HLA class II restricted T cell receptors directed against RAS containing G12R mutations

Abstract

The present invention discloses 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. 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.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application is a divisional application of Chinese patent application No. 202080010373.0, and claims the benefit of U.S. Provisional Patent Application No. 62 / 795,203 filed on January 22, 2019, which is incorporated herein by reference in its entirety.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This invention was made with government support from the National Institutes of Health, National Cancer Institute under Grant No. ZIABC010984. The government has certain rights in this invention.

[0005] Incorporation by Reference of Electronically Submitted Material

[0006] The computer-readable nucleotide / amino acid sequence listing filed concurrently with this document and identified as follows: a 74,093-byte ASCII (text) file named "746666_ST25.txt", dated January 21, 2020, is incorporated herein by reference in its entirety. Background of the Invention

[0007] Some cancers can have very limited treatment options, especially when the cancer becomes metastatic and unresectable. Despite certain advances in treatments such as surgery, chemotherapy, and radiation therapy, the prognosis for many cancers (e.g., pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer, and prostate cancer) can be poor. Therefore, there is an unmet need for additional cancer treatments. Summary of the Invention

[0008] One embodiment of the present invention provides an isolated or purified T cell receptor (TCR), wherein the TCR has antigenic specificity for a mutant human RAS amino acid sequence in which the glycine at position 12 is substituted with arginine, wherein the mutant human RAS amino acid sequence is a mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS), a mutant human Harvey rat sarcoma viral oncogene homolog (HRAS), or a mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence, and wherein position 12 is defined with reference to wild-type (WT) human KRAS protein, WT human HRAS protein, or WT human NRAS protein, respectively.

[0009] 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 following amino acid sequence: (a) all of SEQ ID NOs: 1-3, (b) all of SEQ ID NOs: 4-6, (c) all of SEQ ID NOs: 7-9, (d) all of SEQ ID NOs: 10-12, (e) all of SEQ ID NOs: 1-6, or (f) all of SEQ ID NOs: 7-12.

[0010] Yet another embodiment of the present invention provides an isolated or purified protein comprising at least one polypeptide of the present invention.

[0011] Embodiments of the present invention further provide nucleic acids, recombinant expression vectors, host cells, cell populations, and pharmaceutical compositions related to the TCRs, polypeptides, and proteins of the present invention.

[0012] One embodiment of the present invention provides an isolated or purified nucleic acid comprising, from 5′ to 3′, a first nucleic acid sequence and a second nucleotide sequence, wherein the first and second nucleotide sequences encode the amino acid sequences of the following 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.

[0013] Embodiments of the present invention further provide methods for detecting the presence of cancer in a mammal, methods for treating or preventing cancer in a mammal, methods for inducing an immune response against cancer in a mammal, methods for producing host cells expressing a TCR having antigenic specificity for the peptide of SEQ ID NO: 39, and methods for producing the TCRs, polypeptides, and proteins of the present invention.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The number of interferon gamma (IFNγ) positive spots detected after co-culture of 4270 TCR-transduced cells and autologous DCs / 2 × 10 4 Graph of (2E4) cells. Autologous DCs were pulsed with serial dilutions (ng / mL) of WT RAS peptide (squares) or G12R RAS peptide (circles) prior to co-culture.

[0016] Figure 2Graph showing IFNγ concentrations (pg / mL) detected after co-culturing 4270 TCR-transduced cells with COS7 cells. Prior to co-culture, COS7 cells were transfected with one of the indicated HLA molecules and then pulsed with G12R RAS peptide. HLA-DRA01:01 is DRB1 11:01, DRB1 15:01, DRB3 02:02, DRB4 01:01 and DRB5 The second member of the heterodimer of each of 01.

[0017] Figure 3 Graph showing the number of IFNγ-positive spots / 2E4 cells detected after co-culture of 4268 TCR-transduced cells with autologous DCs. Prior to co-culture, autologous DCs were pulsed with serial dilutions (ng / mL) of WT RAS peptide (squares) or G12R RAS peptide (circles).

[0018] Figure 4 Graph showing IFNγ concentrations (pg / mL) detected after co-culturing 4268 TCR-transduced cells with COS7 cells. COS7 cells were transfected with one of the indicated HLA molecules prior to co-culture and then pulsed with G12R RAS peptide. HLA-DRA01:01 is DRB1 11:01, DRB1 12:01, DRB3 02. DRB4 01 and DRB5 The second member of the heterodimer of each of 01.

[0019] Detailed Description of the Invention

[0020] RAS family proteins belong to a large family of small GTPases. Without being limited to a particular theory or mechanism, it is believed that when mutated, RAS proteins can participate in signal transduction in the early stages of tumor formation in many human cancers. Single amino acid substitutions can activate the protein. The mutant RAS protein product can be constitutively activated. Mutated RAS proteins can be expressed in any of a variety of human cancers (e.g., 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.

[0021] KRAS is also known as the GTPase KRas, V-Ki-Ras2 Kirsten rat sarcoma viral oncogene, or KRAS2. There are two transcript variants of KRAS: KRAS variant A and KRAS variant B. WT KRAS variant A has the amino acid sequence of SEQ ID NO: 17. WT KRAS variant B has the amino acid sequence of SEQ ID NO: 18. Hereinafter, unless otherwise specified, references to "KRAS" (mutated or unmutated (WT)) refer to both variant A and variant B. Upon activation, mutant KRAS binds to guanosine 5'-triphosphate (GTP) and converts GTP to guanosine 5'-diphosphate (GDP).

[0022] 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.

[0023] NRAS is another member of the RAS protein family. NRAS is also known as the GTPase NRas, V-Ras neuroblastoma RAS viral oncogene homolog, or NRAS1. WT NRAS has the amino acid sequence of SEQ ID NO: 20.

[0024] One embodiment of the present invention provides an isolated or purified TCR, wherein the TCR has antigenic 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 an amino acid sequence of mutant human KRAS, mutant human HRAS, or mutant human NRAS, and wherein position 12 is defined with reference to WT human KRAS protein, WT human HRAS protein, or WT human NRAS protein, respectively. Hereinafter, unless otherwise specified, reference to "TCR" also refers to functional portions and functional variants of TCRs.

[0025] 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 WT human KRAS, NRAS, and HRAS proteins each have a length of 188 or 189 amino acid residues and are highly identical to each other. For example, the amino acid sequence of WT human NRAS protein is 86.8% identical to the amino acid sequence of WT human KRAS protein. Amino acid residues 1-86 of WT human NRAS protein and WT human KRAS protein are 100% identical. The amino acid sequence of WT human HRAS protein is 86.3% identical to the amino acid sequence of WT human KRAS protein. Amino acid residues 1-94 of WT human HRAS protein and WT human KRAS protein are 100% identical. Hereinafter, unless otherwise specified, reference to "RAS" (mutated or unmutated (WT)) collectively refers to KRAS, HRAS, and NRAS.

[0026] In one embodiment of the present invention, the mutant human RAS amino acid sequence comprises a human RAS amino acid sequence in which the glycine at position 12 is substituted with an arginine, wherein position 12 is defined with reference to the corresponding WT RAS protein. The WT RAS protein can be any 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, the WT human NRAS protein and the WT human KRAS protein are 100% identical at amino acid residues 1-86, and the WT human HRAS protein and the WT human KRAS protein are 100% identical at amino acid residues 1-94. Thus, the amino acid residue at position 12 of each of the WT KRAS, WT HRAS, and WT NRAS proteins is the same, i.e., a glycine.

[0027] The mutant human RAS amino acid sequence has an arginine substitution for glycine at position 12. In this regard, embodiments of the present invention provide a TCR that is antigenically specific for any human RAS protein, polypeptide, or peptide amino acid sequence containing the G12R mutation.

[0028] Mutations and substitutions of RAS are defined herein with reference to the amino acid sequence of the corresponding WT RAS protein. Thus, mutations and substitutions of RAS are described herein with reference to the following format: the amino acid residue present at a specific position (i.e., position 12) in the WT RAS protein, followed by the position number, followed by the amino acid residue that replaces the residue in the specific mutation or substitution being discussed. A RAS amino acid sequence (e.g., a RAS peptide) may include amino acid residues that are less than all the amino acid residues of the full-length WT RAS protein. Thus, position 12 is defined herein with reference to the WT full-length RAS protein (i.e., any one of SEQ ID NOs: 17-20), and it should be understood that the actual position of the corresponding residue in a specific instance of a RAS amino acid sequence may be different. When the position is defined according to any one of SEQ ID NOs: 17-20, the term "G12" refers to the glycine that is typically present at position 12 of any one of SEQ ID NOs: 17-20, and "G12R" indicates that the glycine that is typically present at position 12 of any one of SEQ ID NOs: 17-20 is replaced by arginine. For example, in a specific example of a RAS amino acid sequence, for example, TEYKLVVVGA G When referring to GVGKSALTIQLI (SEQ ID NO: 25), an exemplary WT KRAS peptide corresponding to contiguous amino acid residues 2 to 24 of SEQ ID NO: 17, "G12R" refers to a substitution of the underlined glycine in SEQ ID NO: 25 with valine, but the actual position of the underlined glycine in SEQ ID NO: 25 is 11. The human RAS amino acid sequence containing the G12R mutation is referred to below as "G12R RAS."

[0029] Examples of full-length RAS proteins containing the G12R mutation are set forth in Table 1 below.

[0030] Table 1

[0031] In one embodiment of the invention, a TCR is antigenically specific for a RAS peptide comprising a G12R mutation as described above, wherein the G12R RAS peptide has any length. In one embodiment of the invention, a G12R RAS peptide has any length suitable for binding to any HLA class II molecule described herein. For example, a TCR may be antigenically specific for a RAS peptide comprising a G12R mutation 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. A G12R RAS peptide may include any contiguous amino acid residues of the mutant RAS protein comprising the G12R mutation. In one embodiment of the invention, the TCR may have antigenic specificity for a RAS peptide containing a G12R mutation 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 foregoing values. An example of a specific peptide containing a G12R mutation that can be recognized by a TCR of the invention is MTEYKLVVVGA R GVGKSALTIQLI (SEQ ID NO: 39). In one embodiment of the invention, the TCR has antigenic specificity for the mutant human RAS amino acid sequence of SEQ ID NO: 39. In one embodiment of the invention, the TCR has no antigenic specificity for the wild-type human RAS amino acid sequence of SEQ ID NO: 40.

[0032] In one embodiment of the present invention, the TCR of the present invention is capable of recognizing G12R RAS presented by an HLA class II molecule. In this regard, the TCR can induce an immune response when bound to G12R RAS in the context of an HLA class II molecule. The TCR of the present invention is capable of recognizing G12R RAS presented by an HLA class II molecule and can bind to HLA class II molecules other than G12R RAS.

[0033] In one 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 HLA-DR α chain is encoded by the HLA-DRA gene. The HLA-DR β chain 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 the HLA-DRB1 gene may 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 one 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 composed of HLA-DRB5:HLA-DRA heterodimers. 01:HLA-DRA 01:01 allele (i.e. HLA-DRB5 01:HLA-DRA 01:01 heterodimer) expression.

[0034] 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 HLA-DQ α chain is encoded by the HLA-DQA1 gene. HLA-DQA1 alleles may include 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 06:01. The HLA-DQ β chain is encoded by the HLA-DQB1 gene. HLA-DQB1 alleles can 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 one 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 composed of HLA-DQA1:HLA-DQB1 heterodimer. 05:05:HLA-DQB1 03:01 allele (i.e. HLA-DQA1 05:05: HLA-DQB1 03:01 heterodimer) expression.

[0035] The TCRs of the present invention may provide any one or more of a variety of advantages, including use in adoptive cell transfer when expressed by cells. G12R RAS is expressed by cancer cells and not by normal, non-cancerous cells. Without being limited to a particular theory or mechanism, it is believed that the TCRs of the present invention advantageously destroy cancer cells while minimizing or eliminating damage to normal, non-cancerous cells, thereby reducing (e.g., by minimizing or eliminating) toxicity. In addition, because G12R mutations may occur early in tumor formation, G12R RAS mutations may be expressed on substantially all patient cancer cells. The TCRs of the present invention may advantageously and successfully treat or prevent G12R RAS-positive cancers that do not respond to other types of treatment (e.g., chemotherapy, surgery, or radiation). In addition, the TCRs of the present invention may recognize G12R RAS with high affinity, which may enable recognition of unmanipulated tumor cells (e.g., tumor cells that have not been treated with interferon (IFN)-γ, cells encoding G12R RAS, and HLA-DRB5). 01:HLA-DRA One or both of 01:01, G12R RAS, and HLA-DQA1 05:05:HLA-DQB1 03:01, or both of them, or pulsed with G12R RAS peptide, or a combination thereof). In addition, HLA-DRB5 The 01 allele is expressed in the human population at a frequency of approximately 18%, and HLA-DQA1 05:05:HLA-DQB1 03:01 is expressed in approximately 11% of Caucasian Americans. Therefore, the TCRs of the present invention can increase the number of cancer patients eligible for immunotherapy to include those expressing HLA-DRB5 01:HLA-DRA 01:01 Alleles and HLA-DQA1 05:05:HLA-DQB1 Patients who have one or both of the 03:01 alleles and may not be candidates for immunotherapy using TCRs that recognize G12R RAS presented by other MHC molecules. Based on the HLA-DRB5 prevalence in various cancers in the United States 01:HLA-DRA 01:01 and HLA-DQA1 05:05:HLA-DQB1 Based on estimates of the frequency of the 03:01 allele and G12R RAS expression, it is estimated that greater than approximately 1,000 pancreatic cancer patients per year are potentially suitable for treatment using a TCR of the invention. For example, the KRAS G12R mutation is expressed in greater than approximately 8% of pancreatic cancer patients.

