mOsm / L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water, or lactated Ringer's solution. In an embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumen.

[0107] For purposes of the present invention, the amount or dose of the TCR material of the invention administered (e.g., number of cells if the TCR material of the invention is one or more cells) should be sufficient to effect, e.g., a therapeutic or prophylactic response in a subject or animal over an appropriate time frame. For example, a dose of the TCR material of the invention should be sufficient to bind a cancer antigen (e.g., G12R RAS) or detect, treat, or prevent cancer for a period of about 2 hours or more from the time of administration, e.g., 12-24 hours or more. In certain embodiments, the period may be even longer. The dose will be determined by the efficacy of the particular TCR material of the invention and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) to be treated.

[0108] Many assays for determining the dose to be administered are known in the art. For purposes of the present invention, a starting dose to be administered to a mammal can be determined using an assay that involves, for example, comparing the extent to which target cells are lysed or IFN-γ is secreted by T cells expressing a given dose of a TCR, polypeptide, or protein of the present invention when administered to the mammal among a set of mammals that have each received different doses of T cells. The extent to which target cells are lysed or IFN-γ is secreted when a particular dose is administered can be assayed by methods known in the art.

[0109] The dose of the TCR material of the invention will also be determined by the existence, nature, and extent of any adverse side effects that may accompany the administration of a particular TCR material of the invention. Typically, the attending physician will determine the dose of the TCR material of the invention for treating each individual patient, taking into account a variety of factors, such as age, weight, general health, diet, sex, the TCR material of the invention to be administered, the route of administration, and the severity of the cancer being treated. In embodiments where the TCR material of the invention is a population of cells, the number of cells administered per injection may be, for example, about 1×10 6 ~Approx. 1×10 12 In certain embodiments, the number of cells may vary from 1×10 6 Fewer than one cell may be administered.

[0110] Those skilled in the art will readily appreciate that the TCR material of the present invention may be modified in any number of ways, resulting in increased therapeutic or prophylactic efficacy of the TCR material of the present invention through such modifications. For example, the TCR material of the present invention may be conjugated to a chemotherapeutic agent, either directly or indirectly via cross-linking. The practice of conjugating compounds to chemotherapeutic agents is known in the art. Those skilled in the art will appreciate that sites of the TCR material of the present invention that are not essential to the function of the TCR material of the present invention are suitable sites for conjugating cross-linking and / or chemotherapeutic agents, as long as the cross-linking and / or chemotherapeutic agent does not interfere with the function of the TCR material of the present invention, i.e., its ability to bind to G12R RAS or detect, treat, or prevent cancer, when conjugated to the TCR material of the present invention.

[0111] It is contemplated that the pharmaceutical compositions, TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, and populations of cells of the invention can be used in methods of treating or preventing cancer. Without being bound to a particular theory, it is believed that the TCRs of the invention specifically bind to G12R RAS, such that the TCR (or the associated polypeptides or proteins of the invention), when expressed by a cell, can mediate an immune response against target cells expressing G12R RAS. In this regard, embodiments of the invention provide a method of treating or preventing cancer in a mammal, comprising administering to the mammal any of the pharmaceutical compositions, TCRs, polypeptides, or proteins described herein, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein, or any host cell or population of cells comprising a recombinant vector encoding any of the TCRs, polypeptides, or proteins described herein, in an amount effective to treat or prevent cancer in the mammal.

[0112]

[0023] Embodiments of the present invention provide a method of inducing an immune response against cancer in a mammal comprising administering to the mammal any of the pharmaceutical compositions, TCRs, polypeptides, or proteins described herein, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein, or any host cell or population of cells comprising a recombinant vector encoding any of the TCRs, polypeptides, or proteins described herein, in an amount effective to induce an immune response against cancer in the mammal.

[0113] Embodiments of the invention provide any of the pharmaceutical compositions, TCRs, polypeptides, or proteins described herein, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein, or any host cell or population of cells comprising a recombinant vector encoding any of the TCRs, polypeptides, or proteins described herein, for use in the treatment or prevention of cancer in a mammal.

[0114] Embodiments of the invention provide any of the pharmaceutical compositions, TCRs, polypeptides, or proteins described herein, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein, or any host cell or population of cells comprising a recombinant vector encoding any of the TCRs, polypeptides, or proteins described herein, for use in inducing an immune response against cancer in a mammal.

[0115] The terms "treat" and "prevent" and words derived therefrom, as used herein, do not necessarily mean 100% or complete treatment or prevention. Rather, there are various degrees of treatment or prevention that one of skill in the art will recognize as having potential benefits or therapeutic effects. In this regard, the method of the present invention can provide any amount or level of treatment or prevention of cancer in a mammal. Furthermore, the treatment or prevention provided by the method of the present invention can include treatment or prevention of one or more pathologies or symptoms of the cancer being treated or prevented. For example, the treatment or prevention can include promoting tumor regression. Also, for purposes herein, "prevention" can include delaying the onset of cancer or a symptom or condition thereof. Alternatively, or in addition, "prevention" can include preventing or delaying the recurrence of cancer or a symptom or condition thereof.

[0116] Also provided is a method of detecting the presence of cancer in a mammal, the method comprising: (i) contacting any of the TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, populations of cells, or pharmaceutical compositions of the invention described herein with a sample comprising one or more cells from a mammal, thereby forming a complex, and detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

[0117] For the present methods of detecting cancer in a mammal, the cellular sample may be a sample comprising whole cells, a lysate thereof, or a fraction of a whole cell lysate, such as a nuclear or cytoplasmic fraction, a total protein fraction, or a nucleic acid fraction.

[0118] For purposes of the present method of detecting cancer, the contacting may be performed in vitro or in vivo in a mammal, preferably in vitro.

[0119] Detection of the complex can also be accomplished through any number of methods known in the art. For example, the TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, or populations of cells of the invention described herein may be labeled with a detectable label, such as a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and an elemental particle (e.g., gold particle), etc.

[0120] For purposes of the methods of the invention in which a host cell or population of cells is administered, the cells may be allogeneic or autologous to the mammal. Preferably, the cells are autologous to the mammal.

