HLA class II restrictive T cell receptors for mutated RAS

Abstract

An isolated or purified T cell receptor (TCR) is disclosed, wherein the TCR has antigen specificity for a mutated Kirsteen rat sarcoma virus oncogene homolog (KRAS) presented by a human leukocyte antigen (HLA) class II molecule. Related polypeptides and proteins, as well as related nucleic acids, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions are also provided. Methods of detecting the presence of cancer in a mammal and methods of treating or preventing cancer in a mammal are also disclosed.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201880060535.4, the filing date of Chinese Patent Application No. 201880060535.4 is September 19, 2018, and the entire content thereof is incorporated herein by reference.

[0002] Cross-reference to related applications

[0003] This patent application claims the benefit of U.S. Provisional Patent Application No. 62 / 560,930, filed on September 20, 2017, which is incorporated herein by reference in its entirety.

[0004] Statement regarding federally sponsored research or development

[0005] This invention was made with government support under Grant No. BC010984 from the National Cancer Institute of the National Institutes of Health. The government has certain rights in this invention.

[0006] Materials submitted electronically are incorporated by reference

[0007] Incorporated herein by reference in its entirety is the computer-readable nucleotide / amino acid sequence listing that is concurrently filed herewith and identified as follows: a 59,753-byte ASCII (text) file named "739664_ST25.txt" and dated September 5, 2018. Background of the invention

[0009] Some cancers can have extremely limited treatment options, especially when the cancer becomes metastatic and unresectable. Despite the progress of treatments such as surgery, chemotherapy, and radiation therapy, the prognosis of many cancers (such as pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer, and prostate cancer) may be poor. Therefore, there is an unmet need for other treatments for cancer.

[0010] Summary of the invention

[0011] Embodiments of the present invention provide an isolated or purified T cell receptor (TCR) that is antigen-specific for a mutant human Ras amino acid sequence presented by a human leukocyte antigen (HLA) class II molecule, wherein the mutant human RAS amino acid sequence is a mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS) amino acid sequence, a mutant human Harvey rat sarcoma viral oncogene homolog (HRAS) amino acid sequence, or a mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence.

[0012] Another embodiment of the present invention provides an isolated or purified polypeptide comprising the functional portion of the TCR of the present invention, wherein the functional portion comprises the following amino acid sequences: (a) all of SEQ ID NO: 1-3, (b) all of SEQ ID NO: 4-6, (c) all of SEQ ID NO: 7-9, (d) all of SEQ ID NO: 10-12, (e) all of SEQ ID NO: 1-6, or (f) all of SEQ ID NO: 7-12.

[0013] Yet another embodiment of the present invention provides an isolated or purified protein comprising at least one polypeptide of the present invention.

[0014] Embodiments of the present invention also provide nucleic acids, recombinant expression vectors, host cells, cell populations, and pharmaceutical compositions related to the TCR, polypeptides, and proteins of the present invention.

[0015] Embodiments of the present invention also provide methods for detecting the presence of cancer in a mammal and methods for treating or preventing cancer in a mammal.

[0016] Brief description of several views in the drawings

[0017] Figure 1 Depicts experimental data (dot plots) showing the detection by flow cytometry of cells stained for isotype (control) or for PD-1 and / or OX40 expression. The values in the histograms represent the percentage of cells expressing PD-1.

[0018] Figure 2 Is a graph showing the number of IFNγ-positive spots per well, which was detected when a pooled culture of effector autologous T cells (culture numbers W1-W16) was co-cultured with target DC pulsed with a pool of indicated 25-mer peptides (PP) or a pool of peptides encoded by a 25-mer tandem minigene (TMG) covering various tumor-specific mutations. Autologous T cells cultured alone or in the presence of dimethyl sulfoxide (DMSO) or OKT3 antibody were used as controls. The boxed symbols (▼) indicate the pooled cultures (7 and 8) of isolated TCR.

[0019] Figure 3 Is a graph showing the number of IFNγ-positive spots / 2×10 4 (2E4) cells, which was detected when autologous T cells of culture number 7 (W7) were co-cultured with autologous DC pulsed with each of the peptides 1-17 (P1-P17) from peptide pool 1 (PP1). Autologous T cells cultured with dimethyl sulfoxide (DMSO) or OKT3 antibody were used as controls.

[0020] Figure 4 It is a graph showing the percentage of effector T cells transduced with the TCR expressing 4-1BB after co-culture with target autologous APC pulsed with KRAS G12V peptide (1 ng / mL) in the presence of HLA-blocking antibodies W6 / 32 (anti-HLA-A, -B, -C), IVA12 (pan-specific, anti-HLA class II), B7 / 21 (anti-HLA-DP), HB55 (anti-HLA-DR), or SPV-L3 (HLA-DQ) (target cells). Effector transduced cells cultured alone, or cultured with DMSO or phorbol myristate acetate (PMA) were used as controls. Effector cells transduced with empty vector (mock) and co-cultured with target autologous APC (pulsed with 1 ng / mL KRAS G12V peptide) were used as another control.