[0036] As used herein, the phrase "antigen specific" means that the TCR can specifically bind to and immunologically recognize G12R RAS with high affinity. For example, after co-culturing 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 foregoing values) or (b) antigen-negative, HLA class II molecule-positive target cells into which a nucleotide sequence encoding G12R RAS has been introduced so as to express G12R RAS, if about 1 × 10 4 About 1× 10 5 If a T cell expressing a TCR secretes 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 foregoing values) of IFN-γ, the TCR can be considered "antigen-specific" for G12R RAS. Cells expressing the TCR of the invention can also secrete IFN-γ after co-culture with antigen-negative, HLA class II molecule-positive target cells pulsed with a higher concentration of G12R RAS peptide. The HLA class II molecule can 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).

[0037] Alternatively or additionally, a TCR can be considered "antigen-specific" for G12R RAS if T cells expressing the TCR secrete at least twice as much IFN-γ as the amount of IFN-γ expressed by a negative control following co-culture with (a) antigen-negative, HLA class II-positive target cells pulsed with a low concentration of a G12R RAS peptide or (b) antigen-negative, HLA class II-positive target cells into which a nucleotide sequence encoding G12R RAS has been introduced such that the target cells express G12R RAS. Negative controls can be, for example, (i) T cells that express a TCR and are co-cultured with (a) antigen-negative, HLA class II-positive target cells pulsed with the same concentration of an irrelevant peptide (e.g., some other peptide having a different sequence from the G12R RAS peptide) or (b) antigen-negative, HLA class II-positive target cells into which a nucleotide sequence encoding an irrelevant peptide has been introduced such that the target cells express the irrelevant peptide; or (ii) untransduced T cells (e.g., derived from PBMCs that do not express a TCR) that are co-cultured with (a) antigen-negative, HLA class II-positive target cells pulsed with the same concentration of a G12R RAS peptide or (b) antigen-negative, HLA class II-positive target cells into which a nucleotide sequence encoding G12R RAS has been introduced such that the target cells express G12R RAS. The HLA class II molecules expressed by the target cells of the negative control are the same as the HLA class II molecules expressed by the target cells co-cultured with the T cells being tested. The HLA class II molecule can 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, such as enzyme-linked immunosorbent assay (ELISA).

[0038] Alternatively or additionally, a TCR can be considered "antigen-specific" for G12R RAS if at least twice as many T cells expressing the TCR secrete IFN-γ as compared to the number of negative control T cells secreting IFN-γ following co-culture with (a) antigen-negative, HLA class II-positive target cells pulsed with a low concentration of G12R RAS peptide or (b) negative, HLA class II-positive target cells into which a nucleotide sequence encoding G12R RAS has been introduced such that the target cells express the G12R RAS antigen. HLA class II molecules, peptide concentrations, and negative controls can be as described herein for other aspects of the invention. The number of cells secreting IFN-γ can be measured by methods known in the art (e.g., ELISPOT).

[0039] Alternatively or additionally, a TCR can be considered "antigen-specific" for G12RRAS if, following stimulation with a target cell expressing G12RRAS, the T cell expressing the TCR upregulates expression of one or more T cell activation markers (as measured, for example, by flow cytometry). Examples of T cell activation markers include 4-1BB, OX40, CD107a, CD69, and cytokines that are upregulated upon antigen stimulation (e.g., tumor necrosis factor (TNF), interleukin (IL)-2, etc.).

[0040] One embodiment of the present invention provides a TCR comprising two polypeptides (i.e., polypeptide chains), such as an alpha (α) chain of a TCR, a beta (β) chain of a TCR, a gamma (γ) chain of a TCR, a delta (δ) chain of a TCR, or a combination thereof. The polypeptide of the TCR of the present invention may include any amino acid sequence, provided that the TCR has antigenic specificity for G12R RAS. In some embodiments, the TCR is non-natural.

[0041] In one embodiment of the invention, a TCR comprises two polypeptide chains, each of which comprises a variable region comprising a complementarity determining region (CDR) 1, CDR2, and CDR3 of the TCR. In one embodiment of the invention, the TCR comprises: a first polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 1 (CDR1 of the α chain of the 4268 TCR), a CDR2 comprising the amino acid sequence of SEQ ID NO: 2 (CDR2 of the α chain of the 4268 TCR), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3 (CDR3 of the α chain of the 4268 TCR); and a second polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 4 (CDR1 of the β chain of the 4268 TCR), a CDR2 comprising the amino acid sequence of SEQ ID NO: 5 (CDR2 of the β chain of the 4268 TCR), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 6 (CDR3 of the β chain of the 4268 TCR).

[0042] In another embodiment of the present invention, the TCR comprises: a first polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 7 (CDR1 of the α chain of the 4270 TCR), a CDR2 comprising the amino acid sequence of SEQ ID NO: 8 (CDR2 of the α chain of the 4270 TCR), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 9 (CDR3 of the α chain of the 4270 TCR); and a second polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 10 (CDR1 of the β chain of the 4270 TCR), a CDR2 comprising the amino acid sequence of SEQ ID NO: 11 (CDR2 of the β chain of the 4270 TCR), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 12 (CDR3 of the β chain of the 4270 TCR).

[0043] In this regard, the TCR of the present invention may include any one or more of the amino acid sequences selected from SEQ ID NOs: 1-12. In one embodiment of the present invention, the TCR includes the following amino acid sequences: (a) all of SEQ ID NOs: 1-3, (b) all of SEQ ID NOs: 4-6, (c) all of SEQ ID NOs: 7-9, (d) all of SEQ ID NOs: 10-12, (e) all of SEQ ID NOs: 1-6, or (f) all of SEQ ID NOs: 7-12. In a particularly preferred embodiment, the TCR includes the following amino acid sequences: (i) all of SEQ ID NOs: 1-6, or (ii) all of SEQ ID NOs: 7-12.

[0044] In one embodiment of the present invention, the TCR comprises the amino acid sequence of the variable region of the TCR comprising the above-mentioned CDRs. In this regard, a TCR may comprise the following amino acid sequences: SEQ ID NO: 13 (variable region of the α chain of the 4268 TCR with an N-terminal signal peptide); SEQ ID NO: 14 (variable region of the β chain of the 4268 TCR with an N-terminal signal peptide); SEQ ID NO: 15 (variable region of the α chain of the 4270 TCR with an N-terminal signal peptide); SEQ ID NO: 16 (variable region of the β chain of the 4270 TCR with an N-terminal signal peptide); SEQ ID NO: 43 (variable region of the α chain of the 4268 TCR without an N-terminal signal peptide); SEQ ID NO: 44 (variable region of the β chain of the 4268 TCR without an N-terminal signal peptide); SEQ ID NO: 41 (variable region of the α chain of the 4270 TCR without an N-terminal signal peptide); SEQ ID NO: 42 (variable region of the β chain of the 4270 TCR without an N-terminal signal peptide); SEQ ID NO: 13 and 14; SEQ ID NOs: 15 and 16; SEQ ID NOs: 41 and 42; or SEQ ID NOs: 43 and 44. Preferably, the TCR comprises the following amino acid sequences: (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.

[0045] The TCR of the present invention may further include an α chain constant region and a β chain constant region. The constant region may be derived from any suitable species, such as humans or mice. In one embodiment of the present invention, the TCR further includes murine α and β chain constant regions or human α and β chain constant regions. As used herein, when referring to any component of a TCR or a TCR described herein (e.g., CDR, variable region, constant region, α chain and / or β chain), the term "murine" or "human" means a TCR (or its components) derived from a mouse or a human, i.e., a TCR (or its components) derived from a mouse T cell or a human T cell or expressed therein at one time.

[0046] One embodiment of the present invention provides a chimeric TCR comprising a human variable region and a murine constant region, wherein the TCR has antigenic specificity for a mutant human RAS amino acid sequence in which the glycine at position 12 is substituted with an arginine. The murine constant region may provide any one or more advantages. For example, the murine constant region may reduce mismatch between the TCR of the present invention and the endogenous TCR of the host cell into which the TCR of the present invention is introduced. Alternatively or additionally, the murine constant region may increase expression of the TCR of the present invention compared to the same TCR with a human constant region. The chimeric TCR may comprise the amino acid sequence of SEQ ID NO: 28 (WT murine α chain constant region), SEQ ID NO: 29 (WT murine β chain constant region), or both SEQ ID NOs: 28 and 29. Preferably, the TCR of the present invention comprises the amino acid sequence of both SEQ ID NOs: 28 and 29. The chimeric TCR may comprise any of the murine constant regions described herein in combination with any of the CDR regions as described herein for other aspects of the present invention. In this regard, the TCR may comprise the following amino acid sequences: (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, a chimeric TCR may comprise any of the murine constant regions described herein in combination with any of the variable regions described herein for other aspects of the invention. In this regard, the TCR may comprise the following amino acid sequences: (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.

[0047] In another embodiment of the invention, the TCR comprises the following amino acid sequence: SEQ ID NO: 34 (4268 TCR alpha chain with WT murine constant region and N-terminal signal peptide), SEQ ID NO: 35 (4268 TCR beta chain with WT murine constant region and N-terminal signal peptide), SEQ ID NO: 36 (4270 TCR alpha chain with WT murine constant region and N-terminal signal peptide), SEQ ID NO: 37 (4270 TCR beta chain with WT murine constant region and N-terminal signal peptide), SEQ ID NO: 49 (4268 TCR alpha chain with WT murine constant region and no N-terminal signal peptide), SEQ ID NO: 50 (4268 TCR beta chain with WT murine constant region and no N-terminal signal peptide), SEQ ID NO: 51 (4270 TCR alpha chain with WT murine constant region and no N-terminal signal peptide), SEQ ID NO: 52 (4270 TCR beta chain with WT murine constant region and without N-terminal signal peptide), both SEQ ID NOs: 34-35, both SEQ ID NOs: 36-37, both SEQ ID NOs: 49-50, or both SEQ ID NOs: 51-52.

[0048] In one embodiment of the invention, the TCR comprises an alpha chain comprising a variable region and a constant region and a beta chain comprising a variable region and a constant region. In this regard, the TCR may include (a) an alpha chain comprising the amino acid sequence of SEQ ID NO: 30 (the alpha chain of the 4268 TCR with an N-terminal signal peptide), 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; (b) a beta chain comprising the amino acid sequence of SEQ ID NO: 31 (the beta chain of the 4268 TCR with an N-terminal signal peptide), wherein SEQ ID NO: (c) an α chain comprising the amino acid sequence of SEQ ID NO: 32 (the α chain of the 4270 TCR with an N-terminal signal peptide), wherein: (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; (d) a β chain comprising the amino acid sequence of SEQ ID NO: 33 (the β chain of the 4270 TCR with an N-terminal signal peptide), wherein SEQ ID (e) both (a) and (b); (f) both (c) and (d); (g) an α chain comprising the amino acid sequence of SEQ ID NO: 45 (the α chain of the 4268 TCR without the N-terminal signal peptide), wherein: (i) X at position 161 of SEQ ID NO: 45 is Thr or Cys; (ii) X at position 225 of SEQ ID NO: 45 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp;(iii) X at position 227 of SEQ ID NO: 45 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 228 of SEQ ID NO: 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (h) a beta chain comprising the amino acid sequence of SEQ ID NO: 46 (the beta chain of 4268 TCR without an N-terminal signal peptide), wherein X at position 178 of SEQ ID NO: 46 is Ser or Cys; (i) an alpha chain comprising the amino acid sequence of SEQ ID NO: 47 (the alpha chain of 4270 TCR without an N-terminal signal peptide), wherein: (i) X at position 168 of SEQ ID NO: 47 is Thr or Cys; (ii) (i) wherein X at position 232 of SEQ ID NO: 47 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (ii) wherein X at position 234 of SEQ ID NO: 47 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iii) wherein X at position 234 of SEQ ID NO: 47 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) 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 (the β chain of the 4270 TCR without the N-terminal signal peptide), 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).

[0049] In one embodiment of the invention, TCR includes a substituted constant region. In this regard, TCR may include the amino acid sequence of any TCR described herein, wherein there is one, two, three or four amino acid substitutions in the constant region of one or both of the α and β chains. Preferably, TCR includes a murine constant region, wherein there is one, two, three or four amino acid substitutions in the murine constant region of one or both of the α and β chains. In a particularly preferred embodiment, TCR includes a murine constant region, wherein there is one, two, three or four amino acid substitutions in the murine constant region of the α chain and one amino acid substitution in the murine constant region of the β chain. In some embodiments, compared to the parent TCR including an unsubstituted (wild type) constant region, the TCR including a substituted constant region advantageously provides one or more of the following advantages: increased G12R RAS +Target recognition, increased host cell expression, reduced endogenous TCR mismatch, and increased anti-tumor activity. Generally, the substituted amino acid sequences of the murine constant regions of the TCR α and β chains of SEQ ID NOs: 26 and 27 correspond to all or a portion of the unsubstituted murine constant region amino acid sequences of SEQ ID NOs: 28 and 29, respectively, wherein SEQ ID NO: 26 has one, two, three, or four amino acid substitutions compared to SEQ ID NO: 28 and SEQ ID NO: 27 has one amino acid substitution compared to SEQ ID NO: 29. In this regard, one embodiment of the invention provides a TCR comprising the following amino acid sequence: (a) SEQ ID NO: 26 (constant region of the α chain), wherein (i) X at position 48 is Thr or Cys; (ii) X at position 112 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 114 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 115 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) SEQ ID NO: 27 (constant region of the β chain), wherein X at position 57 is Ser or Cys; or (c) both SEQ ID NOs: 26 and 27. In one embodiment of the invention, the TCR comprising SEQ ID NO: 26 does not comprise SEQ ID NO: 28 (unsubstituted murine constant region of the α chain). In one embodiment of the invention, the TCR comprising SEQ ID NO: 27 does not comprise SEQ ID NO: 29 (the unsubstituted murine constant region of the beta chain).