[0121] In the context of the methods of the present invention, the cancer may be any of the following: acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vagina, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, hepatic leukemia, and / or esophageal cancer. The cancer may be any cancer, including glioma, Hodgkin's lymphoma, hypopharyngeal cancer, renal cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, oropharynx cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, ureteral cancer, and bladder cancer.Preferred cancers are pancreatic, colorectal, lung, endometrial, ovarian, or prostate cancer.Preferred cancers are pancreatic, colorectal, lung, endometrial, ovarian, or prostate cancer.Preferably, the lung cancer is lung adenocarcinoma, the ovarian cancer is epithelial ovarian cancer, and the pancreatic cancer is pancreatic adenocarcinoma. In an embodiment of the invention, the cancer expresses a mutant human RAS amino acid sequence in which glycine at position 12 is substituted with arginine, the mutant human RAS amino acid sequence being a mutant human KRAS, mutant human HRAS, or mutant human NRAS amino acid sequence, where position 12 is defined by reference to the WT human KRAS, WT human HRAS, or WT human NRAS protein, respectively. The mutant human KRAS, mutant human HRAS, and mutant human NRAS expressed by the cancer may be as described herein for other aspects of the invention.

[0122] The mammal referred to in the method of the present invention may be any mammal. As used herein, the term "mammal" refers to any mammal, including but not limited to rodent mammals, such as mice and hamsters, and lagomorph mammals, such as rabbits. Preferably, the mammal is a mammal of the order Carnivora, including cats and dogs. More preferably, the mammal is a mammal of the order Artiodactyla, including cats and pigs, or Perissodactyla, including horses. Most preferably, the mammal is a mammal of the order Primates, Ceboids or Simoids (monkeys), or Anthropoids (humans and apes). A particularly preferred mammal is a human.

[0123] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. EXAMPLES

[0124] Example 1 This example shows HLA-DRB5 * 01:HLA-DRA * 1 shows the isolation of a TCR with antigen specificity for human KRAS with the G12R mutation presented by the 01:01 heterodimer.

[0125] HLA-DRB5 * 01:HLA-DRA * TCRs with antigen specificity for human KRAS with the G12R mutation presented by the 01:01 heterodimer were isolated from TILs of a metastatic pancreatic cancer patient, patient 4270. TCRs that showed specific recognition for KRAS G12R, but not for WT KRAS, were isolated from TILs.

[0126] To determine the sequence of reactive 4270 TCR, reactive TILs were sorted by fluorescence-activated cell sorting (FACS) based on upregulation of the T cell activation marker 4-1BB. Cells were then lysed and TCR transcripts were sequenced by Sanger sequencing. The amino acid sequences of the variable regions of the alpha and beta chains of the 4270 TCR are shown in Table 5. The CDRs are underlined.

[0127] [Table 5]

[0128] Example 2 This example demonstrates that the 4270 TCR transduced cells isolated in Example 1 specifically recognize the G12R RAS peptide.

[0129] The nucleic acid sequence encoding the G12R RAS-reactive 4270 TCR of Example 1 and comprising a cysteine-substituted LVL-modified murine constant region was cloned into a retroviral expression vector. The α chain murine constant region comprised the amino acid sequence of SEQ ID NO:26, where X at position 48 is Cys, X at position 112 is Leu, X at position 114 is Ile, and X at position 115 is Val. The β chain constant region comprised the amino acid sequence of SEQ ID NO:27, where X at position 57 is Cys. A linker comprising the amino acid sequence RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO:38) was located between the α and β chain constant regions. Allogeneic PBMCs were transduced with the retroviral expression vector.

[0130] Transduced cells were incubated with WT RAS peptide MTEYKLVVVGA G GVGKSALTIQLI (SEQ ID NO: 40) or G12R RAS peptide MTEYKLVVVGA R Autologous DCs were co-cultured overnight with serial dilutions of GVGKSALTIQLI (SEQ ID NO: 39). IFNγ secretion was assessed by enzyme-linked immunospot (ELISpot). The results are shown in FIG. 1.

[0131] As shown in Figure 1, 4270 TCR-transduced cells specifically recognized the G12R RAS peptide.

[0132] Example 3 This example demonstrates that the 4270 TCR transduced cells isolated in Example 1 express HLA-DRB5 * 01:HLA-DRA * We demonstrate recognition of the G12R RAS peptide presented in the context of a 01:01 heterodimer.

[0133] Allogeneic PBMCs were virally transduced with the retroviral expression vector of Example 2. Target COS7 cells were transfected with one of the HLA molecules expressed by patient 4270, as shown in Figure 2. Transfected COS7 cells were then transfected with the G12R RAS peptide MTEYKLVVVGA R GVGKSALTIQLI (SEQ ID NO: 39). 4270 TCR-transduced cells were co-cultured overnight with pulsed, transfected COS7 cells. IFNγ secretion was assessed by ELISA. The results are shown in Figure 2.

[0134] As shown in FIG. 2, the 4270 TCR transduced cells isolated in Example 1 express HLA-DRB5 * 01:HLA-DRA * It specifically recognized the G12R RAS peptide presented in the context of the 01:01 heterodimer.

[0135] Example 4 This example shows HLA-DQA1 * 05:05:HLA-DQB1 * 1 shows the isolation of a TCR with antigen specificity for human KRAS with the G12R mutation presented by the 03:01 heterodimer.

[0136] HLA-DQA1 * 05:05:HLA-DQB1 *A TCR with antigen specificity for human KRAS with the G12R mutation presented by the 03:01 heterodimer was isolated from TILs of a metastatic colon cancer patient, patient 4268. A TCR that showed specific recognition for KRAS G12R, but not WT KRAS, was isolated from the TILs.

[0137] To determine the sequence of reactive 4268 TCR, reactive TILs were FACS sorted based on upregulation of the T cell activation marker 4-1BB. Cells were then lysed and TCR transcripts were sequenced by Sanger sequencing. The amino acid sequences of the variable regions of the alpha and beta chains of the 4268 TCR are shown in Table 6. The CDRs are underlined.

[0138] [Table 6]

[0139] Example 5 This example demonstrates that the 4268 TCR transduced cells isolated in Example 4 specifically recognize the G12R RAS peptide.

