HLA class ii-restricted t cell receptors against ras with g12r mutation

Abstract

To provide a further method for treating cancer.SOLUTION: Disclosed is an isolated or purified T cell receptor (TCR), wherein the TCR has antigenic specificity for a mutated human RAS amino acid sequence with a substitution of glycine at position 12 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 of detecting the presence of cancer in a mammal and methods of treating or preventing cancer in a mammal.SELECTED DRAWING: Figure 1

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 January 22, 2019, which is incorporated herein by reference in its entirety.

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

[0003] Incorporation by Reference of Electronically Submitted Documents The computer-readable nucleotide / amino acid sequence listing, submitted concurrently herewith and identified as follows, is incorporated herein by reference in its entirety: 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 treatments such as surgery, chemotherapy, and radiation therapy, the prognosis for many cancers, such as pancreatic, colorectal, lung, endometrial, ovarian, and prostate cancers, may be poor. Thus, 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), which has antigen specificity for a mutant human RAS amino acid sequence in which glycine at position 12 is substituted with arginine, wherein the mutant human RAS amino acid sequence is 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 present 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 present invention.

[0009] An embodiment of the present invention comprises a first nucleic acid sequence and a second nucleotide sequence in the 5' to 3' direction. and 41; 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 present 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 producing host cells expressing a TCR with antigen specificity for the peptide of SEQ ID NO: 39, and methods of producing the TCRs, polypeptides, and proteins of the invention. [Brief explanation of the drawings]

[0011] [Figure 1] Figure 1 shows the number of interferon-γ (IFNγ)-positive spots detected per 2 × 10 (2E4) cells when 4270 TCR-transduced cells were cocultured with autologous DCs. Prior to coculture, autologous DCs were pulsed with serial dilutions (ng / mL) of WT RAS peptide (squares) or G12R RAS peptide (circles). [Figure 2] This graph shows the concentration of IFNγ (pg / mL) 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 the 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. [Figure 3]Figure 1 shows the number of IFNγ-positive spots detected per 2E4 cells when 4268 TCR-transduced cells were cocultured with autologous DCs. Prior to coculture, autologous DCs were pulsed with serial dilutions (ng / mL) of WT RAS peptide (squares) or G12R RAS peptide (circles). [Figure 4] This graph shows the concentration of IFNγ (pg / mL) 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 the 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 INVENTION

[0012] RAS family proteins belong to a large family of small GTPases. Without being bound by any particular theory or mechanism, it is believed that when mutated, RAS proteins may be involved in signal transduction during 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 pancreatic cancer (e.g., pancreatic carcinoma), 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. Two transcript variants of KRAS exist: 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. It has the amino acid sequence at sequence number 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 known as Harvey rat sarcoma viral oncoprotein, V-Ha-Ras Harvey rat sarcoma viral 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 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 present invention provides an isolated or purified TCR, which has 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, and position 12 is defined by reference to the WT human KRAS, WT human HRAS, or WT human NRAS protein, respectively. Hereinafter, reference to "TCR" also refers to functional portions and functional variants of the TCR, unless otherwise specified.

[0017] The mutant human RAS amino acid sequence may 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 wild-type human KRAS, NRAS, and HRAS proteins are 188 or 189 amino acid residues long, respectively, and are highly identical to one another. For example, the amino acid sequence of the wild-type human NRAS protein is 86.8% identical to that of the wild-type human KRAS protein. Amino acid residues 1-86 of the wild-type human NRAS protein and the wild-type human KRAS protein are 100% identical. The amino acid sequence of the wild-type human HRAS protein is 86.3% identical to that of the wild-type human KRAS protein. Amino acid residues 1-94 of the wild-type human HRAS protein and the wild-type human KRAS protein are 100% identical. Hereinafter, references to "RAS" (mutant or non-mutant (WT)) collectively refer to KRAS, HRAS, and NRAS, unless otherwise specified.

[0018] In an embodiment of the present invention, the mutant human RAS amino acid sequence comprises a human RAS amino acid sequence in which glycine at position 12 is substituted 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. Therefore, the amino acid residue at position 12 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 TCRs with antigen specificity for any human RAS protein, polypeptide, or peptide amino acid sequence having the 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 that 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 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 In the case of 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 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 with the G12R mutation are listed in Table 1 below.

[0022] [Table 1]

[0023] In an embodiment of the present invention, a 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, a TCR may have antigen specificity for a RAS peptide having the 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 comprise any consecutive amino acid residues of a mutant RAS protein containing the G12R mutation. In an embodiment of the present invention, a 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 G12R RAS presented by HLA class II molecules. In this regard, the TCR can elicit an immune response when it binds to G12R RAS within the framework of an HLA class II molecule. The TCR of the present invention can recognize G12R RAS presented by HLA class II molecules and can bind to HLA class II molecules in addition to 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 comprising an α chain and a β chain. The α chain of HLA-DR is encoded by the HLA-DRA gene. The β 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 present 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 comprising 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 beta chain of HLA-DQ is encoded by the HLA-DQB1 gene. HLA-DQB1 alleles include HLA-DQB1 * 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 present 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, the TCRs of the present invention advantageously target the destruction of cancer cells while minimizing or eliminating the destruction of normal non-cancer cells, e.g., by minimizing or eliminating toxicity, thereby Furthermore, because the G12R mutation is likely to occur in the early stages of tumorigenesis, the G12R RAS mutation is likely to be expressed in substantially all of a patient's cancer cells. The TCRs of the present invention can 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 can be used to treat or prevent unmanipulated tumor cells (e.g., tumor cells expressing G12R RAS and HLA-DRB5 that have not been treated with interferon (IFN)-γ). * 01:HLA-DRA * One or both of 01:01, G12R RAS and HLA-DQA1 * 05:05:HLA-DQB1 * The present invention provides highly avid recognition of G12R RAS, which may provide the ability to recognize tumor cells that are not transfected with vectors encoding one or both of the G12R RAS peptides, G12R RAS peptide-pulsed tumor cells, or ... *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 people of Caucasian ethnicity in the United States. Therefore, 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. * 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 over approximately 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 over approximately 8% of pancreatic cancer patients.