[0050] In one embodiment of the present 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. The opposing cysteines in the α and β chains provide a disulfide bond that connects the constant regions of the α and β chains of the substituted TCR to each other and is not present in a TCR comprising an unsubstituted murine constant region. In this regard, the TCR may be a cysteine-substituted TCR, wherein one or both of the native Thr at position 48 (Thr48) in SEQ ID NO: 28 and the native Ser at position 57 (Ser57) in SEQ ID NO: 29 may be substituted with Cys. Preferably, both the native Thr48 of SEQ ID NO: 28 and the native Ser57 of SEQ ID NO: 29 are substituted with Cys. Examples of cysteine-substituted TCR constant region sequences are set forth in Table 2. In one embodiment 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, wherein both SEQ ID NOs: 26 and 27 are as defined in Table 2. In addition to any of the CDRs or variable regions described herein, the cysteine-substituted TCRs of the invention may also comprise a substituted constant region.

[0051] In one embodiment of the invention, the chimeric TCR substituted with cysteine ​​comprises a full-length α chain and a full-length β chain. Examples of chimeric TCR α chain and β chain sequences substituted with cysteine ​​are set forth in Table 2. In one embodiment of the invention, the TCR comprises (i) SEQ ID NO: 30, (ii) SEQ ID NO: 31, (iii) SEQ ID NO: 32, (iv) SEQ ID NO: 33, (v) both SEQ ID NOs: 30 and 31, (vi) both SEQ ID NOs: 32 and 33, (vii) SEQ ID NO: 45, (viii) SEQ ID NO: 46, (ix) SEQ ID NO: 47, (x) SEQ ID NO: 48, (xi) both SEQ ID NOs: 45 and 46, or (xii) both SEQ ID NOs: 47 and 48, wherein SEQ ID NOs: 30-33 and 45-48 are as defined in Table 2.

[0052] Table 2

[0053] In one embodiment of the present invention, the substituted amino acid sequence is substituted with a hydrophobic amino acid in one, two, or three amino acids in the transmembrane (TM) domain of the constant region of one or both of the α and β chains to provide a TCR substituted with a hydrophobic amino acid (also referred to herein as a "LVL-modified TCR"). The hydrophobic amino acid substitution in the TM domain of the TCR can increase the hydrophobicity of the TM domain of the TCR compared to a TCR without a hydrophobic amino acid substitution in the TM domain. In this regard, the TCR is an LVL-modified TCR, wherein one, two, or three of the native Ser112, Met114, and Gly115 of SEQ ID NO: 28 can be independently substituted with Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp, preferably Leu, Ile, or Val. Preferably, the native Ser112, Met114, and Gly115 of SEQ ID NO: 28 are independently substituted with Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp, preferably Leu, Ile, or Val. In one 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, wherein both SEQ ID NOs: 26 and 27 are as defined in Table 3. In addition to any of the CDRs or variable regions described herein, the LVL-modified TCR of the invention may comprise a substituted constant region.

[0054] In one embodiment of the present invention, the LVL-modified TCR comprises a full-length α chain and a full-length β chain. Examples of LVL-modified TCR α chain and β chain sequences are set forth in Table 3. In one embodiment of the invention, the LVL-modified TCR comprises (i) SEQ ID NO: 30, (ii) SEQ ID NO: 31, (iii) SEQ ID NO: 32, (iv) SEQ ID NO: 33, (v) both SEQ ID NOs: 30 and 31, (vi) both SEQ ID NOs: 32 and 33, (vii) SEQ ID NO: 45, (viii) SEQ ID NO: 46, (ix) SEQ ID NO: 47, (x) SEQ ID NO: 48, (xi) both SEQ ID NOs: 45 and 46, or (xii) both SEQ ID NOs: 47 and 48, wherein SEQ ID NOs: 30-33 and 45-48 are as defined in Table 3.

[0055] Table 3

[0056] In one embodiment of the invention, the substituted amino acid sequence comprises a combination of the following substitutions: cysteine ​​substitution in the constant region of one or both of the α and β chains; and substitution of one, two, or three amino acids with a hydrophobic amino acid in the transmembrane (TM) domain of one or both of the constant region α and β chains (also referred to herein as a "cysteine-substituted, LVL-modified TCR"). In this regard, the TCR is a cysteine-substituted, LVL-modified chimeric TCR in which the native Thr48 of SEQ ID NO: 28 is substituted with Cys; one, two, or three of the native Ser112, Met114, and Gly115 of SEQ ID NO: 28 are independently substituted with Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp, preferably Leu, Ile, or Val; and the native Ser57 of SEQ ID NO: 29 is substituted with Cys. Preferably, the native Ser112, Met114, and Gly115 of SEQ ID NO: 28 are all independently substituted with Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp, preferably Leu, Ile, or Val. In one embodiment of the invention, the cysteine-substituted, LVL-modified TCR comprises (i) SEQ ID NO: 26, (ii) SEQ ID NO: 27, or (iii) both SEQ ID NOs: 26 and 27, wherein both SEQ ID NOs: 26 and 27 are as defined in Table 4. In addition to any of the CDRs or variable regions described herein, the cysteine-substituted, LVL-modified TCR of the invention may comprise a substituted constant region.

[0057] In one embodiment, the cysteine-substituted, LVL-modified TCR comprises a full-length α chain and a full-length β chain. In one embodiment of the invention, the cysteine-substituted, LVL-modified TCR comprises (i) SEQ ID NO: 30, (ii) SEQ ID NO: 31, (iii) SEQ ID NO: 32, (iv) SEQ ID NO: 33, (v) both SEQ ID NOs: 30 and 31, (vi) both SEQ ID NOs: 32 and 33, (vii) SEQ ID NO: 45, (viii) SEQ ID NO: 46, (ix) SEQ ID NO: 47, (x) SEQ ID NO: 48, (xi) both SEQ ID NOs: 45 and 46, or (xii) both SEQ ID NOs: 47 and 48, wherein SEQ ID NOs: 30-33 and 45-48 are as defined in Table 4.

[0058] Table 4

[0059] The present invention also provides polypeptides comprising a functional portion of any of the TCRs described herein. As used herein, the term "polypeptide" includes oligopeptides and refers to a single chain of amino acids linked by one or more peptide bonds.

[0060] With respect to the polypeptides of the present invention, a functional portion can be any portion comprising contiguous amino acids of the TCR of which it is a part, provided that the functional portion specifically binds to G12R RAS. The term "functional portion" when used in reference to a TCR refers to any portion or fragment of a TCR of the present invention that retains the biological activity of the TCR of which it is a part (the parent TCR). Functional portions encompass, for example, TCRs that are still able to specifically bind to G12R RAS (e.g., in the context of HLA-DRB5) to a similar degree, the same degree, or a greater degree than the parent TCR. 01:HLA-DRA 01:01 Heterodimer or HLA-DQA1 05:05:HLA-DQB1 03:01 heterodimer) or those portions that detect, treat, or prevent cancer. With reference to a parental TCR, a functional portion can include, for example, about 10%, about 25%, about 30%, about 50%, about 68%, about 80%, about 90%, about 95% or more of the parental TCR.

[0061] The functional portion may include additional amino acids at the amino or carboxyl terminus or both termini of the portion 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., specific binding to G12R RAS and / or ability to detect, treat or prevent cancer, etc.). More desirably, the additional amino acids enhance the biological activity compared to the biological activity of the parent TCR.

[0062] The polypeptide may include a functional portion of either or both of the α and β chains of the TCR of the present invention, for example, a functional portion of one or more of the CDR1, CDR2, and CDR3 of the variable region of the α chain and / or β chain of the TCR of the present invention. In one embodiment of the present invention, the polypeptide may include 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 include 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.

[0063] In this regard, the polypeptide of the present invention may include any one or more of the amino acid sequences selected from SEQ ID NOs: 1-12. In one embodiment of the present invention, the TCR comprises the following amino acid sequences: (a) all of SEQ ID NOs: 1-3, (b) all of SEQ ID NOs: 4-6, (c) all of SEQ ID NOs: 7-9, (d) all of SEQ ID NOs: 10-12, (e) all of SEQ ID NOs: 1-6, or (f) all of SEQ ID NOs: 7-12. In a preferred embodiment, the polypeptide comprises the following amino acid sequence: (i) all of SEQ ID NOs: 1-6, or (ii) all of SEQ ID NOs: 7-12.

[0064] In one embodiment of the present invention, the polypeptide of the present invention may include, for example, the variable region of the TCR of the present invention comprising a combination of the above-mentioned CDR regions. In this regard, the polypeptide may include the following amino acid sequence: (i) SEQ ID NO: 13 (variable region of the α chain), (ii) SEQ ID NO: 14 (variable region of the β chain), (iii) both SEQ ID NOs: 13 and 14, (iv) SEQ ID NO: 15 (variable region of the α chain), (v) SEQ ID NO: 16 (variable region of the β chain), (vi) both SEQ ID NOs: 15 and 16, (vii) SEQ ID NO: 41 (variable region of the α chain), (viii) SEQ ID NO: 42 (variable region of the β chain), (ix) both SEQ ID NOs: 41 and 42, (x) SEQ ID NO: 43 (variable region of the α chain), (xi) SEQ ID NO: 44 (variable region of the β chain), or (xii) both SEQ ID NOs: 43 and 44. Preferably, the polypeptide comprises the following amino acid sequences: (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) SEQ ID NOs: 43 and 44.

[0065] In one embodiment of the invention, the polypeptide of the invention may further include the constant region of the TCR of the invention described above. In this regard, the polypeptide may further include the following amino acid sequence: SEQ ID NO: 28 (WT murine constant region of the α chain), SEQ ID NO: 29 (WT murine constant region of the β chain), SEQ ID NO: 26 (substituted murine constant region of the α chain), SEQ ID NO: 27 (substituted murine constant region of the β chain), SEQ ID NO: 28 and 29, or SEQ ID NO: 26 and 27. Preferably, the polypeptide further includes the amino acid sequence of SEQ ID NO: 26 and 27 or SEQ ID NO: 28 and 29 in combination with any CDR region or variable region described herein for other aspects of the invention. In one embodiment of the invention, one or both of SEQ ID NO: 26 and 27 of the polypeptide is as defined in any one of Tables 2-4.

[0066] In one embodiment of the invention, a polypeptide of the invention may comprise the entire length of the α or β chain of a TCR described herein. In this regard, a polypeptide of the invention may comprise the amino acid sequence of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, both SEQ ID NOs: 30-31, both SEQ ID NOs: 32-33, 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, a polypeptide of the invention may comprise both chains of a TCR described herein.

[0067] For example, a polypeptide of the invention may include (a) an 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; (b) an amino acid sequence of SEQ ID NO: 31, wherein X at position 198 of SEQ ID NO: 31 is Ser or Cys; (c) an amino acid sequence of SEQ ID NO: 32, wherein: (i) X at position 246 of SEQ ID NO: 30 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; (i) 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; (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) the amino acid sequence of SEQ ID NO: 45, wherein: (i) SEQ ID NO: (i) X at position 161 of SEQ ID NO: 45 is Thr or Cys; (ii) X at position 225 of SEQ ID NO: 45 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 227 of SEQ ID NO: 45 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) 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) the amino acid sequence of SEQ ID NO: 47, wherein: (i) X at position 168 of SEQ ID NO: 47 is Thr or Cys; (ii) X at position 232 of SEQ ID NO: 47 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 234 of SEQ ID NO: 47 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 235 of SEQ ID NO: 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (j) 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 one embodiment of the invention, any one or more of SEQ ID NOs: 30-33 and 45-48 of the polypeptide is as defined in any one of Tables 2-4.

[0068] The present invention further provides proteins comprising at least one polypeptide described herein."Protein"means a molecule comprising one or more polypeptide chains.

[0069] In one embodiment, the protein of the present invention may include (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 NOs: 7-9 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NOs: 10-12.

[0070] In another embodiment of the present invention, the protein may include (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.

[0071] The protein of the present invention may further include any of the constant regions described herein for other aspects of the invention. In this regard, in one embodiment of the invention, (i) the first polypeptide chain may further include the amino acid sequence of SEQ ID NO: 26 and the second polypeptide chain may further include the amino acid sequence of SEQ ID NO: 27; or (ii) the first polypeptide chain may further include the amino acid sequence of SEQ ID NO: 28 and the second polypeptide chain may further include the amino acid sequence of SEQ ID NO: 29. In one embodiment 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.