[0140] The nucleic acid sequence encoding the G12R RAS-reactive 4268 TCR of Example 4 and including the cysteine-substituted LVL-modified mouse constant region was cloned into a retroviral expression vector. The α chain mouse constant region comprised the amino acid sequence of SEQ ID NO: 26, where X at position 48 is Cys, X at position 112 is Leu, X at position 114 is Ile, and X at position 115 is Val. The β chain constant region comprised the amino acid sequence of SEQ ID NO: 27, where X at position 57 is Cys. A linker comprising the amino acid sequence RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 38) was located between the α and β chain constant regions. Allogeneic PBMCs were transduced with the retroviral expression vector.

[0141] Transduced cells were incubated with WT RAS peptide MTEYKLVVVGA GGVGKSALTIQLI (SEQ ID NO: 40) or G12R RAS peptide MTEYKLVVVGA R Autologous DCs were co-cultured overnight with serial dilutions of GVGKSALTIQLI (SEQ ID NO: 39) pulsed. IFNγ secretion was assessed by ELIspot. The results are shown in FIG. 3.

[0142] As shown in Figure 3, 4268 TCR-transduced cells specifically recognized the G12R RAS peptide.

[0143] Example 6 This example demonstrates that the 4268 TCR transduced cells isolated in Example 4 express HLA-DQA1 * 05:05:HLA-DQB1 * We demonstrate that the antibody recognizes the G12R RAS peptide presented in the context of the 03:01 heterodimer.

[0144] Allogeneic PBMCs were virally transduced with the retroviral expression vector of Example 5. Target COS7 cells were transfected with one of the HLA molecules expressed by patient 4268, as shown in Figure 4. Transfected COS7 cells were then transfected with the G12R RAS peptide MTEYKLVVVGA R GVGKSALTIQLI (SEQ ID NO: 39). 4268 TCR-transduced cells were co-cultured overnight with pulsed, transfected COS7 cells. IFNγ secretion was assessed by ELISA. The results are shown in FIG. 4.

[0145] As shown in FIG. 4, the 4268 TCR transduced cells isolated in Example 4 express HLA-DQA1 * 05:05:HLA-DQB1 * It specifically recognized the G12R RAS peptide presented in the context of the 03:01 heterodimer.

[0146] All references cited in this specification, including publications, patent applications, and patents, are hereby incorporated by reference as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0147] Use of the terms "a" and "an" and "the" and "at least one" and similar referents in connection with the description of the present invention (particularly in connection with the claims that follow) should be construed to cover both the singular and the plural, unless otherwise specified herein or clearly contradicted by context. Use of the term "at least one" following a list of one or more items (e.g., "at least one of A and B") should be construed to mean one item (A or B) selected from the listed items or any combination of two or more of the listed items (A and B), unless otherwise specified herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including but not limited to"), unless otherwise specified herein or clearly contradicted by context. The recitation of ranges of values ​​herein is intended to serve merely as a shorthand for referring individually to each separate value within the range, and each separate value is incorporated into the specification as if it were individually recited herein, unless otherwise specified herein. All methods described herein can be performed in any suitable order, unless otherwise specified herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to further elucidate the invention, and does not pose a limitation on the scope of the invention, unless otherwise claimed. No language in the specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0148] Preferred embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of the preferred embodiments may become apparent to those of skill in the art upon reading the foregoing description. The inventors expect that such variations will be utilized by those of skill in the art, and the inventors intend that the invention be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or clearly contradicted by context.

Claims

1. An isolated or purified T cell receptor (TCR), said TCR having antigen specificity for a mutant human RAS amino acid sequence in which glycine at position 12 is replaced by arginine; the mutant human RAS amino acid sequence is an amino acid sequence of a mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS), a mutant human Harvey rat sarcoma viral oncogene homolog (HRAS), or a mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS); Position 12 is defined by reference to the wild-type human KRAS, wild-type human HRAS, or wild-type human NRAS protein, respectively; and The TCR is (a) an α-chain CDR1 having the amino acid sequence of SEQ ID NO:1, an α-chain CDR2 having the amino acid sequence of SEQ ID NO:2, an α-chain CDR3 having the amino acid sequence of SEQ ID NO:3, a β-chain CDR1 having the amino acid sequence of SEQ ID NO:4, a β-chain CDR2 having the amino acid sequence of SEQ ID NO:5, and a β-chain CDR3 having the amino acid sequence of SEQ ID NO:6, or (b) an α-chain CDR1 having the amino acid sequence of SEQ ID NO:7, an α-chain CDR2 having the amino acid sequence of SEQ ID NO:8, an α-chain CDR3 having the amino acid sequence of SEQ ID NO:9, a β-chain CDR1 having the amino acid sequence of SEQ ID NO:10, a β-chain CDR2 having the amino acid sequence of SEQ ID NO:11, and a β-chain CDR3 having the amino acid sequence of SEQ ID NO:12; Including, TCR.

2. The TCR of claim 1 , wherein the mutant human RAS amino acid sequence is SEQ ID NO:

39.

3. 3. The TCR of claim 1 or 2, which does not have antigen specificity for the wild-type human RAS amino acid sequence of SEQ ID NO:

40.

4. The TCR of any one of claims 1 to 3, wherein the mutant human RAS amino acid sequence is presented by a human leukocyte antigen (HLA) class II molecule.

5. The TCR of claim 4, wherein the HLA class II molecule is an HLA-DR heterodimer or an HLA-DQ heterodimer.

6. The TCR of claim 4, wherein the HLA class II molecule is an HLA-DRB5:HLA-DRA heterodimer or an HLA-DQA1:HLA-DQB1 heterodimer.

7. The TCR of claim 4, wherein the HLA class II molecule is an HLA-DRB5*01:HLA-DRA*01:01 heterodimer or an HLA-DQA1*05:05:HLA-DQB1*03:01 heterodimer.