[0028] The phrase "antigen specificity" as used herein means that a TCR can specifically bind to and immunologically recognize G12R RAS with high avidity. For example, a TCR can express about 1 x 10 G12R RAS peptides 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 so 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 IFN-γ 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). * 01:HLA-DRA * 01:01 heterodimer or HLA-DQA1 * 05:05:HLA-DQB1 * 03:01 heterodimer).

[0029] Alternatively, or additionally, a TCR is considered to have "antigen specificity" for G12R RAS if T cells expressing that TCR secrete at least two-fold more IFN-γ when co-cultured with either (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 transfected with a nucleotide sequence encoding G12R RAS 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 so that the target cells express the irrelevant peptide, or (ii) non-transduced TCR cells (e.g., derived from PBMCs that do not express 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 so 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 additionally, a TCR may be considered to have "antigen specificity" for G12R RAS if at least two-fold greater numbers of T cells expressing the TCR secrete IFN-γ when cocultured 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, peptide concentration, and negative control may be as described herein for other aspects of the invention. The number of IFN-γ-secreting cells can be measured by methods known in the art, e.g., ELISPOT.

[0031] Alternatively, or additionally, a TCR may be considered to have "antigen specificity" for G12R RAS if T cells expressing that TCR upregulate the expression of one or more T cell activation markers, as measured, for example, by flow cytometry, following stimulation with target cells 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 TCRs 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 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 regions (CDR) 1, CDR2, and CDR3. In an embodiment of the invention, the TCR comprises a first polypeptide chain comprising CDR1 (CDR1 of the α chain of 4268 TCR) comprising the amino acid sequence of SEQ ID NO: 1, CDR2 (CDR2 of the α chain of 4268 TCR) comprising the amino acid sequence of SEQ ID NO: 2, and CDR3 (CDR3 of the α chain of 4268 TCR) comprising the amino acid sequence of SEQ ID NO: 3, and a second polypeptide chain comprising CDR1 (CDR1 of the β chain of 4268 TCR) comprising the amino acid sequence of SEQ ID NO: 4, and CDR2 (CDR3 of the β chain of 4268 TCR) comprising the amino acid sequence of SEQ ID NO: 5. and a second polypeptide chain comprising a CDR2 of the β chain of 268 TCR, and a CDR3 (CDR3 of the β 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 CDR1 (CDR1 of the alpha chain of 4270 TCR) comprising the amino acid sequence of SEQ ID NO: 7, CDR2 (CDR2 of the alpha chain of 4270 TCR) comprising the amino acid sequence of SEQ ID NO: 8, and 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 CDR1 (CDR1 of the beta chain of 4270 TCR) comprising the amino acid sequence of SEQ ID NO: 10, CDR2 (CDR2 of the beta chain of 4270 TCR) comprising the amino acid sequence of SEQ ID NO: 11, and 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 embodiments 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 particularly preferred embodiments, 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 present invention, the TCR comprises the amino acid sequence of a variable region of a TCR comprising the above-mentioned 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 of SEQ ID NOs: 13 and 14; both of SEQ ID NOs: 15 and 16; both of SEQ ID NOs: 41 and 42; or both of 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 derived from any suitable species, such as, for example, human or mouse. In embodiments of the invention, the TCR further comprises mouse alpha and beta chain constant regions or human alpha and beta chain constant regions. 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 region, constant region, alpha chain, and / or beta chain), 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 mutant human RAS amino acid sequence in which glycine at position 12 is substituted 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 an endogenous TCR of a 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 be expressed as SEQ ID NO: 28 (WT mouse α chain constant region). , SEQ ID NO: 29 (WT mouse β chain constant region), or both SEQ ID NOs: 28 and 29. Preferably, the TCRs of the invention comprise the amino acid sequences of both SEQ ID NOs: 28 and 29. Chimeric TCRs 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 sequences 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, chimeric TCRs may comprise any of the mouse 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 sequences of (i) both SEQ ID NOs: 13 and 28; (ii) both SEQ ID NOs: 14 and 29; (iii) both SEQ ID NOs: 15 and 28; (iv) both 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 the group consisting of 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 without N-terminal signal peptide), SEQ ID NO: 50 (beta chain of 4268 TCR with WT mouse constant region and without N-terminal signal peptide), SEQ ID NO: 51 (alpha chain of 4270 TCR with WT mouse constant region and without N-terminal signal peptide), SEQ ID NO: 52 (4270 TCR with WT mouse constant region and without N-terminal signal peptide). TCR β chain), both 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.

[0040] In an embodiment of the invention, the TCR comprises an α chain comprising a variable region and a constant region, and a β 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 (the α chain of a 4268 TCR with an N-terminal signal peptide); or (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 (the α chain of a 4268 TCR with an 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 (iii) an α 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, or Trp; and (iv) an α chain comprising 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 (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, wherein 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 (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 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). the beta chain of the TCR; (k) both (g) and (h); or (l) both (i) and (j).