[0072] Alternatively or additionally, a protein of one embodiment of the present invention may include (a) a first polypeptide 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; (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) a first polypeptide chain comprising SEQ ID NO: (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; (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 of (a) and (b); (f) both of (c) and (d); (g) a first polypeptide chain comprising SEQ ID NO: The amino acid sequence of SEQ ID NO: 45, wherein: (i) X at position 161 of SEQ ID NO: 45 is Thr or Cys; (ii) X at position 225 of SEQ ID NO: 45 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 227 of SEQ ID NO: 45 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 228 of SEQ ID NO: 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp;(h) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 46, wherein X at position 178 of SEQ ID NO: 46 is Ser or Cys; (i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 47, wherein: (i) X at position 168 of SEQ ID NO: 47 is Thr or Cys; (ii) X at position 232 of SEQ ID NO: 47 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 234 of SEQ ID NO: 47 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 235 of SEQ ID NO: 47 is 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 SEQ ID NO: X at position 171 of SEQ ID NO: 48 is Ser or Cys; (k) (g) and (h) both; or (l) (i) and (j) both. In one embodiment of the invention, one or more of SEQ ID NO: 30-33 and 45-48 are as defined in any one of Tables 2-4.

[0073] The protein of the present invention may be a TCR. Alternatively, for example, if the protein comprises a single polypeptide chain comprising the amino acid sequences of both SEQ ID NOs: 30 and 31, or both SEQ ID NOs: 32 and 33, or if the first and / or second polypeptide chains of the protein further comprise other amino acid sequences (e.g., amino acid sequences encoding immunoglobulins or portions thereof), the protein of the present invention may be a fusion protein. In this regard, the present invention also provides fusion proteins comprising at least one polypeptide of the present invention described herein and at least one other polypeptide. The other polypeptide may be present as a separate polypeptide in the fusion protein, or may be present 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 peptide or protein molecule or portion thereof (including but not limited to immunoglobulins, CD3, CD4, CD8, MHC molecules, CD1 molecules (e.g., CD1a, CD1b, CD1c, CD1d), etc.).

[0074] Fusion proteins can include one or more copies of a polypeptide of the present invention and / or one or more copies of other polypeptides. For example, a fusion protein can include 1, 2, 3, 4, 5 or more copies of a polypeptide of the present invention and / or other polypeptides. Suitable methods for preparing fusion proteins are known in the art and include, for example, recombinant methods.

[0075] 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. When a construct comprising the linker peptide is expressed by a host cell, the linker peptide may be cleaved to produce separated α and β chains. In one embodiment of the present invention, the TCR, polypeptide, or protein 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 chain and the β chain.

[0076] The protein of the present invention may be a recombinant antibody or antigen-binding portion thereof comprising at least one polypeptide of the present invention as described herein. As used herein, a "recombinant antibody" refers to a recombinant (e.g., genetically modified) protein comprising at least one polypeptide of the present invention and a polypeptide chain of an antibody or its antigen-binding portion. The polypeptide of an antibody or its antigen-binding portion may be a heavy chain, light chain, variable or constant region of a heavy chain or light chain, a single-chain variable fragment (scFv), or an Fc, Fab, or F(ab)2' fragment, etc. The polypeptide chain of an antibody or its antigen-binding portion may be an isolated polypeptide of a recombinant antibody. Alternatively, the polypeptide chain of an antibody or its antigen-binding portion may be a polypeptide expressed in the same frame (in series) as a polypeptide of the present invention. The polypeptide of an antibody or its antigen-binding portion may be a polypeptide of any antibody or any antibody fragment (including any antibody and antibody fragment described herein).

[0077] The scope of the present invention includes functional variants of the TCR, polypeptide or protein of the present invention as described herein. The term "functional variant" as used herein refers to a TCR, polypeptide or protein having substantial or significant sequence identity or similarity with a parent TCR, polypeptide or protein, and the functional variant retains the biological activity of the TCR, polypeptide or protein as its variant. Functional variants encompass (for example) TCR, polypeptide or protein (parent TCR, polypeptide or protein) as described herein that are still able to specifically bind to G12R RAS (parent TCR has antigen specificity to it or parent polypeptide or protein specifically binds to it) to a similar degree, the same degree or a higher degree compared to the parent TCR, polypeptide or protein. With reference to the parent TCR, polypeptide or protein, functional variants can be (for example) at least about 30%, about 50%, about 75%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or more of the amino acid sequence of the parent TCR, polypeptide or protein, respectively.

[0078] Functional variants may, for example, include the amino acid sequence of a parent TCR, polypeptide, or protein with at least one conservative amino acid substitution. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain 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 include substitutions of one acidic amino acid for another acidic amino acid (e.g., Asp or Glu), substitutions of one amino acid having a non-polar side chain for another amino acid having a non-polar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), substitutions of one basic amino acid for another basic amino acid (Lys, Arg, etc.), substitutions of one amino acid having a polar side chain for another amino acid having a polar side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), and the like.

[0079] Alternatively or additionally, a functional variant may comprise an amino acid sequence having at least one non-conservative amino acid substitution in a parent TCR, polypeptide, or protein. In this case, preferably, 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 enhances the biological activity of the functional variant, such that the biological activity of the functional variant is greater than that of the parent TCR, polypeptide, or protein.

[0080] TCR, polypeptide or protein can be essentially composed of one or more specified amino acid sequences as set forth herein, such that other components of the TCR, polypeptide or protein (e.g., other amino acids) do not substantially change the biological activity of the TCR, polypeptide or protein. In this regard, the TCR, polypeptide or protein of the present invention can, for example, be essentially composed of the following amino acid sequence: 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, SEQ ID NO: 30-31, SEQ ID NO: 32-33, SEQ ID NO: 34-35, or SEQ ID NO: 36-37. Likewise, for example, a TCR, polypeptide, or protein of the invention may consist essentially of the following amino acid sequence: (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. Additionally, a TCR, polypeptide, or protein of the invention may consist essentially of the following amino acid sequence: (a) any one or more of SEQ ID NOs: 1-12; (b) all of SEQ ID NOs: 1-3; (c) all of SEQ ID NOs: 4-6; (d) all of SEQ ID NOs: 7-9; (e) all of SEQ ID NOs: 10-12; (f) all of SEQ ID NOs: 1-6; or (g) all of SEQ ID NOs: 7-12.

[0081] The TCRs, polypeptides, and proteins of the present invention can be of any length, i.e., can include any number of amino acids, provided that the TCR, polypeptide, or protein retains its biological activity (e.g., the ability to specifically bind to G12R RAS; detect cancer in a mammal; or treat or prevent cancer in a mammal, etc.). For example, the length of the polypeptide can range from about 50 to about 5000 amino acids, 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 1000, or more amino acids in length. In this regard, polypeptides of the present invention also include oligopeptides.

[0082] The TCRs, polypeptides, and proteins of the present invention may include 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, dihydro Indole-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.

[0083] The TCRs, polypeptides, and proteins of the invention can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, converted into acid addition salts, and / or optionally dimerized or polymerized, or conjugated, for example, via disulfide bridges.

[0084] The TCRs, polypeptides and / or proteins of the present invention can be obtained by methods known in the art (e.g., de novo synthesis). In addition, the nucleic acids described herein can be used recombinantly and standard recombinant methods can be used to produce polypeptides and proteins. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Alternatively, the TCRs, polypeptides, and / or proteins described herein can be synthesized commercially by companies such as Synpep (Dublin, CA), Peptide Technologies Corp. (Gaithersburg, MD), and Multiple Peptide Systems (San Diego, CA). In this regard, the TCRs, polypeptides, and proteins of the present invention can be synthetic, recombinant, isolated, and / or purified. One embodiment of the present invention provides an isolated or purified TCR, polypeptide, or protein encoded by any nucleic acid or vector described herein for other aspects of the present invention. Another embodiment of the present invention provides an isolated or purified TCR, polypeptide, or protein produced by expressing in a cell any nucleic acid or vector described herein for other aspects of the present invention. Yet another embodiment of the present invention provides a method for producing any TCR, polypeptide, or protein described herein, comprising culturing any host cell or host cell population described herein to produce the TCR, polypeptide, or protein.

[0085] The scope of the present invention includes 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, host cell populations, or antibodies or antigen-binding portions thereof of the present invention. In general, conjugates and methods for synthesizing conjugates are known in the art.

[0086] One embodiment of the present invention provides nucleic acids comprising a nucleotide sequence encoding any TCR, polypeptide or protein described herein. "Nucleic acid" as used herein includes "polynucleotide," "oligonucleotide" and "nucleic acid molecule," and generally refers to a polymer of DNA or RNA, which may be single-stranded or double-stranded, may contain natural, non-natural or altered nucleotides, and may contain natural, non-natural or altered internucleotide linkages (e.g., aminophosphoester linkages or thiophosphate linkages) to replace the phosphodiester found between the nucleotides of unmodified oligonucleotides. In one embodiment, the nucleic acid includes complementary DNA (cDNA). It is generally preferred that the nucleic acid does not include any insertion, deletion, inversion and / or substitution. However, in some cases, as discussed herein, the nucleic acid may suitably include one or more insertions, deletions, inversions and / or substitutions.

[0087] Preferably, the nucleic acids of the present invention are recombinant. As used herein, the term "recombinant" means that (i) the molecule is constructed outside of a living cell by joining a natural or synthetic nucleic acid segment to a nucleic acid molecule that can replicate in a living cell, or (ii) the molecule is derived from replication of that described in (i) above. For the purposes of this article, replication can be in vitro replication or in vivo replication.

[0088] Nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. For example, see Green and Sambrook et al., supra. For example, nucleic acids can be synthesized chemically using natural nucleotides or various modified nucleotides designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed upon hybridization, such as phosphorothioate derivatives and acridine-substituted nucleotides. Examples of modified nucleotides that can be used to generate nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylquenoside, inosine, N- 6 -Isopentenyl adenine, 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, β-D-mannosyl braided glycoside, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6 -isopentenyl adenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, quercetin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine. Alternatively, one or more nucleic acids of the present invention can be purchased from companies such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0089] The nucleic acid may include any nucleotide sequence encoding any TCR, polypeptide, or protein described herein. In one embodiment of the present invention, the nucleic acid includes a codon-optimized nucleotide sequence encoding any TCR, polypeptide, or protein described herein. Without being limited to any particular theory or mechanism, it is believed that codon optimization of the nucleotide sequence increases the translation efficiency of the mRNA transcript. Codon optimization of the nucleotide sequence may involve replacing the natural codon with another codon that encodes the same amino acid but can be translated by tRNA that is more readily available in the cell, thereby increasing translation efficiency. Optimization of the nucleotide sequence can also reduce secondary mRNA structures that interfere with translation, thereby increasing translation efficiency.

[0090] The present invention also provides nucleic acids comprising: a nucleotide sequence complementary to the nucleotide sequence of any nucleic acid described herein; or a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of any nucleic acid described herein.

[0091] Nucleotide sequences that hybridize under stringent conditions preferably hybridize under high stringency conditions. "High stringency conditions" means that the nucleotide sequence specifically hybridizes to the target sequence (the nucleotide sequence of any nucleic acid described herein) in an amount that is detectably stronger than non-specific hybridization. High stringency conditions include conditions that distinguish polynucleotides with strictly complementary sequences or random sequences containing only a few scattered mismatches from just a few smaller regions (e.g., 3-10 bases) that match the nucleotide sequence. Such smaller regions with complementarity are easier to melt than full-length complements of 14-17 or more bases, and high stringency hybridization makes them easily distinguishable. Relatively high stringency conditions include, for example, low salt and / or high temperature conditions, such as provided by about 0.02-0.1 M NaCl or equivalent at a temperature of about 50-70°C. Such high stringency conditions tolerate fewer (if any) mismatches between the nucleotide sequence and the template or target chain and are particularly suitable for detecting the expression of any TCR of the present invention. It should generally be understood that conditions can be made more stringent by adding increased amounts of formamide.

[0092] One embodiment of the present invention also provides a nucleic acid comprising a nucleotide sequence that is at least about 70% or identical (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 can consist essentially of any of the nucleotide sequences described herein.

[0093] One embodiment of the present invention provides an isolated or purified nucleic acid comprising, from 5' to 3', a first nucleic acid sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence encode the following amino acid sequence, respectively: SEQ ID NO: 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.

[0094] In one embodiment of the present invention, the isolated or purified nucleic acid further includes a third nucleotide sequence inserted between the first nucleotide sequence and the second nucleotide sequence, wherein the third nucleotide sequence encodes a cleavable linker peptide. In one embodiment of the present invention, the cleavable linker peptide includes the amino acid sequence of SEQ ID NO: 38.

[0095] 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 one embodiment of the present invention, the recombinant expression vector comprises a nucleotide sequence encoding an α chain, a β chain, and a linker peptide.

[0096] For the purposes of this document, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that includes a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide and allows the expression of the mRNA, protein, polypeptide, or peptide by the cell when the construct is contacted with a cell under conditions sufficient for the expression of the mRNA, protein, polypeptide, or peptide in the host cell. The vectors of the present invention are not natural as a whole. However, a portion of the vector may be natural. The recombinant expression vectors of the present invention may include any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthesized or partially obtained from natural sources, and may contain natural, non-natural, or altered nucleotides. The recombinant expression vector may include natural, non-natural internucleotide linkages, or both types of linkages. Preferably, the non-natural or altered nucleotides or internucleotide linkages do not hinder transcription or replication of the vector.