8. (i) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 13; (ii) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 14; (iii) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 15; (iv) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 16; (v) both an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:13 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:14; (vi) both an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 15 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 16; (vii) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 41; (viii) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 42; (ix) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 43; (x) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 44; (xi) both an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 41 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 42; or (xii) both an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 43 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 44 The TCR of any one of claims 1 to 7, comprising:

9. (a) (i) X at position 48 of SEQ ID NO:26 is Thr or Cys; (ii) X at position 112 of SEQ ID NO:26 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 114 of SEQ ID NO:26 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an α chain constant region comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 26, in which X at position 115 of SEQ ID NO: 26 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) a β-chain constant region comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:27, in which X at position 57 of SEQ ID NO:27 is Ser or Cys; or (c) Both (a) and (b) The TCR of any one of claims 1 to 8, further comprising:

10. (a) (i) X at position 180 of SEQ ID NO: 30 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 30 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 246 of SEQ ID NO: 30 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an alpha chain comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 30, in which X at position 247 of SEQ ID NO: 30 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) a β-strand comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 31, in which X at position 198 of SEQ ID NO: 31 is Ser or Cys; (c) (i) X at position 188 of SEQ ID NO: 32 is Thr or Cys; (ii) X at position 252 of SEQ ID NO: 32 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 254 of SEQ ID NO: 32 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an alpha chain comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 32, in which X at position 255 of SEQ ID NO: 32 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (d) a β-strand comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 33, in which X at position 191 of SEQ ID NO: 33 is Ser or Cys; (e) both (a) and (b); (f) both (c) and (d); (g) (i) X at position 161 of SEQ ID NO: 45 is Thr or Cys; (ii) X at position 225 of SEQ ID NO: 45 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 227 of SEQ ID NO: 45 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an alpha chain comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 45, in which X at position 228 of SEQ ID NO: 45 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (h) a β-chain comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 46, in which X at position 178 of SEQ ID NO: 46 is Ser or Cys; (i) (i) X at position 168 of SEQ ID NO: 47 is Thr or Cys; (ii) X at position 232 of SEQ ID NO: 47 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 234 of SEQ ID NO: 47 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an alpha chain comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 47, in which X at position 235 of SEQ ID NO: 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (j) a β-chain comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 48, in which X at position 171 of SEQ ID NO: 48 is Ser or Cys; (k) both (g) and (h); or (l) Both (i) and (j) The isolated or purified TCR of any one of claims 1 to 9, comprising:

11. 11. An isolated or purified polypeptide comprising a functional portion of a TCR according to any one of claims 1 to 10, said functional portion comprising: (a) an α-chain CDR1 having the amino acid sequence of SEQ ID NO:1, an α-chain CDR2 having the amino acid sequence of SEQ ID NO:2, an α-chain CDR3 having the amino acid sequence of SEQ ID NO:3, a β-chain CDR1 having the amino acid sequence of SEQ ID NO:4, a β-chain CDR2 having the amino acid sequence of SEQ ID NO:5, and a β-chain CDR3 having the amino acid sequence of SEQ ID NO:6, or (b) an α-chain CDR1 having the amino acid sequence of SEQ ID NO:7, an α-chain CDR2 having the amino acid sequence of SEQ ID NO:8, an α-chain CDR3 having the amino acid sequence of SEQ ID NO:9, a β-chain CDR1 having the amino acid sequence of SEQ ID NO:10, a β-chain CDR2 having the amino acid sequence of SEQ ID NO:11, and a β-chain CDR3 having the amino acid sequence of SEQ ID NO:12; Including, the functional portion has antigen specificity for a mutant human RAS amino acid sequence in which glycine at position 12 is replaced by arginine; the mutant human RAS amino acid sequence is a mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS), a mutant human Harvey rat sarcoma viral oncogene homolog (HRAS), or a mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence; and A polypeptide wherein position 12 is defined by reference to wild-type human KRAS, wild-type human HRAS, or wild-type human NRAS protein, respectively.

12. The functional part is (i) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 13; (ii) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 14; (iii) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 15; (iv) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 16; (v) both an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:13 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:14; (vi) both an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 15 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 16; (vii) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 41; (viii) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 42; (ix) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 43; (x) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 44; (xi) both an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 41 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 42; or (xii) both an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 43 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 44 12. The isolated or purified polypeptide of claim 11 .

13. (a) (i) X at position 48 of SEQ ID NO:26 is Thr or Cys; (ii) X at position 112 of SEQ ID NO:26 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 114 of SEQ ID NO:26 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 26, in which X at position 115 of SEQ ID NO: 26 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 27, in which X at position 57 of SEQ ID NO: 27 is Ser or Cys; or (c) Both (a) and (b) 13. The isolated or purified polypeptide of claim 11 or 12, further comprising:

14. (a) (i) X at position 180 of SEQ ID NO: 30 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 30 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 246 of SEQ ID NO: 30 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 30, in which X at position 247 of SEQ ID NO: 30 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 31, in which X at position 198 of SEQ ID NO: 31 is Ser or Cys; (c) (i) X at position 188 of SEQ ID NO: 32 is Thr or Cys; (ii) X at position 252 of SEQ ID NO: 32 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 254 of SEQ ID NO: 32 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 32, in which X at position 255 of SEQ ID NO: 32 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (d) an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 33, in which X at position 191 of SEQ ID NO: 33 is Ser or Cys; (e) both (a) and (b); (f) both (c) and (d); (g) (i) X at position 161 of SEQ ID NO: 45 is Thr or Cys; (ii) X at position 225 of SEQ ID NO: 45 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 227 of SEQ ID NO: 45 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 45, in which X at position 228 of SEQ ID NO: 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (h) an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 46, in which X at position 178 of SEQ ID NO: 46 is Ser or Cys; (i) (i) X at position 168 of SEQ ID NO: 47 is Thr or Cys; (ii) X at position 232 of SEQ ID NO: 47 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 234 of SEQ ID NO: 47 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 47, in which X at position 235 of SEQ ID NO: 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (j) an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 48, in which X at position 171 of SEQ ID NO: 48 is Ser or Cys; (k) both (g) and (h); or (l) Both (i) and (j) The isolated or purified polypeptide according to any one of claims 11 to 13, comprising:

15. An isolated or purified protein comprising a functional portion of a T cell receptor (TCR), comprising: The functional part (a) a first polypeptide chain comprising an α-chain CDR1 having the amino acid sequence of SEQ ID NO:1, an α-chain CDR2 having the amino acid sequence of SEQ ID NO:2, and an α-chain CDR3 having the amino acid sequence of SEQ ID NO:3, and a second polypeptide chain comprising a β-chain CDR1 having the amino acid sequence of SEQ ID NO:4, a β-chain CDR2 having the amino acid sequence of SEQ ID NO:5, and a β-chain CDR3 having the amino acid sequence of SEQ ID NO:6; or (b) a first polypeptide chain comprising an α-chain CDR1 having the amino acid sequence of SEQ ID NO:7, an α-chain CDR2 having the amino acid sequence of SEQ ID NO:8, and an α-chain CDR3 having the amino acid sequence of SEQ ID NO:9, and a second polypeptide chain comprising a β-chain CDR1 having the amino acid sequence of SEQ ID NO:10, a β-chain CDR2 having the amino acid sequence of SEQ ID NO:11, and a β-chain CDR3 having the amino acid sequence of SEQ ID NO:

12. Including, the functional portion has antigen specificity for a mutant human RAS amino acid sequence in which glycine at position 12 is replaced by arginine; the mutant human RAS amino acid sequence is a mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS), a mutant human Harvey rat sarcoma viral oncogene homolog (HRAS), or a mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence; and An isolated or purified protein, wherein position 12 is defined by reference to wild-type human KRAS, wild-type human HRAS, or wild-type human NRAS protein, respectively.

16. (i) the first polypeptide chain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:13, and the second polypeptide chain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:14; (ii) the first polypeptide chain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:15, and the second polypeptide chain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:16; (iii) the first polypeptide chain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:41 and the second polypeptide chain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:42; or (iv) the first polypeptide chain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:43 and the second polypeptide chain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:

44.

16. The isolated or purified protein of claim 15.

17. (a) the first polypeptide chain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:26; (i) X at position 48 of SEQ ID NO:26 is Thr or Cys; (ii) X at position 112 of SEQ ID NO:26 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 114 of SEQ ID NO:26 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) X at position 115 of SEQ ID NO:26 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) the second polypeptide chain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:27, and X at position 57 of SEQ ID NO:27 is Ser or Cys; or (c) Both (a) and (b) 17. The isolated or purified protein of claim 15 or 16,

18. (a) the first polypeptide chain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:30; (i) X at position 180 of SEQ ID NO: 30 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 30 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 246 of SEQ ID NO: 30 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) X at position 247 of SEQ ID NO: 30 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) the second polypeptide chain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:31, and X at position 198 of SEQ ID NO:31 is Ser or Cys; (c) the first polypeptide chain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:32; (i) X at position 188 of SEQ ID NO: 32 is Thr or Cys; (ii) X at position 252 of SEQ ID NO: 32 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 254 of SEQ ID NO: 32 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) X at position 255 of SEQ ID NO: 32 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (d) the second polypeptide chain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:33, and X at position 191 of SEQ ID NO:33 is Ser or Cys; (e) both (a) and (b); (f) both (c) and (d); (g) the first polypeptide chain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:45; (i) X at position 161 of SEQ ID NO: 45 is Thr or Cys; (ii) X at position 225 of SEQ ID NO: 45 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 227 of SEQ ID NO: 45 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) X at position 228 of SEQ ID NO: 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (h) the second polypeptide chain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:46, and wherein X at position 178 of SEQ ID NO:46 is Ser or Cys; (i) the first polypeptide chain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:47; (i) X at position 168 of SEQ ID NO: 47 is Thr or Cys; (ii) X at position 232 of SEQ ID NO: 47 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 234 of SEQ ID NO: 47 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) X at position 235 of SEQ ID NO: 47 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (j) the second polypeptide chain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:48, and wherein X at position 171 of SEQ ID NO:48 is Ser or Cys; (k) both (g) and (h); or (l) Both (i) and (j) The isolated or purified protein according to any one of claims 15 to 17,

19. An isolated or purified nucleic acid comprising a nucleotide sequence encoding a TCR according to any one of claims 1 to 10, a polypeptide according to any one of claims 11 to 14, or a protein according to any one of claims 15 to 18.