[0041] In embodiments 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 mouse constant region with one, two, three, or four amino acid substitution(s) in one or both of the α and β chain mouse constant regions. In particularly preferred embodiments, the TCR comprises a mouse constant region with one, two, three, or four amino acid substitution(s) in the α chain mouse constant region and one amino acid substitution in the β chain mouse constant region. In some embodiments, the TCR comprising the substituted constant region advantageously exhibits a G12R RAS activity compared to a parent TCR comprising an unsubstituted (wild-type) constant region. + and provides one or more of: increased target recognition, increased expression by host cells, reduced mispairing with endogenous TCRs, and increased anti-tumor activity. Generally, the substituted amino acid sequences of the murine constant regions of the TCR α and β chains, SEQ ID NOs:26 and 27, respectively, correspond to all or part 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 present invention provide TCRs comprising: (a) SEQ ID NO: 26 (α chain constant region), 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 GIy, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) SEQ ID NO: 27 (β chain constant region), in which X at position 57 is Ser or Cys; or (c) both of the amino acid sequences of SEQ ID NOs: 26 and 27. In embodiments of the present invention, a TCR comprising SEQ ID NO: 26 does not comprise SEQ ID NO: 28 (unsubstituted murine 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 the constant region of one or both 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 murine constant region. In this regard, the TCR comprises a 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) are substituted with Cys. The cysteine-substituted TCR may be a cysteine-substituted TCR, which may comprise a CDR or variable region as defined in Table 2. Preferably, both the native Thr48 of SEQ ID NO: 28 and the native Ser57 of SEQ ID NO: 29 are replaced with Cys. Examples of constant region sequences for cysteine-substituted TCRs are listed in Table 2. In embodiments of the invention, the cysteine-substituted 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-substituted TCRs of the invention may comprise a replaced constant region in addition to any of the CDRs or variable regions described herein.

[0043] In embodiments 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 embodiments 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 embodiments of the invention, the substituted amino acid sequence comprises the substitution 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 substituted TCR (also referred to herein as an "LVL-modified TCR"). The hydrophobic amino acid substitution(s) in the TM domain of the TCR can increase the hydrophobicity of the TM domain of the TCR compared to a TCR that does not have the hydrophobic amino acid substitution(s) in the TM domain. In this regard, the TCR is an LVL-modified TCR in which one, two, or three of 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. 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 LVL-modified TCR comprises (i) SEQ ID NO: 26, (ii) SEQ ID NO: 27, or (iii) both SEQ ID NOs: 26 and 27, and the LVL-modified TCR comprises (i) SEQ ID NO: 26, (ii) SEQ ID NO: 27, or (iii) both SEQ ID NOs: 26 and 27. are all 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 embodiments 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 LVL-modified TCRs are set forth in Table 3. In embodiments 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, wherein 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 embodiments of the invention, the substituted amino acid sequence comprises a cysteine substitution in one or both of the α and β chain constant regions in combination with substitution(s) of one, two, or three amino acids in the transmembrane (TM) domain(s) of one or both of the α and β chain constant regions with hydrophobic amino acids (also referred to herein as "cysteine-substituted LVL-modified TCRs"). In this regard, the TCR has native Thr48 of SEQ ID NO: 28 replaced with Cys; and one, two, or three of native Ser112, Met114, and Gly115 of SEQ ID NO: 28 replaced, independently, with Ala, Val, Leu, Ile, Pro, Phe, Me and the native Ser57 of SEQ ID NO: 29 is substituted 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 embodiments 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 TCRs of the invention may comprise a substituted constant region in addition to any of the CDRs or variable regions described herein.

[0050] In embodiments, the cysteine-substituted LVL-modified TCR comprises a full-length alpha chain and a full-length beta chain. In embodiments 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, wherein 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 part or fragment of a TCR of the invention that retains the biological activity of the TCR of which it is a part (parent TCR). A functional portion refers to a portion or fragment that retains, for example, to a similar extent, the same extent, or to a greater extent, the parent TCR (e.g., HLA-DRB5 * 01:HLA-DRA * 01:01 heterodimer or HLA-DQA1 * 05:05:HLA-DQB1 *The term "functional portion" encompasses a portion of a TCR that specifically binds to G12R RAS (within the context of a 03:01 heterodimer) or retains the ability to detect, treat, or prevent cancer. With respect to a 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 α chain and β chain of a TCR of the present invention, for example a functional portion comprising one or more of CDR1, CDR2, and CDR3 of the variable region(s) of the α chain and / or β chain of a TCR of the present invention. In an embodiment of the present invention, the polypeptide may comprise the amino acid sequence of SEQ ID NO: 1 (CDR1 of the α chain), SEQ ID NO: 2 (CDR2 of the α chain), SEQ ID NO: 3 (CDR3 of the α chain), SEQ ID NO: 4 (CDR1 of the β chain), SEQ ID NO: 5 (CDR2 of the β chain), SEQ ID NO: 6 (CDR3 of the β chain), or a combination thereof. In another embodiment of the present invention, the polypeptide may comprise the amino acid sequence of SEQ ID NO: 7 (CDR1 of the α chain), SEQ ID NO: 8 (CDR2 of the α chain), SEQ ID NO: 9 (CDR3 of the α chain), SEQ ID NO: 10 (CDR1 of the β chain), SEQ ID NO: 11 (CDR2 of the β chain), SEQ ID NO: 12 (CDR3 of the β chain), 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 polypeptide 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 embodiments of the present invention, the polypeptide of the present invention may comprise, for example, the variable region of a TCR of the present invention comprising a combination of the above-mentioned CDR regions. In this regard, the polypeptide may comprise the amino acid sequence of (i) SEQ ID NO: 13 (α chain variable region), (ii) SEQ ID NO: 14 (β chain variable region), (iii) both SEQ ID NOs: 13 and 14, (iv) SEQ ID NO: 15 (α chain variable region), (v) SEQ ID NO: 16 (β chain variable region), (vi) both SEQ ID NOs: 15 and 16, (vii) SEQ ID NO: 41 (α chain variable region), (viii) SEQ ID NO: 42 (β chain variable region), (ix) both SEQ ID NOs: 41 and 42, (x) SEQ ID NO: 43 (α chain variable region), (xi) SEQ ID NO: 44 (β chain variable region), 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 the 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 NOs: 28 and 29, or both SEQ ID NOs: 26 and 27. Preferably, the polypeptide comprises any of the CDR regions or variable regions described herein with respect to other aspects of the invention. in combination with both the amino acid sequences of SEQ ID NOs: 26 and 27 or both SEQ ID NOs: 28 and 29. In an embodiment of the invention, one or both of SEQ ID NOs: 26 and 27 of the polypeptide are as defined in any one of Tables 2-4.