[0097] The recombinant expression vector of the present invention can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host cell. Suitable vectors include those designed for reproduction and amplification or for expression or for both, such as plasmids and viruses. Vectors can be selected from: 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). Phage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4 and λNM1149 can 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 one embodiment of the present invention, the recombinant expression vector is a transposon or lentiviral vector.

[0098] The recombinant expression vectors of the present invention can be prepared using standard recombinant DNA techniques as described, for example, in Greem and Sambrook et al. Circular or linear expression vector constructs containing replication systems that function in prokaryotic or eukaryotic host cells can be prepared. Replication systems can be derived, for example, from ColE1, 2μ plasmids, lambda, SV40, bovine papilloma virus, and the like.

[0099] Desirably, where appropriate and considering whether the vector is DNA- or RNA-based, recombinant expression vectors include regulatory sequences, such as transcriptional and translational start and stop codons, specific for the host cell type (e.g., bacteria, fungi, plants, or animals) into which the vector is to be introduced.

[0100] The recombinant expression vector may contain one or more marker genes that allow selection of transformed or transfected host cells. Marker genes include biocidal resistance (e.g., resistance to antibiotics, heavy metals, etc.), complementation in auxotrophic host cells (to provide prototrophy), etc. Suitable marker genes for use in the expression vectors of the present invention include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0101] The recombinant expression vector may include a natural or non-natural promoter operably linked to a nucleotide sequence encoding a TCR, polypeptide or protein or a nucleotide sequence complementary to or hybridized to a nucleotide sequence encoding a TCR, polypeptide or protein. Those skilled in the art are familiar with the selection of promoters (e.g., strong, weak, inducible, tissue-specific and developmentally specific). Similarly, those skilled in the art are also familiar with the combination of nucleotide sequence and promoter. The promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, a SV40 promoter, a RSV promoter, and a promoter found in the long terminal repeats of murine stem cell virus.

[0102] The recombinant expression vectors of the present invention can be designed for transient expression, stable expression, or both. Likewise, the recombinant expression vectors can be prepared for constitutive expression or inducible expression.

[0103] In addition, a recombinant expression vector comprising a suicide gene can be prepared. As used herein, the term "suicide gene" refers to a gene that causes cell death in which the suicide gene is expressed. A suicide gene can be a gene that confers sensitivity to a cell agent (e.g., a drug) that expresses the gene and causes cell death when the cell contacts or is exposed to the agent. Suicide genes are known in the art and include, for example, herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, nitroreductase, and inducible caspase 9 gene systems.

[0104] Another embodiment of the present invention further provides a host cell comprising any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that can contain a recombinant expression vector of the present invention. The host cell can be a eukaryotic cell (e.g., a plant, animal, fungal, or algal cell), or can be a prokaryotic cell (e.g., a bacterial or protozoan cell). The host cell can be a cultured cell or a primary cell (i.e., directly isolated from an organism, such as a human). The host cell can be an attached cell or a suspension cell (i.e., a cell grown in suspension). Suitable host cells are known in the art and include, for example, DH5α Escherichia coli ( E. coli ) cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells and the like. For the purpose of amplifying or replicating the recombinant expression vector, the host cell is preferably a prokaryotic cell, such as a DH5α cell. For the purpose of producing a recombinant TCR, polypeptide or protein, the host cell is preferably a mammalian cell. Most preferably, the host cell line is a human cell. Although the host cell can be any cell type, can be derived from any type of tissue and can be any developmental stage, the host cell is preferably a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). More preferably, the host cell line is a T cell. In one embodiment of the present invention, the host cell line is a human lymphocyte. In another embodiment of the present invention, the host cell is selected from: T cells, natural killer T (NKT) cells, invariant natural killer T (iNKT) cells and natural killer (NK) cells. Yet another embodiment of the present invention provides a method for producing a host cell expressing a TCR having antigenic specificity for the peptide of SEQ ID NO: 39, the method comprising contacting the cell with any of the vectors described herein under conditions that allow introduction of the vector into the cell.

[0105] For the purposes of this article, T cells can be any T cells, such as cultured T cells (e.g., primary T cells, or T cells from cultured T cell lines (e.g., Jurkat, SupT1, etc.), or T cells obtained from mammals). If obtained from mammals, T cells can be obtained from a variety of sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells can also be enriched or purified. Preferably, T cells are human T cells. T cells can be of any type of T cell and can be of any developmental stage, including but not limited to CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells (such as Th1 and Th2 cells), CD4 + T cells, CD8 + T cells (such as cytotoxic T cells), tumor infiltrating lymphocytes (TILs), memory T cells (such as central memory T cells and effector memory T cells), naive T cells, etc.

[0106] The present invention also provides a cell colony comprising at least one host cell as described herein. The cell colony can be a heterogeneous colony comprising a host cell containing any of the recombinant expression vectors described herein and at least one other cell (e.g., a host cell (e.g., T cell) or a cell (e.g., B cell, macrophage, neutrophil, red blood cell, hepatocyte, endothelial cell, epithelial cell, muscle cell, brain cell, etc.) excluding any of the recombinant expression vectors). Alternatively, the cell colony can be a substantially homogeneous colony, wherein the colony mainly comprises (e.g., essentially consisting of) a host cell containing a recombinant expression vector. The colony can also be a cloned cell colony, wherein all cells of the colony are clones of a single host cell comprising a recombinant expression vector, such that all cells of the colony comprise a recombinant expression vector. In one embodiment of the present invention, the cell colony is a clonal colony comprising a host cell containing a recombinant expression vector as described herein.

[0107] In one embodiment of the present invention, the number of cells in the population can be rapidly expanded. T cell numbers can be expanded by any of a number of methods known in the art, such as described in, for example, U.S. Patent No. 8,034,334; U.S. Patent No. 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 In one embodiment, T cells are expanded by culturing them with OKT3 antibody, IL-2, and feeder PBMCs (eg, irradiated allogeneic PBMCs).

[0108] The TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, and host cells (including populations thereof) of the present invention can be isolated and / or purified. The term "isolated" as used herein means removed from its natural environment. The term "purified" as used herein means having increased purity, wherein "purity" is a relative term and need not be interpreted as absolute purity. For example, the purity can be at least about 50%, can be greater than about 60%, about 70%, about 80%, about 90%, about 95%, or can be about 100%.

[0109] The TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, and host cells of the present invention (including populations thereof, hereinafter collectively referred to as "TCR materials of the present invention") can be formulated into compositions (e.g., pharmaceutical compositions). In this regard, the present invention provides a pharmaceutical composition comprising any of the TCRs, polypeptides, proteins, nucleic acids, expression vectors, and host cells (including populations thereof) described herein and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention containing any TCR material of the present invention may include more than one TCR material of the present invention (e.g., polypeptides and nucleic acids) or two or more different TCRs. Alternatively, a pharmaceutical composition may include a TCR material of the present invention in combination with another pharmaceutically active agent or drug, such as a chemotherapeutic agent, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, and the like.

[0110] Preferably, the carrier is a pharmaceutically acceptable carrier. For pharmaceutical compositions, the carrier may be any of the conventionally used TCR materials of the present invention. Methods for preparing cocoa administration compositions are known or apparent to those skilled in the art and are described in more detail in, for example, Remington: The Science and Practice of Pharmacy , 22nd ed., Pharmaceutical Press (2012). Preferably, a pharmaceutically acceptable carrier is one that has no deleterious side effects or toxicity under the conditions of use.

[0111] The choice of carrier depends in part on the specific TCR material of the present invention and the specific method for administering the TCR material of the present invention. Therefore, there are various suitable formulations of the pharmaceutical composition of the present invention. Suitable formulations may include any one for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, intratumoral or intraperitoneal administration. More than one route can be used to administer the TCR material of the present invention, and in some cases, a specific route can provide a faster and more effective response than another route.

[0112] Preferably, the TCR material of the present invention is administered by injection (e.g., intravenously). When the TCR material of the present invention is a host cell (or population thereof) expressing a TCR of the present invention, the pharmaceutically acceptable carrier for the cells for injection may include any isotonic carrier (e.g., normal saline (about 0.90% w / v NaCl in water, about 300 mOsm / L NaCl in water, or about 9.0 g NaCl / liter water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water, or Ringer's lactate). In one embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumin.

[0113] For the purposes of the present invention, the amount or dose of the TCR material of the present invention administered (e.g., the number of cells when the TCR material of the present invention is one or more cells) should be sufficient to produce a certain effect (e.g., a therapeutic or prophylactic response) in an individual or animal over an appropriate time frame. For example, the 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 within a period of about 2 hours or longer (e.g., 12 to 24 or more hours) after administration. In certain embodiments, this time period can be even longer. The dosage depends on the efficacy of the specific TCR material of the present invention and the condition of the animal (e.g., human) and the weight of the animal (e.g., human) to be treated.

[0114] Numerous assays are known in the art for determining the dosage to be administered. For the purposes of the present invention, an assay comprising the following process can be used to determine the starting dose to be administered to a mammal: comparing the extent of target cell lysis or IFN-γ secretion by T cells expressing a TCR, polypeptide, or protein of the invention following administration of a given dose of such T cells to a group of mammals, each of which has been administered a different dose of such T cells. The extent of target cell lysis or IFN-γ secretion following administration of a given dose can be analyzed by methods known in the art.

[0115] The dosage of the TCR material of the present invention also depends on the presence, nature, and extent of any adverse side effects that may accompany the administration of a particular TCR material of the present invention. Generally, the attending physician determines the dosage of the TCR material of the present invention to be treated for each individual patient after considering various factors (e.g., age, weight, overall health, diet, sex, the TCR material of the present invention to be administered, the route of administration, and the severity of the cancer being treated). In embodiments where the TCR material of the present invention is a cell population, the number of cells administered per infusion can vary, for example, from about 1 × 10 6 About 1 × 1012 In some embodiments, less than 1 × 10 6 cells.

[0116] It will be readily understood by those skilled in the art that the inventive TCR materials of the present invention can be modified in any number of ways to increase the therapeutic or preventive efficacy of the TCR materials of the present invention via modification. For example, the TCR materials of the present invention may be conjugated to a chemotherapeutic agent directly or indirectly via a bridge. The practice of conjugating compounds to chemotherapeutic agents is known in the art. Those skilled in the art recognize that sites on the TCR materials of the present invention that are not required for the function of the TCR materials of the present invention are suitable sites for attaching bridges and / or chemotherapeutic agents, provided that the bridges and / or chemotherapeutic agents do not interfere with the function of the TCR materials of the present invention (i.e., being able to bind to G12R RAS or detecting, treating or preventing cancer) after being attached to the TCR materials of the present invention.

[0117] The pharmaceutical compositions, TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, and cell populations of the present invention are expected to be useful in methods for treating or preventing cancer. Without being limited to a particular theory, it is believed that the TCRs of the present invention specifically bind to G12R RAS, such that the TCRs (or related polypeptides or proteins of the present invention) are capable of mediating an immune response against target cells expressing G12R RAS when expressed by cells. In this regard, one embodiment of the present invention provides a method for treating or preventing cancer in a mammal, comprising administering to the mammal any pharmaceutical composition, TCR, polypeptide, or protein described herein, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any TCR, polypeptide, or protein described herein, or any host cell or cell population comprising a recombinant vector encoding any TCR, polypeptide, or protein described herein, in an amount effective to treat or prevent cancer in the mammal.

[0118] One embodiment of the present invention provides a method for inducing an immune response against cancer in a mammal, comprising administering to the mammal any pharmaceutical composition, TCR, polypeptide or protein described herein, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any TCR, polypeptide or protein described herein, or any host cell or cell population comprising a recombinant vector encoding any TCR, polypeptide or protein described herein, in an amount effective to induce an immune response against cancer in the mammal.

[0119] One embodiment of the present invention provides any pharmaceutical composition, TCR, polypeptide or protein described herein, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any TCR, polypeptide or protein described herein, or any host cell or cell population comprising a recombinant vector encoding any TCR, polypeptide or protein described herein, for use in treating or preventing cancer in a mammal.

[0120] One embodiment of the present invention provides any pharmaceutical composition, TCR, polypeptide or protein described herein, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any TCR, polypeptide or protein described herein, or any host cell or cell population comprising a recombinant vector encoding any TCR, polypeptide or protein described herein, for use in inducing an immune response against cancer in a mammal.

[0121] As used herein, the terms "treat" and "prevent" and words derived therefrom do not necessarily mean 100% or complete treatment or prevention. Rather, there are different degrees of treatment or prevention that one skilled in the art would recognize as potentially beneficial or therapeutic. In this regard, the methods of the present invention can provide any level of treatment or prevention of any amount of cancer in a mammal. In addition, the treatment or prevention provided by the methods of the present invention can include treating or preventing one or more conditions or symptoms of the cancer being treated or prevented. For example, treatment or prevention can include promoting tumor regression. Likewise, for purposes herein, "prevention" can encompass delaying the onset of cancer or its symptoms or conditions. Alternatively or in addition, "prevention" can encompass preventing or delaying the onset of cancer or its symptoms or conditions.

[0122] Also provided are methods for detecting the presence of cancer in a mammal, comprising (i) contacting a sample comprising one or more cells from the mammal with any of the TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, cell populations, or pharmaceutical compositions of the invention described herein, thereby forming a complex, and detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

[0123] In the method of the present invention for detecting cancer in a mammal, the cell sample may be a sample comprising whole cells, a lysate thereof, or a portion of a whole cell lysate (eg, a nuclear or cytoplasmic fraction, a total protein fraction, or a nucleic acid fraction).