20. 1. An isolated or purified nucleic acid comprising, in a 5' to 3' orientation, a first nucleotide sequence and a second nucleotide sequence, (a) a first nucleotide sequence encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:13 and includes an alpha chain CDR1 having the amino acid sequence of SEQ ID NO:1, an alpha chain CDR2 having the amino acid sequence of SEQ ID NO:2, and an alpha chain CDR3 having the amino acid sequence of SEQ ID NO:3; and a second nucleotide sequence encoding an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:14 and includes a β-chain CDR1 having the amino acid sequence of SEQ ID NO:4, a β-chain CDR2 having the amino acid sequence of SEQ ID NO:5, and a β-chain CDR3 having the amino acid sequence of SEQ ID NO:6; (b) a first nucleotide sequence encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:14 and includes a β-chain CDR1 having the amino acid sequence of SEQ ID NO:4, a β-chain CDR2 having the amino acid sequence of SEQ ID NO:5, and a β-chain CDR3 having the amino acid sequence of SEQ ID NO:6; and a second nucleotide sequence encoding an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:13 and includes an alpha chain CDR1 having the amino acid sequence of SEQ ID NO:1, an alpha chain CDR2 having the amino acid sequence of SEQ ID NO:2, and an alpha chain CDR3 having the amino acid sequence of SEQ ID NO:3; (c) the first nucleotide sequence encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:15 and includes an alpha chain CDR1 having the amino acid sequence of SEQ ID NO:7, an alpha chain CDR2 having the amino acid sequence of SEQ ID NO:8, and an alpha chain CDR3 having the amino acid sequence of SEQ ID NO:9; and a second nucleotide sequence encoding an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 16 and includes a β-chain CDR1 having the amino acid sequence of SEQ ID NO: 10, a β-chain CDR2 having the amino acid sequence of SEQ ID NO: 11, and a β-chain CDR3 having the amino acid sequence of SEQ ID NO: 12; (d) a first nucleotide sequence encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:16 and includes a β-chain CDR1 having the amino acid sequence of SEQ ID NO:10, a β-chain CDR2 having the amino acid sequence of SEQ ID NO:11, and a β-chain CDR3 having the amino acid sequence of SEQ ID NO:12; and a second nucleotide sequence encoding an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:15 and includes an alpha chain CDR1 having the amino acid sequence of SEQ ID NO:7, an alpha chain CDR2 having the amino acid sequence of SEQ ID NO:8, and an alpha chain CDR3 having the amino acid sequence of SEQ ID NO:9; (e) the first nucleotide sequence encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:30 and includes an alpha chain CDR1 having the amino acid sequence of SEQ ID NO:1, an alpha chain CDR2 having the amino acid sequence of SEQ ID NO:2, and an alpha chain CDR3 having the amino acid sequence of SEQ ID NO:3; and a second nucleotide sequence encoding an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:31 and includes a β-chain CDR1 having the amino acid sequence of SEQ ID NO:4, a β-chain CDR2 having the amino acid sequence of SEQ ID NO:5, and a β-chain CDR3 having the amino acid sequence of SEQ ID NO:6; (f) a first nucleotide sequence encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:31 and includes a β-chain CDR1 having the amino acid sequence of SEQ ID NO:4, a β-chain CDR2 having the amino acid sequence of SEQ ID NO:5, and a β-chain CDR3 having the amino acid sequence of SEQ ID NO:6; and a second nucleotide sequence encoding an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:30 and includes an alpha chain CDR1 having the amino acid sequence of SEQ ID NO:1, an alpha chain CDR2 having the amino acid sequence of SEQ ID NO:2, and an alpha chain CDR3 having the amino acid sequence of SEQ ID NO:3; (g) a first nucleotide sequence encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:32 and includes an alpha chain CDR1 having the amino acid sequence of SEQ ID NO:7, an alpha chain CDR2 having the amino acid sequence of SEQ ID NO:8, and an alpha chain CDR3 having the amino acid sequence of SEQ ID NO:9; and a second nucleotide sequence encoding an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:33 and includes a β-chain CDR1 having the amino acid sequence of SEQ ID NO:10, a β-chain CDR2 having the amino acid sequence of SEQ ID NO:11, and a β-chain CDR3 having the amino acid sequence of SEQ ID NO:12; (h) a first nucleotide sequence encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:33 and includes a β-chain CDR1 having the amino acid sequence of SEQ ID NO:10, a β-chain CDR2 having the amino acid sequence of SEQ ID NO:11, and a β-chain CDR3 having the amino acid sequence of SEQ ID NO:12; and a second nucleotide sequence encoding an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:32 and includes an alpha chain CDR1 having the amino acid sequence of SEQ ID NO:7, an alpha chain CDR2 having the amino acid sequence of SEQ ID NO:8, and an alpha chain CDR3 having the amino acid sequence of SEQ ID NO:9; (i) a first nucleotide sequence encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:34 and includes an alpha chain CDR1 having the amino acid sequence of SEQ ID NO:1, an alpha chain CDR2 having the amino acid sequence of SEQ ID NO:2, and an alpha chain CDR3 having the amino acid sequence of SEQ ID NO:3; and a second nucleotide sequence encoding an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:35 and includes a β-chain CDR1 having the amino acid sequence of SEQ ID NO:4, a β-chain CDR2 having the amino acid sequence of SEQ ID NO:5, and a β-chain CDR3 having the amino acid sequence of SEQ ID NO:6; (j) a first nucleotide sequence encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:35 and includes a β-chain CDR1 having the amino acid sequence of SEQ ID NO:4, a β-chain CDR2 having the amino acid sequence of SEQ ID NO:5, and a β-chain CDR3 having the amino acid sequence of SEQ ID NO:6; and a second nucleotide sequence encoding an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:34 and includes an alpha chain CDR1 having the amino acid sequence of SEQ ID NO:1, an alpha chain CDR2 having the amino acid sequence of SEQ ID NO:2, and an alpha chain CDR3 having the amino acid sequence of SEQ ID NO:3; (k) a first nucleotide sequence encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:36 and includes an alpha chain CDR1 having the amino acid sequence of SEQ ID NO:7, an alpha chain CDR2 having the amino acid sequence of SEQ ID NO:8, and an alpha chain CDR3 having the amino acid sequence of SEQ ID NO:9; and a second nucleotide sequence encoding an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:37 and includes a β-chain CDR1 having the amino acid sequence of SEQ ID NO:10, a β-chain CDR2 having the amino acid sequence of SEQ ID NO:11, and a β-chain CDR3 having the amino acid sequence of SEQ ID NO:12; (l) a first nucleotide sequence encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:37 and includes a β chain CDR1 having the amino acid sequence of SEQ ID NO:10, a β chain CDR2 having the amino acid sequence of SEQ ID NO:11, and a β chain CDR3 having the amino acid sequence of SEQ ID NO:12; and a second nucleotide sequence encoding an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:36 and includes an alpha chain CDR1 having the amino acid sequence of SEQ ID NO:7, an alpha chain CDR2 having the amino acid sequence of SEQ ID NO:8, and an alpha chain CDR3 