[0060] In embodiments of the present invention, polypeptides of the present invention may comprise the full-length α or β chain of a TCR described herein. In this regard, polypeptides of the present 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, polypeptides of the present invention may comprise both chains of a TCR described herein.

[0061] For example, the polypeptide of the present invention can 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, (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, Leu 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) 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) 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: 30-33 and 45-48 of the polypeptide are as defined in any one of Tables 2-4.

[0062] The present invention further provides proteins 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 the amino acid sequence of any of SEQ ID NOs: 1 to 3 and a second polypeptide chain comprising the amino acid sequence of any of SEQ ID NOs: 4 to 6; or (b) a first polypeptide chain comprising the amino acid sequence of any of SEQ ID NOs: 7 to 9 and a second polypeptide chain comprising the amino acid sequence of any 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] 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 may comprise: (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) a first polypeptide chain comprising 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) 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) (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, P; and (iv) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 47, wherein X at position 235 of SEQ ID NO: 47 is Gly, 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 present invention may be a TCR. Alternatively, the protein of the present invention may be a fusion protein, for example, if the protein comprises a single polypeptide chain comprising the amino acid sequences of both SEQ ID NOs: 30 and 31, both SEQ ID NOs: 32 and 33, or if the first and / or second polypeptide chain(s) of the protein further comprise another amino acid sequence, e.g., an amino acid sequence encoding an immunoglobulin or a portion thereof. In this regard, the present invention also provides fusion proteins comprising at least one of the polypeptides of the present invention described herein together with at least one other polypeptide. The other polypeptide may be present as a separate protein of the fusion protein or as a polypeptide expressed in frame (in tandem) with one of the polypeptides of the present 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 can include one or more copies of a polypeptide of the invention and / or one or more copies of another polypeptide. For example, a fusion protein can include 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 present invention, the TCRs, polypeptides, and proteins of the present invention may be expressed as a single protein comprising a linker peptide connecting the α chain and the β chain. In this regard, the TCRs, polypeptides, and proteins of the present invention may further comprise a linker peptide. The linker peptide may advantageously facilitate expression of the recombinant TCR, polypeptide, and / or protein 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 a host cell, the linker peptide may be cleaved, resulting in separated α and β chains. In embodiments of the present invention, the TCRs, polypeptides, or proteins may comprise an amino acid sequence comprising a full-length α chain, a full-length β chain, and a linker peptide located between the α and β chains.

[0070] The protein of the present invention may be a recombinant antibody or antigen-binding portion thereof comprising at least one of the polypeptides of the present 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 present invention and an antibody polypeptide chain, or antigen-binding portion thereof. The antibody polypeptide or antigen-binding portion thereof may be an antibody heavy chain, light chain, variable or constant region of the heavy or light chain, a single-chain variable region (scFv), or an Fc, Fab, or F(ab)2' fragment, etc. The antibody polypeptide chain or antigen-binding portion thereof may be present as separate polypeptides in the recombinant antibody. Alternatively, the antibody polypeptide chain or antigen-binding portion thereof may be present in-frame (in tandem) with a polypeptide of the present invention. The antibody polypeptide or antigen-binding portion thereof may be the polypeptide of any antibody or any antibody fragment, 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 the amino acid sequence of a parent TCR, polypeptide, or protein having 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 having particular physical and / or chemical properties is exchanged for another amino acid having the same chemical or physical properties. For example, conservative amino acid substitutions may be the substitution of an acidic amino acid for another acidic amino acid (e.g., Asp or Glu), an amino acid having a nonpolar side chain for another amino acid having a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid for another basic amino acid (e.g., Lys, Arg), an amino acid having a polar side chain for another amino acid having a polar side chain (e.g., Asn, Cys, Gln, Ser, Thr, Tyr, etc.), etc.

[0073] Alternatively, or in addition, a functional variant may comprise the amino acid sequence of a parent TCR, polypeptide, or protein with at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. Preferably, the non-conservative amino acid substitution can enhance 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 a 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 present 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 present 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 be any one 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 the amino acid sequences of SEQ ID NOs: 7 to 12.

[0075] The TCRs, polypeptides, and proteins of the present invention may be of any length, i.e., 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, polypeptides may range from about 50 to about 5,000 amino acids in length, e.g., 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 1,000 or more amino acids in length. In this regard, polypeptides of the present 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, 2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-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 present 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, for example, de novo synthesis. Polypeptides and proteins can also be produced recombinantly using standard recombinant methods and the nucleic acids described herein. See, e.g., 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. Embodiments of the invention provide isolated or purified TCRs, polypeptides, or proteins 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 isolated or purified TCRs, polypeptides, or proteins encoded by any of the nucleic acids or vectors described herein with respect to other aspects of the invention when expressed in a cell. and an isolated or purified TCR, polypeptide, or protein resulting from the production of the TCR, polypeptide, or protein. Yet another embodiment of the present 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 so as to produce the TCR, polypeptide, or protein.