[0021] Figure 5 It is a graph showing the following results: (i) the number of IFN-γ / 2×10 4 cells measured by ELISPOT; and (ii) the percentage of mTCRβ+CD8+4-1BB+ cells measured by flow cytometry, both of which were measured when the T cells transduced with the TCR of Example 2 were co-cultured with autologous APC (4148MB) or APC from donors with DRB101:01 or DRB107:01 haplotypes and pulsed with KRAS G12V peptide or WT KRAS peptide. Effector cells were co-cultured with APC from HLA-DRB1 positive donors (“DRB mismatch”) as a control. Effector cells cultured alone, or cultured with DMSO or phorbol myristate acetate-ionomycin (PMA:Iono) were used as other controls.

[0022] Figure 6 It is a graph showing the following results: (i) the number of IFN-γ / 2×10 4 cells (hatched bars) measured by ELISPOT; and (ii) the percentage of cells expressing 4-1BB and / or OX40 (black bars) measured by flow cytometry, both of which were measured when the T cells transduced with the TCR of Example 2 were co-cultured with autologous DC pulsed with the cell lysates of tumor cell lines expressing one of the following KRAS G12 mutations: G12R, G12C, G12D, or G12V. Transduced cells co-cultured with autologous DC pulsed with the cell lysates of tumor cell lines expressing WT KRAS were used as controls. Transduced cells cultured alone or cultured with PMA or DMSO were used as other controls.

[0023] Figure 7is a graph showing the percentage of mTCRβ+CD8+4-1BB+ cells measured by flow cytometry, which was measured when TCR-transduced T cells of Example 2 were co-cultured overnight with autologous DCs pulsed with the indicated concentrations of KRAS G12V peptide (triangle) or WT KRAS peptide (square).

[0024] Figure 8 is a graph showing the number of IFN-γ / 2×10 4 cells measured by ELISPOT, which was measured when TCR-transduced T cells of Example 2 were co-cultured with autologous DCs pulsed with the peptides in Table 9 at the indicated concentrations.

[0025] Figure 9 Depicts experimental data (dot plot) showing the percentage of cells expressing murine TCRβ chain and 4-1BB, which was determined when cells transduced with MSGV-1-retrovirus encoding KRAS G12C TCR were co-cultured with DMSO (control) or DCs loaded with the indicated WT KRAS or KRAS G12C peptide at the indicated concentrations. The dot plot indicates the percentage of the following cells: mTCRβ+ / 4-1BB- (upper left quadrant (Q1)); mTCRβ+ / 4-1BB+ (upper right quadrant (Q2)); mTCRβ- / 4-1BB+ (lower right quadrant (Q3)); mTCRβ- / 4-1BB- (lower left quadrant (Q4)), and shows as follows (percentages in parentheses): DMSO: Q1 (71.0), Q2 (0.96), Q3 (0.20), Q4 (27.9). WT 10 μg / ml: Q1 (64.5), Q2 (4.27), Q3 (0.43), Q4 (30.8). WT 1 μg / ml: Q1 (70.6), Q2 (1.13), Q3 (0.20), Q4 (28.1). G12C 10 μg / ml: Q1 (13.6), Q2 (51.7), Q3 (1.61), Q4 (33.0). G12C 1 μg / ml: Q1 (19.7), Q2 (46.9), Q3 (1.67), Q4 (31.7).

[0026] Figure 10 is a graph showing the percentage of cells expressing CD3 and 4-1BB, which was measured after blocking membrane MHC-II molecules using antibodies against HLA-DQ, DR, DP or antibodies against all HLA-DQ, DR and DP, and then co-culturing TCR-transduced T cells against the KRAS G12C with autologous DCs matched to a single HLA-DRB15:01 or HLA-DRB11:01 allele and pulsed with KRAS G12CDetermination was performed after co - culture of autologous DCs or allogeneic DCs pulsed with 24 - mer peptides. Transduced cells co - cultured with DCs pulsed with WT KRAS peptides were used as a control. Transduced cells co - cultured with PMA / ion were used as another control. DETAILED DESCRIPTION OF THE INVENTION

[0028] RAS family proteins belong to the large family of small GTPases. Without being limited to a particular theory or mechanism, it is believed that upon mutation, RAS proteins can be involved in signal transduction in the early stages of tumor formation in many human cancers. A single amino acid substitution can activate the protein. The mutant RAS protein product can be constitutively activated. Mutant RAS proteins can be expressed in any of a variety of human cancers (such as pancreatic cancer (e.g., pancreatic carcinoma), colorectal cancer, lung cancer (e.g., lung adenocarcinoma), endometrial cancer, ovarian cancer (e.g., epithelial ovarian cancer), and prostate cancer). Human RAS family proteins include Kirsten rat sarcoma viral oncogene homolog (KRAS), Harvey rat sarcoma viral oncogene homolog (HRAS), and neuroblastoma rat sarcoma viral oncogene homolog (NRAS).