[0124] For the purpose of the methods of the present invention for detecting cancer, contacting can occur in vitro or in vivo in a mammal. Preferably, contacting is performed in vitro.

[0125] Likewise, the complex can be detected by any quantitative means known in the art. For example, the TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, or cell populations of the invention described herein can be labeled with a detectable marker, such as a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

[0126] For the purposes of the methods of the invention in which a host cell or cell population is administered, the cells may be allogeneic or autologous to the mammal. Preferably, the cells are autologous to the mammal.

[0127] For the purposes of the present invention, the cancer may be any cancer, including any of the following: acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, anal canal cancer or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer or pleural cancer, nasal cancer, nasal cavity cancer or middle ear cancer, oral cancer, vaginal cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumors, glioma, Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, Preferably, the cancer is pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer, or prostate cancer. Preferably, the cancer is pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer, or prostate cancer. Preferably, the lung cancer is lung adenocarcinoma, the ovarian cancer is epithelial ovarian cancer, and the pancreatic cancer is pancreatic adenocarcinoma. In one embodiment of the present invention, the cancer expresses a mutant human RAS amino acid sequence in which the glycine at position 12 is substituted with an arginine, wherein the mutant human RAS amino acid sequence is a mutant human KRAS, mutant human HRAS, or mutant human NRAS amino acid sequence, and wherein position 12 is defined with reference to a 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 can be as described herein for other aspects of the present invention.

[0128] The mammals mentioned 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 mammals of the order Rodentia (e.g., mice and hamsters) and mammals of the order Lagomorpha (e.g., rabbits). Preferably, the mammal is from the order Carnivora, including Felidae (cats) and Canidae (dogs). More preferably, the mammal is from the order Artiodactyla (including Bovidae (cattle) and Suidae (pigs)) or Perissodactyla (including Equine (horses)). Most preferably, the mammal is from the order Primates, Simians or Monkeys (monkeys) or Anthropoids (humans and apes). A particularly preferred mammal is a human.

[0129] This application also relates to the following embodiments: Embodiment 1. An isolated or purified T cell receptor (TCR), wherein the TCR has antigenic 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 a mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS), a mutant human Harvey rat sarcoma viral oncogene homolog (HRAS), or a mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence, and Position 12 is defined with reference to wild-type human KRAS protein, wild-type human HRAS protein or wild-type human NRAS protein, respectively.

[0130] Embodiment 2. The TCR of embodiment 1, wherein the mutant human RAS amino acid sequence is SEQ ID NO: 39.

[0131] Embodiment 3. The TCR of embodiment 1 or 2, wherein the TCR has no antigenic specificity for the wild-type human RAS amino acid sequence of SEQ ID NO: 40.

[0132] Embodiment 4. The TCR of any one of Embodiments 1 to 3, wherein the mutant human RAS amino acid sequence is presented by a human leukocyte antigen (HLA) class II molecule.

[0133] Embodiment 5. The TCR of Embodiment 4, wherein the HLA class II molecule is an HLA-DR heterodimer or an HLA-DQ heterodimer.

[0134] Embodiment 6. The TCR of embodiment 4, wherein the HLA class II molecule is an HLA-DRB5:HLA-DRA heterodimer or an HLA-DQA1:HLA-DQB1 heterodimer.

[0135] Embodiment 7. The TCR of embodiment 4, wherein the HLA class II molecule is HLA-DRB5 01:HLA-DRA 01:01 Heterodimer or HLA-DQA1 05:05:HLA-DQB1 03:01Heterodimer.

[0136] Embodiment 8. The TCR of any one of embodiments 1 to 7, comprising the following amino acid sequence: (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.

[0137] Embodiment 9. The TCR of any one of embodiments 1 to 8, comprising the following amino acid sequence: (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.

[0138] Embodiment 10. The TCR of any one of embodiments 1 to 9, further comprising: (a) an α chain constant region comprising the amino acid sequence of SEQ ID NO: 26, wherein: (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, Ile, Pro, Phe, Met, or Trp; (iii) X at position 114 of SEQ ID NO: 26 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 115 of SEQ ID NO: 26 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) a beta chain constant region 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).

[0139] Embodiment 11. The isolated or purified TCR of any one of Embodiments 1 to 10, comprising: (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; (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; (c) an α chain comprising the amino acid sequence of SEQ ID NO: 32, wherein: (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; (d) a β 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) an α chain comprising the amino acid sequence of SEQ ID NO: 45, wherein: (i) X at position 161 of SEQ ID NO: 45 is Thr or Cys; (ii) X at position 225 of SEQ ID NO: 45 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 227 of SEQ ID NO: 45 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 228 of SEQ ID NO: 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (h) a beta 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) an α chain comprising the amino acid sequence of SEQ ID NO: 47, wherein: (i) X at position 168 of SEQ ID NO: 47 is Thr or Cys; (ii) X at position 232 of SEQ ID NO: 47 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 234 of SEQ ID NO: 47 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 235 of SEQ ID NO: 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (j) a β 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).

[0140] Embodiment 12. An isolated or purified polypeptide comprising a functional portion of the TCR of any one of embodiments 1 to 11, wherein the functional portion comprises the following amino acid sequence: (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.

[0141] Embodiment 13. The isolated or purified polypeptide of embodiment 12, wherein the functional portion comprises the following amino acid sequence: (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.

[0142] Embodiment 14. The isolated or purified polypeptide of embodiment 12 or 13, further comprising: (a) the amino acid sequence of SEQ ID NO: 26, wherein: (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, Ile, Pro, Phe, Met, or Trp; (iii) X at position 114 of SEQ ID NO: 26 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 115 of SEQ ID NO: 26 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) the 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).

[0143] Embodiment 15. The isolated or purified polypeptide of any one of embodiments 12-14, comprising: (a) 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; (b) the amino acid sequence of SEQ ID NO: 31, wherein X at position 198 of SEQ ID NO: 31 is Ser or Cys; (c) the amino acid sequence of SEQ ID NO: 32, wherein: (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; (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) the amino acid sequence of SEQ ID NO: 45, wherein: (i) X at position 161 of SEQ ID NO: 45 is Thr or Cys; (ii) X at position 225 of SEQ ID NO: 45 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 227 of SEQ ID NO: 45 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) 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) the amino acid sequence of SEQ ID NO: 47, wherein: (i) X at position 168 of SEQ ID NO: 47 is Thr or Cys; (ii) X at position 232 of SEQ ID NO: 47 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 234 of SEQ ID NO: 47 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 235 of SEQ ID NO: 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (j) 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).

[0144] Embodiment 16. An isolated or purified protein comprising at least one polypeptide according to any one of embodiments 12 to 15.

[0145] Embodiment 17. The isolated or purified protein of embodiment 16, comprising: (a) a first polypeptide chain comprising the amino acid sequence of SEQ ID NOs: 1 to 3 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NOs: 4 to 6; or (b) a first polypeptide chain comprising the amino acid sequence of SEQ ID NOs: 7 to 9 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NOs: 10 to 12.

[0146] Embodiment 18. The isolated or purified protein of embodiment 16 or 17, comprising: (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.

[0147] Embodiment 19. The isolated or purified protein of any one of Embodiments 16 to 18, further comprising: (a) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 26, wherein: (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, Ile, Pro, Phe, Met, or Trp; (iii) X at position 114 of SEQ ID NO: 26 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 115 of SEQ ID NO: 26 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) 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).

[0148] Embodiment 20. The isolated or purified protein of any one of embodiments 16 to 19, comprising: (a) a first polypeptide 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; (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) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 32, wherein: (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; (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) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 45, wherein: (i) X at position 161 of SEQ ID NO: 45 is Thr or Cys; (ii) X at position 225 of SEQ ID NO: 45 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 227 of SEQ ID NO: 45 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 228 of SEQ ID NO: 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (h) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 46, wherein X at position 178 of SEQ ID NO: 46 is Ser or Cys; (i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 47, wherein: (i) X at position 168 of SEQ ID NO: 47 is Thr or Cys; (ii) X at position 232 of SEQ ID NO: 47 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 234 of SEQ ID NO: 47 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 235 of SEQ ID NO: 47 is 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).

[0149] Embodiment 21. An isolated or purified nucleic acid comprising a nucleotide sequence encoding the TCR of any one of embodiments 1 to 11, the polypeptide of any one of embodiments 12 to 15, or the protein of any one of embodiments 16 to 20.

[0150] Embodiment 22. An isolated or purified nucleic acid comprising, from 5′ to 3′, a first nucleic acid sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence encode the following amino acid sequence, respectively: SEQ ID NO: 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.

[0151] Embodiment 23. The isolated or purified nucleic acid of embodiment 22, further comprising a third nucleotide sequence inserted between the first nucleotide sequence and the second nucleotide sequence, wherein the third nucleotide sequence encodes a cleavable linker peptide.

[0152] Embodiment 24. The isolated or purified nucleic acid of Embodiment 23, wherein the cleavable linker peptide comprises the amino acid sequence of SEQ ID NO: 38.

[0153] Embodiment 25. A recombinant expression vector comprising the nucleic acid of any one of embodiments 21 to 24.

[0154] Embodiment 26. The recombinant expression vector according to embodiment 25, which is a transposon or lentiviral vector.

[0155] Embodiment 27. An isolated or purified TCR, polypeptide, or protein encoded by the nucleic acid of any one of embodiments 21 to 24 or the vector of embodiment 25 or 26.

[0156] Embodiment 28. An isolated or purified TCR, polypeptide, or protein produced by expressing the nucleic acid of any one of Embodiments 21 to 24 or the vector of Embodiment 25 or 26 in a cell.

[0157] Embodiment 29. A method of producing a host cell expressing a TCR having antigenic specificity for a peptide of SEQ ID NO: 39, the method comprising contacting the cell with the vector of embodiment 25 or 26 under conditions permitting introduction of the vector into the cell.

[0158] Embodiment 30. An isolated or purified host cell comprising the nucleic acid of any one of embodiments 21 to 24 or the recombinant expression vector of embodiment 25 or 26.

[0159] Embodiment 31. The host cell of embodiment 30, wherein the cell line is a human lymphocyte.

[0160] Embodiment 32. The host cell of embodiment 30 or 31, wherein the cell is selected from a T cell, a natural killer T (NKT) cell, an invariant natural killer T (iNKT) cell, and a natural killer (NK) cell.

[0161] Embodiment 33. An isolated or purified cell population comprising the host cell of any one of embodiments 30 to 32.

[0162] Embodiment 34. A method of producing a TCR as described in any one of embodiments 1 to 11, 27 or 28, a polypeptide as described in any one of embodiments 12 to 15, 27 or 28, or a protein as described in any one of embodiments 16 to 20, 27 or 28, the method comprising culturing a host cell as described in any one of embodiments 30 to 32 or a population of host cells as described in embodiment 33, thereby producing the TCR, the polypeptide or the protein.

[0163] Embodiment 35. A pharmaceutical composition comprising (a) a TCR as described in any one of embodiments 1 to 11, 27 or 28, a polypeptide as described in any one of embodiments 12 to 15, 27 or 28, or a protein as described in any one of embodiments 16 to 20, 27 or 28, a nucleic acid as described in any one of embodiments 21 to 24, a recombinant expression vector as described in embodiment 25 or 26, a host cell as described in any one of embodiments 30 to 32, or a cell population as described in embodiment 33, and (b) a pharmaceutically acceptable carrier.

[0164] Embodiment 36. A method of detecting the presence of cancer in a mammal, the method comprising: (a) contacting a sample comprising cancer cells with a TCR as described in any one of embodiments 1 to 11, 27 or 28, a polypeptide as described in any one of embodiments 12 to 15, 27 or 28, or a protein as described in any one of embodiments 16 to 20, 27 or 28, a nucleic acid as described in any one of embodiments 21 to 24, a recombinant expression vector as described in embodiment 25 or 26, a host cell as described in any one of embodiments 30 to 32, a cell population as described in embodiment 33, or a pharmaceutical composition as described in embodiment 35, thereby forming a complex; and (b) detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

[0165] Embodiment 37. The TCR of any one of embodiments 1 to 11, 27 or 28, the polypeptide of any one of embodiments 12 to 15, 27 or 28, or the protein of any one of embodiments 16 to 20, 27 or 28, the nucleic acid of any one of embodiments 21 to 24, the recombinant expression vector of embodiment 25 or 26, the host cell of any one of embodiments 30 to 32, the cell population of embodiment 33, or the pharmaceutical composition of embodiment 35, for use in inducing an immune response to cancer in a mammal.

[0166] Embodiment 38. The TCR of any one of embodiments 1 to 11, 27 or 28, the polypeptide of any one of embodiments 12 to 15, 27 or 28, or the protein of any one of embodiments 16 to 20, 27 or 28, the nucleic acid of any one of embodiments 21 to 24, the recombinant expression vector of embodiment 25 or 26, the host cell of any one of embodiments 30 to 32, the cell population of embodiment 33, or the pharmaceutical composition of embodiment 35, for use in treating or preventing cancer in a mammal.

[0167] Embodiment 39. The method of embodiment 36 or the TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, cell population, or pharmaceutical composition for use according to embodiment 37 or 38, wherein the cancer expresses 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 a mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS), a mutant human Harvey rat sarcoma viral oncogene homolog (HRAS), or a mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence, and Position 12 is defined with reference to wild-type human KRAS protein, wild-type human HRAS protein or wild-type human NRAS protein, respectively.