[0079] Also included within the scope of the present invention are conjugates, e.g., bioconjugates, comprising any of the TCRs, polypeptides, or proteins (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 of the present invention. Conjugates, and generally methods for synthesizing conjugates, are known in the art.

[0080] Embodiments of the present invention provide nucleic acids comprising nucleotide sequences 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 polymers of DNA or RNA, which may be single- or double-stranded, contain natural, non-natural, or modified nucleotides, and may contain natural, non-natural, or modified internucleotide linkages, e.g., phosphoramidate or phosphorothioate linkages, in place of the phosphodiesters found between nucleotides in unmodified oligonucleotides. In embodiments, nucleic acids include complementary DNA (cDNA). It is generally preferred that nucleic acids do not contain any insertions, deletions, inversions, and / or substitutions. However, in some instances, it may be preferable for nucleic acids to contain one or more insertions, deletions, inversions, and / or substitutions, as discussed herein.

[0081] Preferably, the nucleic acids of the present invention are recombinant. As used herein, the term "recombinant" refers to (i) a molecule constructed outside a living cell by joining natural or synthetic nucleic acid segments into a nucleic acid molecule capable of replicating in the living cell, or (ii) a molecule obtained by replication of the above (i). For purposes herein, 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 et al., supra. For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or various 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-galactosylquefine, inosine, N-acetyl-3-methyl-4-methyl-5-methyl-1-methyl-2 ... 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 Examples of suitable nucleic acids include, but are not limited to, 2,6-diaminopurine, 3-(3-amino-3-N-2-carboxypropyl)uracil, 2,6-diaminopurine ...

[0083] The nucleic acid may include any nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein. In embodiments of the present invention, the nucleic acid includes a codon-optimized nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein. Without being bound by 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. Optimizing 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 nucleic acids comprising a nucleotide sequence complementary to the nucleotide sequence of any of the nucleic acids described herein, or a nucleotide sequence 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. "Highly stringent conditions" means 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 nonspecific hybridization. Highly stringent conditions include conditions that distinguish polynucleotides with exact complementary sequences, or those containing only a few scattered mismatches, from random sequences that happen to have a few small regions (e.g., 3-10 bases) of nucleotide sequence match. Such small regions of complementarity melt more easily than full-length complements of 14-17 bases or more, making them readily distinguishable by highly stringent hybridization. Relatively highly stringent conditions include low-salt and / or high-temperature conditions, such as those provided by about 0.02-0.1 M NaCl or equivalent at a temperature 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] Embodiments of the present invention also provide nucleic acids 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 present invention is an isolated or purified nucleic acid comprising, in 5' to 3' direction, a first nucleic acid sequence and a second nucleotide sequence, wherein the first and second nucleotide sequences are set forth, respectively, in 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 nucleic acids encoding the amino acid sequences of 52 and 51.

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

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

[0090] For purposes herein, the term "recombinant expression vector" refers to a genetically engineered oligonucleotide or polynucleotide construct that contains a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide, and that is capable of causing a host cell to express the mRNA, protein, polypeptide, or peptide when the vector is contacted with the cell under conditions sufficient to cause expression of the mRNA, protein, polypeptide, or peptide in the cell. The vectors of the present invention are not naturally occurring as a whole. However, portions of the vector may be naturally occurring. The recombinant expression vectors of the present invention may contain 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 contain naturally occurring internucleotide linkages, non-naturally occurring internucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or modified nucleotides or internucleotide linkages do not interfere with 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, 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 an 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 present invention can be prepared using standard recombinant DNA techniques, for example, as described in Green and Sambrook et al., supra. Expression vector constructs, either 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, λ, SV40, bovine papilloma virus, etc.

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

[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 comprise a native or non-native promoter operably linked to a nucleotide sequence encoding a TCR, polypeptide, or protein, or to a nucleotide sequence complementary to or hybridizing to a nucleotide sequence encoding a TCR, polypeptide, or protein. For example, 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, combinations of nucleotide sequences and promoters are also 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 a cytomegalovirus (CMV) promoter, SV40 promoter, RSV promoter, and promoters found in the long terminal repeat of 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 engineered to contain a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the death of cells 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 the gene is expressed, causing the cell to die when contacted with or exposed to the agent. Suicide genes are known in the art and include, for example, the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, nitroreductase, and 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 capable of containing 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 protozoan. The host cell may be a cultured cell or a primary cell, i.e., a cell isolated directly from an organism, such as a human. The host cell may be an adherent cell or a 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, etc. 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 of any cell type, may originate from any type of tissue, and may be at any stage of development. The host cells may be any number of cells, but are preferably peripheral blood lymphocytes (PBLs) or peripheral blood mononuclear cells (PBMCs). More preferably, the host cells are T cells. In an embodiment of the invention, the host cells are human lymphocytes. In another embodiment of the invention, the host cells are selected from the group consisting of T cells, natural killer T (NKT) cells, invariant natural killer T (iNKT) cells, and natural killer (NK) cells. Yet another embodiment of the invention provides a method of generating a host cell expressing a TCR having antigen-specificity for the peptide of SEQ ID NO: 39, comprising contacting the cell with any of the vectors described herein under conditions that allow for the introduction of the vector into the cell.