[0029] KRAS is also known as GTPase KRas, V - Ki - Ras2 Kirsten rat sarcoma viral oncogene, or KRAS2. There are two transcript variants of KRAS: KRAS variant A and KRAS variant B. Wild - type (WT) KRAS variant A has the amino acid sequence of SEQ ID NO:17. Wild - type (WT) KRAS variant B has the amino acid sequence of SEQ ID NO:18. Hereinafter, unless otherwise specified, reference to "KRAS" (mutated or unmutated (WT)) refers to variant A and variant B. Upon activation, mutant KRAS binds guanosine - 5'-triphosphate (GTP) and converts GTP to guanosine - 5′-diphosphate (GDP).

[0030] HRAS is another member of the RAS protein family. HRAS is also known as Harvey rat sarcoma 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.

[0031] NRAS is yet another member of the RAS protein family. NRAS is also known as GTPase NRas, V - Ras neuroblastoma RAS viral oncogene homolog, or NRAS1. WT NRAS has the amino acid sequence of SEQ ID NO:20.

[0032] Embodiments of the present invention provide an isolated or purified TCR that is antigen - specific for a mutant human RAS amino acid sequence (hereinafter "mutant RAS") presented by a human leukocyte antigen (HLA) class II molecule, wherein the mutant human RAS amino acid sequence is a mutant human KRAS, mutant human HRAS, or mutant human NRAS amino acid sequence. Hereinafter, unless otherwise specified, the "TCR" mentioned also refers to the functional part and functional variants of the TCR.

[0033] The TCRs of the present invention can be antigen - specific for any mutant human RAS protein, polypeptide, or peptide amino acid sequence. In embodiments of the present invention, the mutant human RAS amino acid sequence is 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 - 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 that of WT human KRAS protein. The 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 that of WT human KRAS protein. The amino acid residues 1 - 94 of WT human HRAS protein and WT human KRAS protein are 100% identical. Hereinafter, unless otherwise specified, the "RAS" (mutant or unmutated (WT)) mentioned collectively refers to KRAS, HRAS, and NRAS.

[0034] In embodiments of the present invention, the mutant human RAS amino acid sequence includes a WT RAS amino acid sequence in which glycine at position 12 is substituted, wherein position 12 is defined by reference to the WT RAS protein respectively. The WT RAS protein can be any one of WT KRAS protein (SEQ ID NO:17 or 18), WT HRAS protein (SEQ ID NO:19), or WT NRAS protein (SEQ ID NO:20), because as explained above, the amino acid residues 1 - 86 of WT human NRAS protein and WT human KRAS protein are 100% identical, and the amino acid residues 1 - 94 of WT human HRAS protein and WT human KRAS protein are 100% identical. Thus, the amino acid residue at position 12 in each of WT KRAS, WT HRAS, and WT NRAS proteins is the same, that is, glycine.

[0035] The glycine at position 12 of the WT RAS amino acid sequence can be replaced with any amino acid residue other than glycine. In an embodiment of the present invention, the replacement is replacement of the glycine at position 12 of the WT RAS amino acid sequence with valine or cysteine. In this regard, embodiments of the present invention provide TCRs that are antigen - specific for any WT RAS protein, polypeptide, or peptide amino acid sequence having a G12V mutation or a G12C mutation.

[0036] In the present context, mutations and substitutions of RAS are defined by reference to the amino acid sequence of the WT RAS protein. Thus, in the present context, mutations and substitutions of RAS are described by reference to the amino acid residue present at a particular position in the WT RAS protein, followed by the position number, followed by the amino acid residue that replaces that residue for the particular mutation or substitution being discussed. A RAS amino acid sequence (such as a RAS peptide) can include amino acid residues that are less than all of the amino acid residues of the full - length WT RAS protein. Thus, in the present context, position 12 is defined by reference to the full - length WT 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 particular instance of the RAS amino acid sequence can vary. When the position is as defined by any one of SEQ ID NOs: 17 - 20, the term "G12" refers to the glycine that is normally present at position 12 of any one of SEQ ID NOs: 17 - 20, and "G12V" indicates that the glycine that is normally present at position 12 of any one of SEQ ID NOs: 17 - 20 is replaced by valine. For example, when a particular instance of the RAS amino acid sequence is (e.g.) TEYKLVVVGA G GVGKSALTIQLI (SEQ ID NO: 29) (an exemplary WT KRAS peptide corresponding to consecutive amino acid residues 2 to 24 of SEQ ID NO: 17), "G12V" refers to the underlined glycine in SEQ ID NO: 29 being replaced by valine, even though the actual position of the underlined glycine in SEQ ID NO: 29 is 11.