[0168] Embodiment 40. The method of embodiment 39 or the TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, cell population or pharmaceutical composition for the use of embodiment 39, wherein the mutant human RAS amino acid sequence is a mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS) amino acid sequence.

[0169] Embodiment 41. The method of embodiment 39 or the TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, cell population or pharmaceutical composition for the use of embodiment 39, wherein the mutant human RAS amino acid sequence is a mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence.

[0170] Embodiment 42. The method of embodiment 39 or the TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, cell population or pharmaceutical composition for the use of embodiment 39, wherein the mutant human RAS amino acid sequence is a mutant human Harvey rat sarcoma viral oncogene homolog (HRAS) amino acid sequence.

[0171] Embodiment 43. The method of any one of embodiments 36 and 39 to 42, or the TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, cell population, or pharmaceutical composition for use as described in any one of embodiments 38 to 42, wherein the cancer is pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer, or prostate cancer.

[0172] The following examples further illustrate the invention but, of course, should not be construed as limiting its scope in any way.

[0173] Example 1

[0174] This example shows that the G12R mutation is present in HLA-DRB5 01:HLA-DRA 01:01Isolation of antigen-specific TCRs for heterodimer-presented human KRAS.

[0175] TILs isolated from a patient with metastatic pancreatic cancer (patient 4270) harbored a G12R mutation and were expressed by HLA-DRB5 01:HLA-DRA 01:01 Heterodimer-presented human KRAS has antigen-specific TCRs. TCRs were isolated from TILs that showed specific recognition for KRAS G12R but not for WT KRAS.

[0176] To sequence the reactive 4270 TCRs, reactive TILs were sorted by fluorescence-activated cell sorting (FACS) based on the upregulation of the T cell activation marker 4-1BB. Subsequently, the cells were lysed, and Sanger sequencing was performed on the TCR transcripts. The amino acid sequences of the 4270 TCR α and β chain variable regions are shown in Table 5. The CDRs are underlined.

[0177] Table 5

[0178] Example 2

[0179] This example demonstrates that cells transduced with the 4270 TCR isolated in Example 1 specifically recognize the G12R RAS peptide.

[0180] The nucleic acid sequence encoding the G12R RAS-reactive 4270 TCR of Example 1, comprising a cysteine-substituted, LVL-modified murine constant region, was cloned into a retroviral expression vector. The α chain murine constant region comprises the amino acid sequence of SEQ ID NO: 26, wherein X at position 48 is Cys, X at position 112 is Leu, X at position 114 is Ile, and X at position 115 is Val. The β chain constant region comprises the amino acid sequence of SEQ ID NO: 27, wherein X at position 57 is Cys. A linker comprising the amino acid sequence of RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 38) is positioned between the α chain constant region and the β chain variable region. Allogeneic PBMCs were transduced using the retroviral expression vector.

[0181] Transduced cells were treated with the WT RAS peptide MTEYKLVVVGA G GVGKSALTIQLI (SEQ ID NO: 40) or G12RRAS peptide MTEYKLVVVGA R Autologous DCs pulsed with serial dilutions of GVGKSALTIQLI (SEQ ID NO: 39) were co-cultured overnight. IFNγ secretion was assessed by enzyme-linked immunospot (ELISpot). The results are shown in Figure 1 middle.

[0182] like Figure 1As shown in , cells transduced with the 4270 TCR specifically recognized the G12R RAS peptide.

[0183] Example 3

[0184] This example demonstrates that the cells transduced with the 4270 TCR isolated in Example 1 recognize and present HLA-DRB5 01:HLA-DRA 01:01G12R RAS peptide in the context of heterodimers.

[0185] Allogeneic PBMCs were virally transduced using the retroviral expression vector of Example 2. Figure 2 Target COS7 cells were transfected with one of the HLA molecules in the G12R RAS peptide MTEYKLVVVGA. R COS7 cells were pulsed with GVGKSALTIQLI (SEQ ID NO: 39). 4270 TCR-transduced cells were co-cultured with pulsed, transfected COS7 cells overnight. IFNγ secretion was assessed by ELISA. The results are shown in Figure 2 middle.

[0186] like Figure 2 As shown in Example 1, the cells transduced with 4270 TCRs isolated in Example 1 specifically recognized the HLA-DRB5 01:HLA-DRA 01:01G12R RAS peptide in the context of heterodimers.

[0187] Example 4

[0188] This example shows that the G12R mutation is present in HLA-DQA1 05:05:HLA-DQB1 03:01 Isolation of antigen-specific TCRs for heterodimer-presented human KRAS.

[0189] TILs isolated from a patient with metastatic colon cancer (patient 4268) harbored a G12R mutation and were expressed by HLA-DQA1. 05:05:HLA-DQB1 03:01 Heterodimer-presented human KRAS has antigen-specific TCRs. TCRs were isolated from TILs that showed specific recognition for KRAS G12R but not for WT KRAS.

[0190] To sequence the reactive 4268 TCRs, reactive TILs were FACS sorted based on the upregulation of the T cell activation marker 4-1BB. Subsequently, the cells were lysed and Sanger sequencing was performed on the TCR transcripts. The amino acid sequences of the 4268 TCR α and β chain variable regions are shown in Table 6. The CDRs are underlined.

[0191] Table 6

[0192] Example 5

[0193] This example demonstrates that cells transduced with the 4268 TCR isolated in Example 4 specifically recognize the G12R RAS peptide.

[0194] The nucleic acid sequence encoding the G12R RAS-reactive 4268 TCR of Example 4, comprising a cysteine-substituted, LVL-modified murine constant region, was cloned into a retroviral expression vector. The α chain murine constant region comprises the amino acid sequence of SEQ ID NO: 26, wherein X at position 48 is Cys, X at position 112 is Leu, X at position 114 is Ile, and X at position 115 is Val. The β chain constant region comprises the amino acid sequence of SEQ ID NO: 27, wherein X at position 57 is Cys. A linker comprising the amino acid sequence of RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 38) is positioned between the α chain constant region and the β chain variable region. Allogeneic PBMCs were transduced using the retroviral expression vector.

[0195] The transduced cells were treated with the WT RAS peptide MTEYKLVVVGA G GVGKSALTIQLI (SEQ ID NO: 40) or G12R RAS peptide MTEYKLVVVGA R Autologous DCs pulsed with serial dilutions of GVGKSALTIQLI (SEQ ID NO: 39) were co-cultured overnight. IFNγ secretion was assessed by ELIspot. The results are shown in Figure 3 middle.

[0196] like Figure 3 As shown in , cells transduced with the 4268 TCR specifically recognized the G12R RAS peptide.

[0197] Example 6

[0198] This example demonstrates that cells transduced with the 4268 TCR isolated in Example 4 recognize HLA-DQA1 05:05:HLA-DQB1 03:01G12R RAS peptide in the context of heterodimers.

[0199] Allogeneic PBMCs were virally transduced using the retroviral expression vector of Example 5. Figure 4 Target COS7 cells were transfected with one of the HLA molecules in the G12R RAS peptide MTEYKLVVVGA. R COS7 cells were pulsed with GVGKSALTIQLI (SEQ ID NO: 39). 4268 TCR-transduced cells were co-cultured with pulsed, transfected COS7 cells overnight. IFNγ secretion was assessed by ELISA. The results are shown in Figure 4 middle.

[0200] like Figure 4 As shown in the Figure 4, cells transduced with the 4268 TCR isolated in Example 4 specifically recognized HLA-DQA1 05:05:HLA-DQB1 03:01G12R RAS peptide in the context of heterodimers.

[0201] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0202] In the context of describing the present invention (especially in the context of the following claims), the use of the terms "a" and "an," "the," and "at least one," and similar referents are intended to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The term "at least one" followed by a list of one or more items (e.g., "at least one of A and B") is intended to mean one item (A or B) selected from the listed items or a combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are intended to be interpreted as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise indicated herein. Recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated into the specification as if it were individually recited herein. Unless otherwise indicated herein or otherwise clearly contradicted by the context, all methods described herein can be implemented in any suitable order. Unless otherwise claimed, the use of any and all examples or exemplary language (such as (for example)) provided herein is intended only to better illustrate the invention and does not impose a limitation on the scope of the invention. No language in this specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0203] The preferred embodiments of the present invention are described herein, including the best mode known to the inventor for implementing the present invention. Those skilled in the art will understand variations of those preferred embodiments after reading the foregoing description. The inventors expect that those skilled in the art will appropriately adopt such variations, and the inventors expect to practice the present invention in other ways than those specifically described herein. Therefore, the present invention includes all modifications and equivalents of the objects listed in the accompanying claims as allowed by applicable law. In addition, unless otherwise indicated herein or otherwise clearly contradicted by the context, the present invention encompasses any combination of the above elements in all possible variations thereof.

Claims

1. Use of one or more host cells in the preparation of a medicament for detecting, treating or preventing cancer in a mammal, wherein the one or more host cells are allogeneic or autologous to the mammal and contain a recombinant expression vector containing a nucleic acid comprising a nucleotide sequence encoding a T cell receptor (TCR), wherein the TCR has antigenic specificity for the mutant human RAS amino acid sequence shown in SEQ ID NO: 39, and wherein the TCR comprises: (a) the α chain CDR1 with an amino acid sequence as set forth in SEQ ID NO: 1, the α chain CDR2 with an amino acid sequence as set forth in SEQ ID NO: 2, the α chain CDR3 with an amino acid sequence as set forth in SEQ ID NO: 3, the β chain CDR1 with an amino acid sequence as set forth in SEQ ID NO: 4, the β chain CDR2 with an amino acid sequence as set forth in SEQ ID NO: 5, and the β chain CDR3 with an amino acid sequence as set forth in SEQ ID NO: 6; or (b) the α chain CDR1 with the amino acid sequence set forth in SEQ ID NO: 7, the α chain CDR2 with the amino acid sequence set forth in SEQ ID NO: 8, the α chain CDR3 with the amino acid sequence set forth in SEQ ID NO: 9, the β chain CDR1 with the amino acid sequence set forth in SEQ ID NO: 10, the β chain CDR2 with the amino acid sequence set forth in SEQ ID NO: 11, and the β chain CDR3 with the amino acid sequence set forth in SEQ ID NO:

12.

2. The method of claim 1, wherein the TCR has no antigenic specificity for the wild-type human RAS amino acid sequence of SEQ ID NO:

40.

3. The use according to claim 1 or 2, wherein the mutant human RAS amino acid sequence is presented by a human leukocyte antigen (HLA) class II molecule.

4. The use according to claim 3, wherein the HLA class II molecule is an HLA-DR heterodimer or an HLA-DQ heterodimer.

5. The use according to claim 3, wherein the HLA class II molecule is an HLA-DRB5:HLA-DRA heterodimer or an HLA-DQA1:HLA-DQB1 heterodimer.

6. The method of claim 3, wherein the HLA class II molecule is HLA-DRB5 01: HLA-DRA 01:01 Heterodimer or HLA-DQA1 05:05:HLA-DQB1 03:01Heterodimer.

7. The method of claim 1 or 2, wherein the TCR comprises: (i) both the amino acid sequence of SEQ ID NO: 13 and the amino acid sequence of SEQ ID NO: 14, (ii) both the amino acid sequence of SEQ ID NO: 15 and the amino acid sequence of SEQ ID NO: 16, (iii) both the amino acid sequence of SEQ ID NO: 41 and the amino acid sequence of SEQ ID NO: 42, or (iv) both the amino acid sequence of SEQ ID NO: 43 and the amino acid sequence of SEQ ID NO:

44.

8. The use according to claim 1 or 2, wherein the TCR further comprises: (a) an α chain constant region comprising the amino acid sequence of SEQ ID NO: 26, wherein: (i) X at position 48 of SEQ ID NO: 26 is Cys; (ii) X at position 112 of SEQ ID NO: 26 is Leu; (iii) X at position 114 of SEQ ID NO: 26 is He; and (iv) X at position 115 of SEQ ID NO: 26 is Val; (b) a beta chain constant region comprising the amino acid sequence of SEQ ID NO: 27, wherein X at position 57 of SEQ ID NO: 27 is Cys; or (c) Both (a) and (b).

9. The method of claim 1 or 2, wherein the TCR comprises: (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 Cys; (ii) X at position 244 of SEQ ID NO: 30 is Leu; (iii) X at position 246 of SEQ ID NO: 30 is He; and (iv) X at position 247 of SEQ ID NO: 30 is Val; (b) a beta chain comprising the amino acid sequence of SEQ ID NO: 31, wherein X at position 198 of SEQ ID NO: 31 is Cys; (c) an α chain comprising the amino acid sequence of SEQ ID NO: 32, wherein: (i) X at position 188 of SEQ ID NO: 32 is Cys; (ii) X at position 252 of SEQ ID NO: 32 is Leu; (iii) X at position 254 of SEQ ID NO: 32 is Ile; and (iv) X at position 255 of SEQ ID NO: 32 is Val; (d) a beta chain comprising the amino acid sequence of SEQ ID NO: 33, wherein X at position 191 of SEQ ID NO: 33 is Cys; (e) both (a) and (b); (f) both (c) and (d); (g) an α chain comprising the amino acid sequence of SEQ ID NO: 45, wherein: (i) X at position 161 of SEQ ID NO: 45 is Cys; (ii) X at position 225 of SEQ ID NO: 45 is Leu; (iii) X at position 227 of SEQ ID NO: 45 is Ile; and (iv) X at position 228 of SEQ ID NO: 45 is Val; (h) a beta chain comprising the amino acid sequence of SEQ ID NO: 46, wherein X at position 178 of SEQ ID NO: 46 is Cys; (i) comprising an α chain with the amino acid sequence of SEQ ID NO: 47, wherein: (i) X at position 168 of SEQ ID NO: 47 is Cys; (ii) X at position 232 of SEQ ID NO: 47 is Leu; (iii) X at position 234 of SEQ ID NO: 47 is He; and (iv) X at position 235 of SEQ ID NO: 47 is Val; (j) a β chain comprising the amino acid sequence of SEQ ID NO: 48, wherein X at position 171 of SEQ ID NO: 48 is Cys; (k) both (g) and (h); or (l) Both (i) and (j).