[0099] For purposes herein, a T cell may be any T cell, for example, a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., 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 at any stage of development, for example, 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 (TILs), 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 are populations of cells comprising at least one host cell 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 cells other than T cells, e.g., B cells, macrophages, neutrophils, erythrocytes, hepatocytes, endothelial cells, epithelial cells, muscle cells, brain cells, etc. Alternatively, the population of cells may be a substantially homogeneous population comprising primarily host cells comprising (e.g., consisting essentially of) the recombinant expression vector. The population may also be a clonal population of cells, in which all cells in the population are clones of a single host cell comprising the recombinant expression vector, such that all cells in the population comprise the recombinant expression vector. In one embodiment of the present invention, the population of cells is a clonal population comprising host cells comprising the recombinant expression vectors described herein.

[0101] In embodiments of the present invention, the number of cells within a population can be rapidly expanded. Expansion of T cells can be achieved by any of a number of methods known in the art, such as those 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 cells is carried out 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, 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 (collectively hereinafter 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 pharmaceutically 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 nucleic acid, or two or more different TCRs. Alternatively, a pharmaceutical composition may include the TCR material of the invention in combination with another pharmaceutically active agent(s) or drug(s), e.g., a chemotherapeutic agent, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, and the like.

[0104] Preferably, the carrier is a pharmaceutically acceptable carrier. For pharmaceutical compositions, the carrier may be any of those conventionally used for the particular TCR material of the invention under consideration. Methods of 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 ndEd., Pharmaceutical Press (2012). Preferably, a pharmaceutically acceptable carrier is one that has no detrimental side effects or toxicity under the conditions of use.

[0105] The choice of carrier will be determined in part by the specific TCR material of the invention, as well as the specific method used to administer the TCR material of the invention. Accordingly, there are a variety of suitable formulations of the pharmaceutical compositions of the 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 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 present invention is administered, for example, by intravenous injection. When the TCR material of the present invention is a host cell (or population thereof) expressing a TCR of the present invention, a pharmaceutically acceptable carrier for the cells for injection may include any isotonic carrier, such as 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 embodiments, the pharmaceutically acceptable carrier is supplemented with human serum albumen.

[0107] For purposes of the present invention, the amount or dose (e.g., number of cells if the TCR material of the present invention is one or more cells) of the TCR material of the present invention administered should be sufficient to produce, e.g., a therapeutic or prophylactic response in the subject or animal over an appropriate time frame. For example, a dose of the TCR material of the present invention should be sufficient to bind to 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 present invention and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) being treated.

[0108] Many assays for determining the dose to be administered are known in the art. For purposes of the present invention, for example, a set of mammals each receiving different doses of T cells may be used. In particular, a starting dose for administration to a mammal can be determined using an assay that involves comparing the extent to which target cells are lysed or IFN-γ is secreted by T cells expressing a TCR, polypeptide, or protein of the invention when such T cells are administered to a mammal at a given dose. 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 various factors, e.g., 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 infusion may be, for example, about 1 x 10 6 ~Approx. 1×10 12 In certain embodiments, the number of cells can vary from 1 x 10 to 1 x 10 or more. 6Fewer than one cell may be administered.

[0110] Those skilled in the art will readily appreciate that the TCR materials of the present invention may be modified in any number of ways, thereby increasing the therapeutic or prophylactic efficacy of the TCR materials of the present invention through such modifications. For example, the TCR materials 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 recognize that sites on the TCR materials of the present invention that are not essential to the function of the TCR materials of the present invention are suitable sites for attaching cross-linking and / or chemotherapeutic agents, so long as the cross-linking and / or chemotherapeutic agent, when attached to the TCR materials of the present invention, does not interfere with the function of the TCR materials of the present invention, i.e., their ability to bind to G12R RAS or detect, treat, or prevent cancer.

[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 by theory, it is believed that the TCRs of the invention specifically bind to G12R RAS, such that, when expressed by a cell, the TCR (or related polypeptides or proteins of the invention) can mediate an immune response against target cells expressing G12R RAS. In this regard, embodiments of the invention provide methods 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] Embodiments of the present invention provide methods 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] An embodiment of the present invention relates to a pharmaceutical composition, a TCR, a polypeptide, or a protein as described herein, for use in the treatment or prevention of cancer in a mammal. The present invention also provides 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.

[0114] Embodiments of the present 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," as well as derivatives thereof, as used herein, do not necessarily mean 100%, i.e., 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 methods of the present invention can provide any amount or level of cancer treatment or prevention in a mammal. Furthermore, the treatment or prevention provided by the methods of the present invention can include treatment or prevention of one or more conditions or symptoms of the cancer being treated or prevented. For example, 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 additionally, "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 methods of the present invention for 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, contacting may be performed in vitro or in vivo in a mammal, preferably in vitro.

[0119] Detection of the complex can also be achieved 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).

[0120] For purposes of the methods of the present 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] For the purposes of the present invention, cancer may be any of 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, or The cancer may be any cancer, including cancer of the cavity, vagina, vulva, chronic lymphocytic leukemia, chronic myeloid carcinoma, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, glioma, Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, oropharyngeal cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal, omental, and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney 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 cancers. 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 present invention, the cancer expresses a mutant human RAS amino acid sequence in which glycine at position 12 is substituted with arginine, and the mutant human RAS amino acid sequence is 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. 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 present invention.

[0122] The mammal referred to in the methods 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, which includes cats and dogs. More preferably, the mammal is a mammal of the order Artiodactyla, which includes cattle and pigs, or Perissodactyla, which includes 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 present invention but, of course, should not be construed as in any way limiting its scope. [Example]

[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 carrying the G12R mutation, presented by the 01:01 heterodimer, were isolated from TILs of a patient with metastatic pancreatic cancer (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 TCRs, 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 were We demonstrate that it specifically recognizes RAS peptides.