[0037] Examples of full - length RAS proteins having G12V or G12C mutations are shown in Table 1 below.

[0038] Table 1

[0039] Mutated full-length RAS protein SEQ ID NO: G12V KRAS variant A 21 G12V KRAS variant B 22 G12V HRAS 23 G12V NRAS 24 G12C KRAS variant A 25 G12C KRAS variant B 26 G12C HRAS 27 G12C NRAS 28

[0040] In embodiments of the present invention, the TCR has antigen specificity for RAS peptides having the above-described G12V or G12C mutation, wherein the mutated RAS peptide can have any length. In embodiments of the present invention, the mutated RAS peptide has any length suitable for binding to any HLA class II molecule described herein. For example, the TCR can have antigen specificity for an RAS peptide having a G12V or G12C mutation, the RAS peptide having a length of from about 11 to about 30 amino acid residues, from about 12 to about 24 amino acid residues, or from about 18 to about 20 amino acid residues. The mutated RAS peptide can comprise any contiguous amino acid residues of the mutated RAS protein that contain the G12V or G12C mutation. In embodiments of the present invention, the TCR can have antigen specificity for an RAS peptide having a G12V or G12C mutation, the mutated RAS peptide having the following lengths: 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 the range of any two of the foregoing values. Examples of specific peptides each having a G12V mutation that can be recognized by the G12V TCR of the present invention are shown in Table 9.

[0041] In embodiments of the present invention, the TCRs of the present invention are capable of recognizing mutated RAS presented by HLA class II molecules. In this regard, the TCR can initiate an immune response upon binding to the mutated RAS within the context of the HLA class II molecule. The TCRs of the present invention are capable of recognizing mutated RAS presented by HLA class II molecules and can also bind to the HLA class II molecule in addition to the mutated RAS.

[0042] In an embodiment of the present invention, the HLA class II molecule is an HLA-DR molecule. The HLA-DR molecule is a heterodimer of an α chain and a β chain. The HLA-DRα chain can be encoded by the HLA-DRA gene. The HLA-DRβ chain can be encoded by the HLA-DRB1 gene, the HLA-DRB3 gene, the HLA-DRB4 gene, or the HLA-DRB5 gene. The HLA-DR molecule can be any HLA-DR molecule. Examples of the HLA-DR molecule can 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, and HLA-DR16. Preferably, the HLA-DR molecule is HLA-DR7 or HLA-DR11.

[0043] In an embodiment of the present invention, the HLA class II molecule is an HLA-DRB1 molecule. The HLA-DRB1 molecule can be any HLA-DRB1 molecule. Examples of the HLA-DRB1 molecule can include but are not limited to: HLA-DRB1*01:01, HLA-DRB1*01:02, HLA-DRB1*01:03, HLA-DRB1*03:01, HLA-DRB1*04:01, HLA-DRB1*04:02, HLA-DRB1*04:03, HLA-DRB1*04:04, HLA-DRB1*04:05, HLA-DRB1*04:07, HLA-DRB1*07:01, HLA-DRB1*08:01, HLA-DRB1*08:03, HLA-DRB1*09:01, HLA-DRB1*10:01, HLA-DRB1*11:01, HLA-DRB1*11:03, HLA-DRB1*11:04, HLA-DRB1*12:01, HLA-DRB1*13:01, HLA-DRB1*13:02, HLA-DRB1*13:03, HLA-DRB1*14:01, HLA-DRB1*15:01, HLA-DRB1*15:02, and HLA-DRB1*16:01. Preferably, the HLA class II molecule is an HLA-DRB1*07:01 molecule or an HLA-DRB1*11:01 molecule.

[0044] The TCRs of the present invention can provide any one or more of a number of advantages, including for adoptive cell transfer when expressed by cells. Mutant 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 target the destruction of cancer cells while minimizing or eliminating the destruction of normal, non-cancerous cells, thereby reducing toxicity (e.g., minimizing or eliminating it). In addition, the TCRs of the present invention can advantageously and successfully treat or prevent mutant RAS-positive cancers that are unresponsive to other types of treatment (e.g., chemotherapy, surgery, or radiation). For example, the KRAS G12V mutation is expressed in approximately 27% and approximately 8% of patients with pancreatic cancer and colorectal cancer, respectively, and the KRAS G12C mutation is expressed in approximately 15% of patients with lung cancer. Additionally, the TCRs of the present invention can provide highly affinity recognition of mutant RAS, which can provide the ability to recognize untreated tumor cells (e.g., tumor cells not treated with interferon (IFN)-γ, not transfected with a vector encoding one or both of mutant RAS and HLA-DRB1*07:01, mutant RAS and HLA-DRB1*11:01, not pulsed with a RAS peptide having a G12V mutation, not pulsed with a RAS peptide having a G12C mutation, or a combination thereof). Furthermore, in the United States, the HLA-DRB1*07:01 and HLA-DRB1*11:01 alleles are expressed in approximately 25% and approximately 10.5% of Caucasian individuals, respectively. Thus, the TCRs of the present invention can increase the number of cancer patients eligible for immunotherapy to include patients expressing one or both of the HLA-DRB1*07:01 and HLA-DRB1*11:01 alleles, who may not be eligible for immunotherapy using TCRs that recognize RAS presented by other MHC molecules.