10. Use of one or more host cells in the preparation of a medicament for detecting, treating or preventing cancer in a mammal, wherein the one or more host cells are allogeneic or autologous to the mammal and comprise a recombinant expression vector comprising a nucleic acid comprising a nucleotide sequence encoding a functional portion of a T cell receptor (TCR), The functional portion has antigenic specificity for the mutant human RAS amino acid sequence shown in SEQ ID NO: 39 and comprises the following amino acid sequence: (a) the α chain CDR1 with an amino acid sequence as set forth in SEQ ID NO: 1, the α chain CDR2 with an amino acid sequence as set forth in SEQ ID NO: 2, the α chain CDR3 with an amino acid sequence as set forth in SEQ ID NO: 3, the β chain CDR1 with an amino acid sequence as set forth in SEQ ID NO: 4, the β chain CDR2 with an amino acid sequence as set forth in SEQ ID NO: 5, and the β chain CDR3 with an amino acid sequence as set forth in SEQ ID NO: 6; or (b) the α chain CDR1 with the amino acid sequence set forth in SEQ ID NO: 7, the α chain CDR2 with the amino acid sequence set forth in SEQ ID NO: 8, the α chain CDR3 with the amino acid sequence set forth in SEQ ID NO: 9, the β chain CDR1 with the amino acid sequence set forth in SEQ ID NO: 10, the β chain CDR2 with the amino acid sequence set forth in SEQ ID NO: 11, and the β chain CDR3 with the amino acid sequence set forth in SEQ ID NO:

12.

11. The use according to claim 10, wherein the functional part comprises: (i) the amino acid sequence of SEQ ID NO: 13, (ii) the amino acid sequence of SEQ ID NO: 14, (iii) the amino acid sequence of SEQ ID NO: 15, (iv) the amino acid sequence of SEQ ID NO: 16, (v) both the amino acid sequence of SEQ ID NO: 13 and the amino acid sequence of SEQ ID NO: 14, (vi) both the amino acid sequence of SEQ ID NO: 15 and the amino acid sequence of SEQ ID NO: 16, (vii) the amino acid sequence of SEQ ID NO: 41, (viii) the amino acid sequence of SEQ ID NO: 42, (ix) the amino acid sequence of SEQ ID NO: 43, (x) the amino acid sequence of SEQ ID NO: 44, (xi) both the amino acid sequence of SEQ ID NO: 41 and the amino acid sequence of SEQ ID NO: 42, or (xii) both the amino acid sequence of SEQ ID NO: 43 and the amino acid sequence of SEQ ID NO:

44.

12. The method according to claim 10 or 11, wherein the functional part further comprises: (a) the amino acid sequence of SEQ ID NO: 26, wherein: (i) X at position 48 of SEQ ID NO: 26 is Cys; (ii) X at position 112 of SEQ ID NO: 26 is Leu; (iii) X at position 114 of SEQ ID NO: 26 is He; and (iv) X at position 115 of SEQ ID NO: 26 is Val; (b) the amino acid sequence of SEQ ID NO: 27, wherein X at position 57 of SEQ ID NO: 27 is Cys; or (c) Both (a) and (b).

13. The method of claim 10 or 11, wherein the polypeptide comprises: (a) the amino acid sequence of SEQ ID NO: 30, wherein: (i) X at position 180 of SEQ ID NO: 30 is Cys; (ii) X at position 244 of SEQ ID NO: 30 is Leu; (iii) X at position 246 of SEQ ID NO: 30 is He; and (iv) X at position 247 of SEQ ID NO: 30 is Val; (b) the amino acid sequence of SEQ ID NO: 31, wherein X at position 198 of SEQ ID NO: 31 is Cys; (c) the amino acid sequence of SEQ ID NO: 32, wherein: (i) X at position 188 of SEQ ID NO: 32 is Cys; (ii) X at position 252 of SEQ ID NO: 32 is Leu; (iii) X at position 254 of SEQ ID NO: 32 is Ile; and (iv) X at position 255 of SEQ ID NO: 32 is Val; (d) the amino acid sequence of SEQ ID NO: 33, wherein X at position 191 of SEQ ID NO: 33 is Cys; (e) both (a) and (b); (f) both (c) and (d); (g) the amino acid sequence of SEQ ID NO: 45, wherein: (i) X at position 161 of SEQ ID NO: 45 is Cys; (ii) X at position 225 of SEQ ID NO: 45 is Leu; (iii) X at position 227 of SEQ ID NO: 45 is Ile; and (iv) X at position 228 of SEQ ID NO: 45 is Val; (h) the amino acid sequence of SEQ ID NO: 46, wherein X at position 178 of SEQ ID NO: 46 is Cys; (i) the amino acid sequence of SEQ ID NO: 47, wherein: (i) X at position 168 of SEQ ID NO: 47 is Cys; (ii) X at position 232 of SEQ ID NO: 47 is Leu; (iii) X at position 234 of SEQ ID NO: 47 is He; and (iv) X at position 235 of SEQ ID NO: 47 is Val; (j) the amino acid sequence of SEQ ID NO: 48, wherein X at position 171 of SEQ ID NO: 48 is Cys; (k) both (g) and (h); or (l) Both (i) and (j).

14. Use of one or more host cells in the preparation of a medicament for detecting, treating or preventing cancer in a mammal, wherein the one or more host cells are allogeneic or autologous to the mammal and contain a recombinant expression vector comprising a nucleic acid comprising a nucleotide sequence encoding a protein having antigenic specificity for the mutant human RAS amino acid sequence of SEQ ID NO:39 and comprising: (a) a first polypeptide chain comprising the amino acid sequence of SEQ ID NOs: 1 to 3 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NOs: 4 to 6; or (b) a first polypeptide chain comprising the amino acid sequence of SEQ ID NOs: 7 to 9 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NOs: 10 to 12.

15. The use according to claim 14, wherein: (i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 13 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 14; (ii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 15 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 16; (iii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 41 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 42; or (iv) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 43 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:

44.

16. The use according to claim 14 or 15, wherein: (a) The first polypeptide chain further comprises the amino acid sequence of SEQ ID NO: 26, wherein: (i) X at position 48 of SEQ ID NO: 26 is Cys; (ii) X at position 112 of SEQ ID NO: 26 is Leu; (iii) X at position 114 of SEQ ID NO: 26 is He; and (iv) X at position 115 of SEQ ID NO: 26 is Val; (b) the second polypeptide chain further comprises the amino acid sequence of SEQ ID NO: 27, wherein X at position 57 of SEQ ID NO: 27 is Cys; or (c) Both (a) and (b).

17. The use according to claim 14 or 15, wherein: (a) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 30, wherein: (i) X at position 180 of SEQ ID NO: 30 is Cys; (ii) X at position 244 of SEQ ID NO: 30 is Leu; (iii) X at position 246 of SEQ ID NO: 30 is He; and (iv) X at position 247 of SEQ ID NO: 30 is Val; (b) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 31, wherein X at position 198 of SEQ ID NO: 31 is Cys; (c) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 32, wherein: (i) X at position 188 of SEQ ID NO: 32 is Cys; (ii) X at position 252 of SEQ ID NO: 32 is Leu; (iii) X at position 254 of SEQ ID NO: 32 is Ile; and (iv) X at position 255 of SEQ ID NO: 32 is Val; (d) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 33, wherein X at position 191 of SEQ ID NO: 33 is Cys; (e) both (a) and (b); (f) both (c) and (d); (g) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 45, wherein: (i) X at position 161 of SEQ ID NO: 45 is Cys; (ii) X at position 225 of SEQ ID NO: 45 is Leu; (iii) X at position 227 of SEQ ID NO: 45 is Ile; and (iv) X at position 228 of SEQ ID NO: 45 is Val; (h) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 46, wherein X at position 178 of SEQ ID NO: 46 is Cys; (i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 47, wherein: (i) X at position 168 of SEQ ID NO: 47 is Cys; (ii) X at position 232 of SEQ ID NO: 47 is Leu; (iii) X at position 234 of SEQ ID NO: 47 is He; and (iv) X at position 235 of SEQ ID NO: 47 is Val; (j) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 48, wherein X at position 171 of SEQ ID NO: 48 is Cys; (k) both (g) and (h); or (l) Both (i) and (j).

18. Use of one or more host cells in the preparation of a medicament for detecting, treating or preventing cancer in a mammal, wherein the one or more host cells are allogeneic or autologous to the mammal and comprise a recombinant expression vector comprising a nucleic acid comprising, from 5' to 3', a first nucleotide sequence and a second nucleotide sequence, wherein the first nucleotide sequence and the second nucleotide sequence encode the following amino acid sequence, respectively: SEQ ID NO: 13 and 14; 14 and 13; 15 and 16; 16 and 15; 30 and 31; 31 and 30; 32 and 33; 33 and 32; 41 and 42; 42 and 41; 43 and 44; 44 and 43; 45 and 46; 46 and 45; 47 and 48; or 48 and 47, wherein: (i) X at position 180 of SEQ ID NO: 30 is Cys, X at position 244 of SEQ ID NO: 30 is Leu, X at position 246 of SEQ ID NO: 30 is Ile, and X at position 247 of SEQ ID NO: 30 is Val; (ii) X at position 198 of SEQ ID NO: 31 is Cys; (iii) X at position 188 of SEQ ID NO: 32 is Cys, X at position 252 of SEQ ID NO: 32 is Leu, X at position 254 of SEQ ID NO: 32 is Ile, and X at position 255 of SEQ ID NO: 32 is Val; (iv) X at position 191 of SEQ ID NO: 33 is Cys; (v) X at position 161 of SEQ ID NO: 45 is Cys, X at position 225 of SEQ ID NO: 45 is Leu, X at position 227 of SEQ ID NO: 45 is Ile, and X at position 228 of SEQ ID NO: 45 is Val; (vi) X at position 178 of SEQ ID NO: 46 is Cys; (vii) X at position 168 of SEQ ID NO: 47 is Cys, X at position 232 of SEQ ID NO: 47 is Leu, X at position 234 of SEQ ID NO: 47 is Ile, and X at position 235 of SEQ ID NO: 47 is Val; and (viii) X at position 171 of SEQ ID NO: 48 is Cys.

19. The use according to claim 18, wherein the nucleic acid further comprises a third nucleotide sequence inserted between the first nucleotide sequence and the second nucleotide sequence, wherein the third nucleotide sequence encodes a cleavable linker peptide.

20. The use of claim 19, wherein the cleavable linker peptide comprises the amino acid sequence of SEQ ID NO:

38.

21. The use according to any one of claims 1-2, 10-11, 14-15 and 19-21, wherein the recombinant expression vector is a transposon or a lentiviral vector.

22. The use of any one of claims 1-2, 10-11, 14-15, and 19-21, wherein the one or more host cells are allogeneic to the mammal.

23. The use of any one of claims 1-2, 10-11, 14-15, and 19-21, wherein the one or more host cells are autologous to the mammal.

24. The use of any one of claims 1-2, 10-11, 14-15, and 19-21, wherein the one or more host cells are human lymphocytes.

25. The use of any one of claims 1-2, 10-11, 14-15, and 19-21, wherein the one or more host cells are selected from T cells, natural killer T (NKT) cells, invariant natural killer T (iNKT) cells, and natural killer (NK) cells.

26. The use of any one of claims 1-2, 10-11, 14-15, and 19-21, wherein the cancer expresses a mutant human RAS amino acid sequence in which the glycine at position 12 is substituted with arginine, wherein the mutant human RAS amino acid sequence is a mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS), a mutant human Harvey rat sarcoma viral oncogene homolog (HRAS), or a mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence, and Position 12 is defined with reference to wild-type human KRAS protein, wild-type human HRAS protein or wild-type human NRAS protein, respectively.

27. The use according to claim 26, wherein the mutant human RAS amino acid sequence is a mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS) amino acid sequence.

28. The use of claim 26, wherein the mutant human RAS amino acid sequence is a mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence.

29. The use according to claim 26, wherein the mutant human RAS amino acid sequence is a mutant human Harvey rat sarcoma viral oncogene homolog (HRAS) amino acid sequence.

30. The use of any one of claims 1-2, 10-11, 14-15, and 19-21, wherein the cancer is pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer, or prostate cancer.

31. An isolated or purified T cell receptor (TCR), wherein the TCR has antigenic specificity for a mutated human RAS amino acid sequence as shown in SEQ ID NO: 21, 22, 23 or 24.

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