[0129] A nucleic acid sequence encoding the G12R RAS-reactive 4270 TCR of Example 1, including 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, in which X at position 48 was Cys, X at position 112 was Leu, X at position 114 was Ile, and X at position 115 was Val. The β chain constant region comprised the amino acid sequence of SEQ ID NO: 27, in which X at position 57 was Cys. A linker comprising the amino acid sequence RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 38) was positioned between the α chain constant region and the β chain constant region. Allogeneic PBMCs were transduced with the retroviral expression vector.

[0130] Transduced cells were transduced with WT RAS peptide MTEYKLVVVGA G GVGKSALTIQLI (SEQ ID NO: 40) or G12R RAS peptide MTEYKLVVVGA R The cells were co-cultured overnight with autologous DCs pulsed with serial dilutions of GVGKSALTIQLI (SEQ ID NO: 39). IFNγ secretion was assessed by enzyme-linked immunospot (ELISpot). The results are shown in Figure 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. The 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 Figure 2, the 4270 TCR transduced cells isolated in Example 1 expressed 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 * TCRs with antigen specificity for human KRAS with the G12R mutation, presented by the 03:01 heterodimer, were isolated from TILs of a patient with metastatic colon cancer (patient 4268). TCRs that showed specific recognition for KRAS G12R, but not for WT KRAS, were isolated from TILs.

[0137] To determine the sequence of the reactive 4268 TCR, reactive TILs were sorted by 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 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 were We demonstrate that it specifically recognizes RAS peptides.

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

[0141] Transduced cells were transduced with WT RAS peptide MTEYKLVVVGA G GVGKSALTIQLI (SEQ ID NO: 40) or G12R RAS peptide MTEYKLVVVGA R The cells were co-cultured overnight with autologous DCs pulsed with serial dilutions of GVGKSALTIQLI (SEQ ID NO: 39). IFNγ secretion was assessed by ELIspot. The results are shown in Figure 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 G12R RAS peptide is recognized when presented in the context of a 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. The 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 Figure 4.

[0145] As shown in Figure 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] In connection with the description of the present invention (particularly in connection with the claims which follow), use of the terms "a," "an," "the," and "at least one," and similar referents 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") refers to one item (A or B) selected from the listed items or any combination of two or more of the listed items, unless otherwise specified herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed to mean a combination of (A and B). 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. The recitation of ranges of values herein, unless otherwise specified herein, is merely intended to serve as a shorthand method for individually referring to each separate value within the range, and each separate value is incorporated into the specification as if it were individually listed 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 any non-claimed element as essential to the practice of the invention.

[0148] Preferred embodiments of the present 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 ordinary skill in the art upon reading the foregoing description. The inventors anticipate that such variations will be utilized by those skilled in the art, and the inventors intend that the invention may 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. Furthermore, 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), wherein the TCR has 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 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); TCRs where position 12 is defined by reference to wild-type human KRAS, wild-type human HRAS, or wild-type human NRAS proteins, respectively.

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

8. (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 The TCR of any one of claims 1 to 7, comprising the amino acid sequence:

9. (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; (vi) both SEQ ID NOs: 15 and 16; (vii) SEQ ID NO: 41, (viii) SEQ ID NO: 42, (ix) SEQ ID NO: 43, (x) SEQ ID NO: 44, (xi) both SEQ ID NOs: 41 and 42; or (xii) both SEQ ID NOs: 43 and 44 The TCR of any one of claims 1 to 8, comprising the amino acid sequence:

10. (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 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 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 9, further comprising:

11. (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 the amino acid sequence of SEQ ID NO: 30, wherein 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 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 α 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 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; (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 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) 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; (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 the amino acid sequence of SEQ ID NO: 47, wherein 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 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 10, comprising:

12. 12. An isolated or purified polypeptide comprising a functional portion of a TCR according to any one of claims 1 to 11, wherein said functional portion comprises: (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 A polypeptide comprising the amino acid sequence of

13. The functional moiety is (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; (vi) both SEQ ID NOs: 15 and 16; (vii) SEQ ID NO: 41, (viii) SEQ ID NO: 42, (ix) SEQ ID NO: 43, (x) SEQ ID NO: 44, (xi) both SEQ ID NOs: 41 and 42; or (xii) both SEQ ID NOs: 43 and 44 13. The isolated or purified polypeptide of claim 12, comprising the amino acid sequence(s) of:

14. (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) X at position 115 of SEQ ID NO: 26 is Gly, Ala, Val, Leu, or Ile. , Pro, Phe, Met, or Trp; (b) 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) 14. The isolated or purified polypeptide of claim 12 or 13, further comprising:

15. (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) 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, He, 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, 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) 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, He, Pro, Phe, Met, or Trp; (d) 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) 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) 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) 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, He, 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) The isolated or purified polypeptide of any one of claims 12 to 14, comprising:

16. An isolated or purified protein comprising at least one of the polypeptides according to any one of claims 12 to 15.

17. (a) a first polypeptide chain comprising the amino acid sequence of SEQ ID NOs: 1-3 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NOs: 4-6; or (b) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 7-9 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 10-12; 17. The isolated or purified protein of claim 16, comprising:

18. (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.

18. The isolated or purified protein of claim 16 or 17, comprising:

19. (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) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 26, wherein X at position 115 of SEQ ID NO: 26 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (b) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 27, wherein X at position 57 of SEQ ID NO: 27 is Ser or Cys; or (c) Both (a) and (b) 19. The isolated or purified protein of any one of claims 16 to 18, further comprising:

20. (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) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 30, wherein X at position 247 of SEQ ID NO: 30 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (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, 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) 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, He, 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, 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) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 45, wherein X at position 228 of SEQ ID NO: 45 is Gly, Ala, Val, Leu, He, 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) (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) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 47, wherein X at position 235 of SEQ ID NO: 47 is Gly, Ala, Val, Leu, He, 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) 20. The isolated or purified protein of any one of claims 16 to 19, comprising:

21. An isolated or purified nucleic acid comprising a nucleotide sequence encoding a TCR according to any one of claims 1 to 11, a polypeptide according to any one of claims 12 to 15, or a protein according to any one of claims 16 to 20.