[0045] As used herein, the phrase "antigen specific" means that the TCR is capable of specifically binding with high affinity and immunologically recognizing mutant RAS. For example, if, when co-cultured with (a) antigen-negative, HLA class II molecule-positive target cells pulsed with a low concentration of mutant RAS peptide (e.g., from 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 that have been introduced with a nucleotide sequence encoding mutant RAS such that the target cells express mutant RAS, about 1×10 4 to about 1×10 5If a TCR-expressing T cell 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 to have “antigen specificity” for the mutant RAS. The cells expressing the TCR of the present invention can also secrete IFN-γ when co-cultured with antigen-negative, HLA class II molecule-positive target cells pulsed with a higher concentration of mutant RAS peptide. The HLA class II molecule can be any HLA class II molecule described herein (e.g., HLA-DRB1*07:01 molecule or HLA-DRB1*11:01 molecule).

[0046] Optionally or alternatively, if, when co-cultured with (a) antigen-negative, HLA class II molecule-positive target cells pulsed with a low concentration of mutant RAS peptide or (b) antigen-negative, HLA class II molecule-positive target cells into which the nucleotide sequence encoding mutant RAS has been introduced such that the target cells express mutant RAS, a TCR-expressing T cell secretes at least twice the amount of IFN-γ expressed by a negative control, the TCR can be considered to have “antigen specificity” for the mutant RAS. The negative control can be, for example, (i) a TCR-expressing T cell co-cultured with: (a) antigen-negative, HLA class II molecule-positive target cells pulsed with the same concentration of an irrelevant peptide (e.g., some other peptide having a different sequence from the mutant RAS peptide), or (b) antigen-negative, HLA class II molecule-positive target cells into which the nucleotide sequence encoding an irrelevant peptide has been introduced such that the target cells express the irrelevant peptide; or (ii) an untransduced T cell (e.g., derived from PBMCs that do not express TCR) co-cultured with: (a) antigen-negative, HLA class II molecule-positive target cells pulsed with the same concentration of mutant RAS peptide, or (b) antigen-negative, HLA class II molecule-positive target cells into which the nucleotide sequence encoding mutant RAS has been introduced such that the target cells express mutant RAS. The HLA class II molecule expressed by the target cells of the negative control is the same as the HLA class II molecule expressed by the target cells co-cultured with the T cells being tested. The HLA class II molecule can be any HLA class II molecule described herein (e.g., HLA-DRB1*07:01 molecule or HLA-DRB1*11:01 molecule). IFN-γ secretion can be measured by methods known in the art (e.g., enzyme-linked immunosorbent assay, ELISA).

[0047] Optionally or alternatively, if, upon co - culture with (a) antigen - negative, HLA class II - positive target cells pulsed with a low concentration of mutant RAS peptide or (b) antigen - negative, HLA class II - positive target cells into which the nucleotide sequence encoding mutant RAS has been introduced such that the target cells express mutant RAS, at least twice the number of TCR - expressing T cells secrete IFN - γ compared to the number of negative control T cells that secrete IFN - γ, the TCR can be considered to have "antigen specificity" for mutant RAS. The HLA class II molecule, peptide concentration, and negative control 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).

[0048] Optionally or alternatively, if TCR - expressing T cells up - regulate the expression of one or more T - cell activation markers, as measured (e.g., by flow cytometry) after stimulation with target cells expressing mutant RAS, the TCR can be considered to have "antigen specificity" for mutant RAS. Examples of T - cell activation markers include 4 - 1BB, OX40, CD107a, CD69, and cytokines (e.g., tumor necrosis factor (TNF), interleukin (IL) - 2, etc.) that are up - regulated upon antigen stimulation.

[0049] Embodiments of the invention provide TCRs that comprise two polypeptides (i.e., polypeptide chains, such as the α - chain of the TCR, the β - chain of the TCR, the γ - chain of the TCR, the δ - chain of the TCR, or combinations thereof). The polypeptides of the TCRs of the invention can comprise any amino acid sequence, provided that the TCR has antigen specificity for mutant RAS.