having the amino acid sequence of SEQ ID NO:9; (m) a first nucleotide sequence encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:41, and includes an alpha chain CDR1 having the amino acid sequence of SEQ ID NO:7, an alpha chain CDR2 having the amino acid sequence of SEQ ID NO:8, and an alpha chain CDR3 having the amino acid sequence of SEQ ID NO:9; and a second nucleotide sequence encoding an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:42 and includes a β-chain CDR1 having the amino acid sequence of SEQ ID NO:10, a β-chain CDR2 having the amino acid sequence of SEQ ID NO:11, and a β-chain CDR3 having the amino acid sequence of SEQ ID NO:12; (n) a first nucleotide sequence encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:42 and includes a β chain CDR1 having the amino acid sequence of SEQ ID NO:10, a β chain CDR2 having the amino acid sequence of SEQ ID NO:11, and a β chain CDR3 having the amino acid sequence of SEQ ID NO:12; and a second nucleotide sequence encoding an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:41 and includes an alpha chain CDR1 having the amino acid sequence of SEQ ID NO:7, an alpha chain CDR2 having the amino acid sequence of SEQ ID NO:8, and an alpha chain CDR3 having the amino acid sequence of SEQ ID NO:9; (o) a first nucleotide sequence encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:43 and includes an alpha chain CDR1 having the amino acid sequence of SEQ ID NO:1, an alpha chain CDR2 having the amino acid sequence of SEQ ID NO:2, and an alpha chain CDR3 having the amino acid sequence of SEQ ID NO:3; and a second nucleotide sequence encoding an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:44 and includes a β-chain CDR1 having the amino acid sequence of SEQ ID NO:4, a β-chain CDR2 having the amino acid sequence of SEQ ID NO:5, and a β-chain CDR3 having the amino acid sequence of SEQ ID NO:6; (p) a first nucleotide sequence encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:44 and includes a β-chain CDR1 having the amino acid sequence of SEQ ID NO:4, a β-chain CDR2 having the amino acid sequence of SEQ ID NO:5, and a β-chain CDR3 having the amino acid sequence of SEQ ID NO:6; and a second nucleotide sequence encoding an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:43 and includes an alpha chain CDR1 having the amino acid sequence of SEQ ID NO:1, an alpha chain CDR2 having the amino acid sequence of SEQ ID NO:2, and an alpha chain CDR3 having the amino acid sequence of SEQ ID NO:3; (q) a first nucleotide sequence encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:45 and includes an alpha chain CDR1 having the amino acid sequence of SEQ ID NO:1, an alpha chain CDR2 having the amino acid sequence of SEQ ID NO:2, and an alpha chain CDR3 having the amino acid sequence of SEQ ID NO:3; and a second nucleotide sequence encoding an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:46 and includes a β-chain CDR1 having the amino acid sequence of SEQ ID NO:4, a β-chain CDR2 having the amino acid sequence of SEQ ID NO:5, and a β-chain CDR3 having the amino acid sequence of SEQ ID NO:6; (r) a first nucleotide sequence encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:46 and includes a β-chain CDR1 having the amino acid sequence of SEQ ID NO:4, a β-chain CDR2 having the amino acid sequence of SEQ ID NO:5, and a β-chain CDR3 having the amino acid sequence of SEQ ID NO:6; and a second nucleotide sequence encoding an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:45 and includes an alpha chain CDR1 having the amino acid sequence of SEQ ID NO:1, an alpha chain CDR2 having the amino acid sequence of SEQ ID NO:2, and an alpha chain CDR3 having the amino acid sequence of SEQ ID NO:3; (s) a first nucleotide sequence encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:47 and includes an alpha chain CDR1 having the amino acid sequence of SEQ ID NO:7, an alpha chain CDR2 having the amino acid sequence of SEQ ID NO:8, and an alpha chain CDR3 having the amino acid sequence of SEQ ID NO:9; and a second nucleotide sequence encoding an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:48 and includes a β-chain CDR1 having the amino acid sequence of SEQ ID NO:10, a β-chain CDR2 having the amino acid sequence of SEQ ID NO:11, and a β-chain CDR3 having the amino acid sequence of SEQ ID NO:12; (t) a first nucleotide sequence encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:48 and includes a β chain CDR1 having the amino acid sequence of SEQ ID NO:10, a β chain CDR2 having the amino acid sequence of SEQ ID NO:11, and a β chain CDR3 having the amino acid sequence of SEQ ID NO:12; and a second nucleotide sequence encoding an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:47 and includes an alpha chain CDR1 having the amino acid sequence of SEQ ID NO:7, an alpha chain CDR2 having the amino acid sequence of SEQ ID NO:8, and an alpha chain CDR3 having the amino acid sequence of SEQ ID NO:9; (u) a first nucleotide sequence encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:49 and includes an alpha chain CDR1 having the amino acid sequence of SEQ ID NO:1, an alpha chain CDR2 having the amino acid sequence of SEQ ID NO:2, and an alpha chain CDR3 having the amino acid sequence of SEQ ID NO:3; and a second nucleotide sequence encoding an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:50 and includes a β-chain CDR1 having the amino acid sequence of SEQ ID NO:4, a β-chain CDR2 having the amino acid sequence of SEQ ID NO:5, and a β-chain CDR3 having the amino acid sequence of SEQ ID NO:6; (v) the first nucleotide sequence encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:50 and includes a β-chain CDR1 having the amino acid sequence of SEQ ID NO:4, a β-chain CDR2 having the amino acid sequence of SEQ ID NO:5, and a β-chain CDR3 having the amino acid sequence of SEQ ID NO:6; and a second nucleotide sequence encoding an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:49 and includes an alpha chain CDR1 having the amino acid sequence of SEQ ID NO:1, an alpha chain CDR2 having the amino acid sequence of SEQ ID NO:2, and an alpha chain CDR3 having the amino acid sequence of SEQ ID NO:3; (w) a first nucleotide sequence encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:51 and includes an alpha chain CDR1 having the amino acid sequence of SEQ ID NO:7, an alpha chain CDR2 having the amino acid sequence of SEQ ID NO:8, and an alpha chain CDR3 having the amino acid sequence of SEQ ID NO:9; and the second nucleotide sequence encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:52 and includes a β-chain CDR1 having the amino acid sequence of SEQ ID NO:10, a β-chain CDR2 having the amino acid sequence of SEQ ID NO:11, and a β-chain CDR3 having the amino acid sequence of SEQ ID NO:12; or (x) a first nucleotide sequence encodes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:52 and includes a β chain CDR1 having the amino acid sequence of SEQ ID NO:10, a β chain CDR2 having the amino acid sequence of SEQ ID NO:11, and a β chain CDR3 having the amino acid sequence of SEQ ID NO:12; and a second nucleotide sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:51 and that encodes an amino acid sequence comprising an alpha chain CDR1 having the amino acid sequence of SEQ ID NO:7, an alpha chain CDR2 having the amino acid sequence of SEQ ID NO:8, and an alpha chain CDR3 having the amino acid sequence of SEQ ID NO:9; Isolated or purified nucleic acid.