22. An isolated or purified nucleic acid comprising, in the 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.

23. 23. The isolated or purified nucleic acid of claim 22, further comprising a third nucleotide sequence interposed between the first and second nucleotide sequences, said third nucleotide sequence encoding a cleavable linker peptide.

24. 24. The isolated or purified nucleic acid of claim 23, wherein the cleavable linker peptide comprises the amino acid sequence of SEQ ID NO:

38.

25. A recombinant expression vector comprising the nucleic acid of any one of claims 21 to 24.

26. 26. The recombinant expression vector of claim 25, which is a transposon or lentiviral vector.

27. An isolated or purified TCR, polypeptide, or protein encoded by the nucleic acid of any one of claims 21 to 24 or the vector of claim 25 or 26.

28. An isolated or purified TCR, polypeptide, or protein obtained as a result of intracellular expression of the nucleic acid according to any one of claims 21 to 24 or the vector according to claim 25 or 26.

29. 27. A method for producing a host cell expressing a TCR having antigen specificity for the peptide of SEQ ID NO: 39, comprising contacting the cell with a vector described in claim 25 or 26 under conditions that allow introduction of the vector into the cell.

30. An isolated or purified host cell comprising a nucleic acid according to any one of claims 21 to 24 or a recombinant expression vector according to claim 25 or 26.

31. 31. The host cell of claim 30, which is a human lymphocyte.

32. 32. The host cell of claim 30 or 31, selected from the group consisting of T cells, natural killer T (NKT) cells, invariant natural killer T (iNKT) cells, and natural killer (NK) cells.

33. 33. A population of isolated or purified cells comprising a host cell according to any one of claims 30 to 32.

34. 34. A method of producing a TCR according to any one of claims 1 to 11, 27 or 28, a polypeptide according to any one of claims 12 to 15, 27 or 28, or a protein according to any one of claims 16 to 20, 27 or 28, the method comprising culturing a host cell according to any one of claims 30 to 32 or a population of host cells according to claim 33 so that the TCR, polypeptide or protein is produced.

35. 34. A pharmaceutical composition comprising: (a) a TCR according to any one of claims 1 to 11, 27, or 28, a polypeptide according to any one of claims 12 to 15, 27, or 28, or a protein according to any one of claims 16 to 20, 27, or 28, a nucleic acid according to any one of claims 21 to 24, a recombinant expression vector according to claim 25 or 26, a host cell according to any one of claims 30 to 32, or a population of cells according to claim 33; and (b) a pharmaceutically acceptable carrier.

36. 1. A method for detecting the presence of cancer in a mammal, comprising: (a) treating a sample containing cancer cells with a TCR according to any one of claims 1 to 11, 27, or 28, a TCR according to any one of claims 12 to 15, 27, or 28, or a protein according to any one of claims 16 to 20, 27 or 28, a nucleic acid according to any one of claims 21 to 24, a recombinant expression vector according to claim 25 or 26, a host cell according to any one of claims 30 to 32, a population of cells according to claim 33, or a pharmaceutical composition according to claim 35, thereby forming a complex; (b) detecting the complex; and Including, The method, wherein detection of said complex indicates the presence of cancer in said mammal.

37. 36. A TCR according to any one of claims 1 to 11, 27 or 28, a polypeptide according to any one of claims 12 to 15, 27 or 28, or a protein according to any one of claims 16 to 20, 27 or 28, a nucleic acid according to any one of claims 21 to 24, a recombinant expression vector according to claim 25 or 26, a host cell according to any one of claims 30 to 32, a population of cells according to claim 33, or a pharmaceutical composition according to claim 35, for use in inducing an immune response against cancer in a mammal.

38. 36. A TCR according to any one of claims 1 to 11, 27 or 28, a polypeptide according to any one of claims 12 to 15, 27 or 28, or a protein according to any one of claims 16 to 20, 27 or 28, a nucleic acid according to any one of claims 21 to 24, a recombinant expression vector according to claim 25 or 26, a host cell according to any one of claims 30 to 32, a population of cells according to claim 33, or a pharmaceutical composition according to claim 35, for use in the treatment or prevention of cancer in a mammal.

39. 39. The method of claim 36, or the TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, population of cells, or pharmaceutical composition for use according to claim 37 or 38, 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); TCRs where position 12 is defined by reference to wild-type human KRAS, wild-type human HRAS, or wild-type human NRAS proteins, respectively.

40. 40. The method of claim 39, or the TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, population of cells, or pharmaceutical composition for use in accordance with claim 39, wherein the mutant human RAS amino acid sequence is a mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS) amino acid sequence.

41. 40. The method of claim 39, or the TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, population of cells, or pharmaceutical composition for use in accordance with claim 39, wherein the mutant human RAS amino acid sequence is a mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence.

42. 40. The method of claim 39, or the TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, population of cells, or pharmaceutical composition for use in accordance with claim 39, wherein the mutant human RAS amino acid sequence is a mutant human Harvey rat sarcoma viral oncogene homolog (HRAS) amino acid sequence.

43. 43. The method of any one of claims 36 and 39-42, or the TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, population of cells, or pharmaceutical composition for use in accordance with any one of claims 38-42, wherein the cancer is pancreatic, colorectal, lung, endometrial, ovarian, or prostate cancer.

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