[0050] In embodiments of the invention, the TCR comprises two polypeptide chains, each of which comprises a variable region containing the complementarity - determining regions (CDRs) 1, 2, and 3 of the TCR. In embodiments of the invention, the TCR comprises a first polypeptide chain and a second polypeptide chain. The first polypeptide chain comprises a CDR1 (CDR1 of the α - chain) having the amino acid sequence of SEQ ID NO:1, a CDR2 (CDR2 of the α - chain) having the amino acid sequence of SEQ ID NO:2, and a CDR3 (CDR3 of the α - chain) having the amino acid sequence of SEQ ID NO:3. The second polypeptide chain comprises a CDR1 (CDR1 of the β - chain) having the amino acid sequence of SEQ ID NO:4, a CDR2 (CDR2 of the β - chain) having the amino acid sequence of SEQ ID NO:5, and a CDR3 (CDR3 of the β - chain) having the amino acid sequence of SEQ ID NO:6.

[0051] In another embodiment of the present invention, the TCR comprises a first polypeptide chain and a second polypeptide chain. The first polypeptide chain comprises a CDR1 (CDR1 of the α chain) having the amino acid sequence of SEQ ID NO:7, a CDR2 (CDR2 of the α chain) having the amino acid sequence of SEQ ID NO:8, and a CDR3 (CDR3 of the α chain) having the amino acid sequence of SEQ ID NO:9. The second polypeptide chain comprises a CDR1 (CDR1 of the β chain) having the amino acid sequence of SEQ ID NO:10, a CDR2 (CDR2 of the β chain) having the amino acid sequence of SEQ ID NO:11, and a CDR3 (CDR3 of the β chain) having the amino acid sequence of SEQ ID NO:12.

[0052] In this regard, the TCR of the present invention may comprise any one or more of the amino acid sequences selected from SEQ ID NO:1-12. In an embodiment of the present invention, the TCR comprises the following amino acid sequences: (a) all of SEQ ID NO:1-3, (b) all of SEQ ID NO:4-6, (c) all of SEQ ID NO:7-9, (d) all of SEQ ID NO:10-12, (e) all of SEQ ID NO:1-6, or (f) all of SEQ ID NO:7-12. In a particularly preferred embodiment, the TCR comprises the following amino acid sequences: (i) all of SEQ ID NO:1-6 or (ii) all of SEQ ID NO:7-12.

[0053] In an embodiment of the present invention, the TCR comprises the amino acid sequence of the variable region of the TCR containing the above CDRs. In this regard, the TCR may comprise the following amino acid sequences: SEQ ID NO:13 (variable region of the α chain); SEQ ID NO:14 (variable region of the β chain); SEQ ID NO:15 (variable region of the α chain); SEQ ID NO:16 (variable region of the β chain); both SEQ ID NO:13 and 14; or both SEQ ID NO:15 and 16. Preferably, the TCR comprises the following amino acid sequences: (i) both SEQ ID NO:13 and 14, or (ii) both SEQ ID NO:15 and 16.

[0054] The TCR of the present invention may also include an alpha chain constant region and a beta chain constant region. The constant region may be derived from any suitable species (e.g., human or murine). In embodiments of the present invention, the TCR further includes a murine alpha chain and beta chain constant region or a human alpha chain and beta chain constant region. As used herein, when referring to the TCR or any component of the TCR described herein (e.g., complementarity determining region (CDR), variable region, constant region, alpha chain, and / or beta chain), the term "murine" or "human" means that the TCR (or its component) is derived from a mouse or a human, respectively, i.e., the TCR (or its component) is respectively derived from or once expressed by a murine T cell or a human T cell.

[0055] Embodiments of the present invention provide a chimeric TCR comprising a human variable region and a murine constant region, wherein the TCR is antigen - specific for a mutant human RAS amino acid sequence presented by an HLA class II molecule. The murine constant region may provide any one or more advantages. For example, the murine constant region may reduce the mis - pairing of the TCR of the present invention with the endogenous TCR of the host cell into which the TCR of the present invention is introduced. Optionally or additionally, compared to the same TCR having a human constant region, the murine constant region may increase the expression of the TCR of the present invention. The chimeric TCR may include the amino acid sequence of SEQ ID NO:32 (wild - type (WT) murine alpha chain constant region), SEQ ID NO:33 (WT murine beta chain constant region), or both SEQ ID NO:32 and 33. Preferably, the TCR of the present invention includes the amino acid sequences of both SEQ ID NO:32 and 33. The chimeric TCR may include any combination of any murine constant region described herein and any CDR region as described herein for other aspects of the present invention. In this regard, the TCR may include the following amino acid sequences: (a) all of SEQ ID NO:1 - 3 and 32; (b) all of SEQ ID NO:4 - 6 and 33; (c) all of SEQ ID NO:7 - 9 and 32; (d) all of SEQ ID NO:10 - 12 and 33; (e) all of SEQ ID NO:1 - 6 and 32 - 33; or (f) all of SEQ ID NO:7 - 12 and 32 - 33. In another embodiment of the present invention, the chimeric TCR may include any combination of any murine constant region described herein and any variable region as described herein for other aspects of the present invention. In this regard, the TCR may include the following amino acid sequences: (i) both SEQ ID NO:13 and 32; (ii) both SEQ ID NO:14 and 33; (iii) both SEQ ID NO:15 and 32; (iv) both SEQ ID NO:16 and 33; (v) all of SEQ ID NO:13 - 14 and 32 - 33; or (vi) all of SEQ ID NO:15 - 16 and 32 - 33.