21. 21. The isolated or purified nucleic acid of claim 20, further comprising a third nucleotide sequence interposed between the first and second nucleotide sequences, said third nucleotide sequence encoding a cleavable linker peptide.

22. 22. The isolated or purified nucleic acid of claim 21, wherein the cleavable linker peptide comprises the amino acid sequence of SEQ ID NO:

38.

23. A recombinant expression vector comprising the nucleic acid according to any one of claims 19 to 22.

24. 24. The recombinant expression vector of claim 23, which is a transposon or lentiviral vector.

25. 25. An isolated or purified TCR, polypeptide, or protein encoded by a nucleic acid according to any one of claims 19 to 22 or a vector according to claim 23 or 24.

26. An isolated or purified TCR, polypeptide, or protein obtained as a result of intracellular expression of a nucleic acid according to any one of claims 19 to 22 or a vector according to claim 23 or 24.

27. A method for generating a host cell expressing a TCR having antigen specificity for a peptide of SEQ ID NO: 39, comprising contacting the cell with a vector described in claim 23 or 24 under conditions that allow introduction of the vector into the cell.

28. 25. An isolated or purified host cell comprising a nucleic acid according to any one of claims 19 to 22 or a recombinant expression vector according to claim 23 or 24.

29. 29. The host cell of claim 28 which is a human lymphocyte.

30. 30. The host cell of claim 28 or 29, selected from the group consisting of T cells, natural killer T (NKT) cells, invariant natural killer T (iNKT) cells, and natural killer (NK) cells.

31. 31. A population of isolated or purified cells comprising a host cell according to any one of claims 28 to 30.

32. 32. A method of producing a TCR according to any one of claims 1 to 10, 25 or 26, a polypeptide according to any one of claims 11 to 14, 25 or 26, or a protein according to any one of claims 15 to 18, 25 or 26, the method comprising culturing a host cell according to any one of claims 28 to 30 or a population of host cells according to claim 31 such that the TCR, polypeptide or protein is produced.

33. 19. A pharmaceutical composition comprising: (a) a TCR according to any one of claims 1 to 10, 25 or 26, a polypeptide according to any one of claims 11 to 14, 25 or 26, or a protein according to any one of claims 15 to 18, 25 or 26, a nucleic acid according to any one of claims 19 to 22, a recombinant expression vector according to claim 23 or 24, a host cell according to any one of claims 28 to 30, or a population of cells according to claim 31; and (b) a pharma- ceutical acceptable carrier.

34. 1. A method for detecting the presence of cancer cells in a sample from a mammal, comprising: (a) contacting said sample comprising said cancer cells with a TCR according to any one of claims 1 to 10, 25 or 26, a polypeptide according to any one of claims 11 to 14, 25 or 26, or a protein according to any one of claims 15 to 18, 25 or 26, a nucleic acid according to any one of claims 19 to 22, a recombinant expression vector according to claims 23 or 24, a host cell according to any one of claims 28 to 30, a population of cells according to claim 31, or a pharmaceutical composition according to claim 33, thereby forming a complex; (b) detecting the complex; and Including, The method, wherein detection of the complex indicates the presence of cancer cells in the sample.

35. An agent for inducing an immune response against cancer in a mammal, comprising a TCR according to any one of claims 1 to 10, 25 or 26, a polypeptide according to any one of claims 11 to 14, 25 or 26, or a protein according to any one of claims 15 to 18, 25 or 26, a nucleic acid according to any one of claims 19 to 22, a recombinant expression vector according to claim 23 or 24, a host cell according to any one of claims 28 to 30, a population of cells according to claim 31, or a pharmaceutical composition according to claim 33.

36. An agent for treating or preventing cancer in a mammal, comprising a TCR according to any one of claims 1 to 10, 25 or 26, a polypeptide according to any one of claims 11 to 14, 25 or 26, or a protein according to any one of claims 15 to 18, 25 or 26, a nucleic acid according to any one of claims 19 to 22, a recombinant expression vector according to claim 23 or 24, a host cell according to any one of claims 28 to 30, a population of cells according to claim 31, or a pharmaceutical composition according to claim 33.

37. 35. The method of claim 34, wherein the cancer expresses a mutant human RAS amino acid sequence in which glycine at position 12 is substituted with arginine; the mutant human RAS amino acid sequence is an amino acid sequence of a mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS), a mutant human Harvey rat sarcoma viral oncogene homolog (HRAS), or a mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS); 35. The method of claim 34, wherein position 12 is defined by reference to wild-type human KRAS, wild-type human HRAS, or wild-type human NRAS proteins, respectively.

38. 38. The method of claim 37, wherein the mutant human RAS amino acid sequence is a mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS) amino acid sequence.

39. 38. The method of claim 37, wherein the mutant human RAS amino acid sequence is a mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence.

40. 38. The method of claim 37, wherein the mutant human RAS amino acid sequence is a mutant human Harvey rat sarcoma viral oncogene homolog (HRAS) amino acid sequence.

41. 41. The method of any one of claims 34 and 37-40, wherein the cancer is pancreatic, colorectal, lung, endometrial, ovarian, or prostate cancer.

42. the cancer expresses a mutant human RAS amino acid sequence in which glycine at position 12 is replaced by arginine; the mutant human RAS amino acid sequence is an amino acid sequence of a mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS), a mutant human Harvey rat sarcoma viral oncogene homolog (HRAS), or a mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS); 37. The agent of claim 35 or 36, wherein position 12 is defined by reference to wild-type human KRAS, wild-type human HRAS, or wild-type human NRAS proteins, respectively.

43. The method of claim 42, wherein the mutant human RAS amino acid sequence is a mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS) amino acid sequence.

44. The method of claim 42, wherein the mutant human RAS amino acid sequence is a mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence.

45. The method of claim 42, wherein the mutant human RAS amino acid sequence is a mutant human Harvey rat sarcoma viral oncogene homolog (HRAS) amino acid sequence.

46. The method of any one of claims 35, 36 and 42 to 45, wherein the cancer is pancreatic, colorectal, lung, endometrial, ovarian, or prostate cancer.

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