[0056] In another embodiment of the present invention, the TCR comprises the following amino acid sequences: SEQ ID NO:38 (α chain with WT murine constant region), SEQ ID NO:39 (β chain with WT murine constant region), SEQ ID NO:40 (α chain with WT murine constant region), SEQ ID NO:41 (β chain with WT murine constant region), SEQ ID NO:38-39 both or SEQ ID NO:40-41 both.

[0057] In an embodiment of the present invention, the TCR comprises an α chain containing a variable region and a constant region and a β chain containing a variable region and a constant region. In this regard, the TCR may comprise (a) an α chain comprising the amino acid sequence of SEQ ID NO:34, wherein: (i) X at position 179 of SEQ ID NO:34 is Thr or Cys; (ii) X at position 243 of SEQ ID NO:34 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 245 of SEQ ID NO:34 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 246 of SEQ ID NO:34 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) a β chain comprising the amino acid sequence of SEQ ID NO:35, wherein X at position 189 of SEQ ID NO:35 is Ser or Cys; (c) an α chain comprising the amino acid sequence of SEQ ID NO:36, wherein: (i) X at position 180 of SEQ ID NO:36 is Thr or Cys; (ii) X at position 244 of SEQ ID NO:36 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 246 of SEQ ID NO:36 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 247 of SEQ ID NO:36 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (d) a β chain comprising the amino acid sequence of SEQ ID NO:37, wherein X at position 194 of SEQ ID NO:37 is Ser or Cys; (e) both (a) and (b); or (f) both (c) and (d).

[0058] In embodiments of the present invention, the TCR comprises a substituted constant region. In this regard, the TCR can comprise one, two, three, or four amino acid substitutions in the constant region of the amino acid sequence of any TCR described herein in one or both of the α and β chains. Preferably, the TCR comprises a murine constant region having 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, the TCR comprises a murine constant region having 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 a parental TCR comprising an unsubstituted (wild-type) constant region, the TCR comprising a substituted constant region advantageously provides one or more of the following: increased recognition of mutant RAS + target, increased expression in host cells, reduced mispairing with endogenous TCRs, and increased anti-tumor activity. Generally, the substituted amino acid sequences SEQ ID NO:30 and 31 of the murine constant regions of the TCR α and β chains respectively correspond to all or a portion of the unsubstituted murine constant region amino acid sequences SEQ ID NO:32 and 33, wherein SEQ ID NO:30 has one, two, three, or four amino acid substitutions compared to SEQ ID NO:32 and SEQ ID NO:31 has one amino acid substitution compared to SEQ ID NO:33. In this regard, embodiments of the present invention provide a TCR comprising the following amino acid sequences: (a) SEQ ID NO:30 (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:31 (constant region of the β chain), wherein X at position 57 is Ser or Cys; or (c) both SEQ ID NO:30 and 31. In embodiments of the present invention, the TCR comprising SEQ ID NO:30 does not comprise SEQ ID NO:32 (unsubstituted murine constant region of the α chain). In embodiments of the present invention, the TCR comprising SEQ ID NO:31 does not comprise SEQ ID NO:33 (unsubstituted murine constant region of the β chain).

[0059] In embodiments of the present invention, the substituted constant region comprises a cysteine substitution in the constant region of one or both of the α-chain and the β-chain to provide a cysteine-substituted TCR. The opposing cysteines in the α-chain and the β-chain provide a disulfide bond that links the constant regions of the α-chain and the β-chain of the substituted TCR and that is not present in TCRs comprising unsubstituted murine constant regions. In this regard, the TCR can be a cysteine-substituted TCR in which one or both of the native Thr (Thr48) at position 48 of SEQ ID NO:32 and the native Ser (Ser57) at position 57 of SEQ ID NO:33 can be substituted with Cys. Preferably, both the native Thr48 of SEQ ID NO:32 and the native Ser57 of SEQ ID NO:33 are substituted with Cys. Examples of cysteine-substituted TCR constant region sequences are shown in Table 2. In embodiments of the present invention, the cysteine-substituted TCR comprises (i) SEQ ID NO:30, (ii) SEQ ID NO:31, or (iii) both SEQ ID NO:30 and 31, where both SEQ ID NO:30 and 31 are as defined in Table 2. In addition to any CDRs or variable regions described herein, the cysteine-substituted TCRs of the present invention can comprise substituted constant regions.

[0060] In embodiments of the present invention, the cysteine-substituted chimeric TCR comprises a full-length α-chain and a full-length β-chain. Examples of cysteine-substituted chimeric TCR α-chain and β-chain sequences are shown in Table 2. In embodiments of the present invention, the TCR comprises (i) SEQ ID NO:34, (ii) SEQ ID NO:35, (iii) SEQ ID NO:36, (iv) SEQ ID NO:37, (v) both SEQ ID NO:34 and 35, or (vi) both SEQ ID NO:36 and 37, where all of SEQ ID NO:34-37 are as defined in Table 2.

[0061] Table 2

[0062]

[0063] In embodiments of the present invention, the substituted amino acid sequence comprises a substitution of one, two, or three amino acids in a transmembrane (TM) domain in the constant region of one or both of the α-chain and the β-chain having a hydrophobic amino acid, thereby providing a TCR substituted with a hydrophobic amino acid (also referred to herein as a "LVL-modified TCR"). Compared to a TCR lacking a hydrophobic amino acid substitution in the TM domain, the hydrophobic amino acid substitution in the TM domain of the TCR can increase the hydrophobicity of the TM domain of the TCR. In this regard, the TCR is a LVL-modified TCR, wherein one, two, or three of the native Ser112, Met114, and Gly115 of SEQ ID NO:32 can be independently substituted with Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; preferably substituted with Leu, Ile, or Val. Preferably, all three of the native Ser112, Met114, and Gly115 of SEQ ID NO:32 can be independently substituted with Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; preferably substituted with Leu, Ile, or Val. In embodiments of the present invention, the LVL-modified TCR comprises (i) SEQ ID NO:30, (ii) SEQ ID NO:31, or (iii) both SEQ ID NO:30 and 31, wherein both SEQ ID NO:30 and 31 are defined as in Table 3. In addition to any CDR or variable region described herein, the LVL-modified TCR of the present invention may comprise a substituted constant region.

[0064] In embodiments of the present invention, the LVL-modified TCR comprises a full-length α-chain and a full-length β-chain. Examples of the LVL-modified TCR α-chain and β-chain sequences are shown in Table 3. In embodiments of the present invention, the LVL-modified TCR comprises (i) SEQ ID NO:34, (ii) SEQ ID NO:35, (iii) SEQ ID NO:36, (iv) SEQ ID NO:37, (v) both SEQ ID NO:34 and 35, or (vi) both SEQ ID NO:36 and 37, wherein all of SEQ ID NO:34-37 are defined as in Table 3.

[0065] Table 3

[0066]

[0067]

[0068]

[0069] In an embodiment of the present invention, the substituted amino acid sequence comprises a combination of cysteine substitutions in the constant region of one or both of the α-chain and the β-chain, and comprises substitutions of one, two, or three amino acids in the transmembrane (TM) domain in the constant region of one or both of the α-chain and the β-chain having a hydrophobic amino acid (also referred to herein as "cysteine-substituted, LVL-modified TCR"). In this regard, the TCR is a cysteine-substituted, LVL-modified chimeric TCR, wherein the native Thr48 of SEQ ID NO: 32 is substituted with Cys; one, two, or three of the native Ser112, Met114, and Gly115 of SEQ ID NO: 32 are independently substituted with Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; preferably substituted with Leu, Ile, or Val; and the native Ser57 of SEQ ID NO: 33 is substituted with Cys. Preferably, all three of the native Ser112, Met114, and Gly115 of SEQ ID NO: 32 can be independently substituted with Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; preferably substituted with Leu, Ile, or Val. In an embodiment of the present invention, the cysteine-substituted, LVL-modified TCR comprises (i) SEQ ID NO: 30, (ii) SEQ ID NO: 31, or (iii) both SEQ ID NO: 30 and 31, wherein both SEQ ID NO: 30 and 31 are defined as in Table 4. In addition to any CDR or variable region described herein, the cysteine-substituted, LVL-modified TCR of the present invention may comprise a substituted constant region.

[0070] In an embodiment, the cysteine-substituted, LVL-modified TCR comprises a full-length α-chain and a full-length β-chain. In an embodiment of the present invention, the cysteine-substituted, LVL-modified TCR comprises (i) SEQ ID NO: 34, (ii) SEQ ID NO: 35, (iii) SEQ ID NO: 36, (iv) SEQ ID NO: 37, (v) both SEQ ID NO: 34 and 35, or (vi) both SEQ ID NO: 36 and 37, wherein all of SEQ ID NO: 34-37 are defined as in Table 4.

[0071] Table 4

[0072]

[0073]

[0074]

[0075] The present invention also provides a polypeptide comprising a functional portion of any TCR described herein. As used herein, the term "polypeptide" includes oligopeptides and refers to a single chain of amino acids joined by one or more peptide bonds.

[0076] For the polypeptides of the present invention, the functional portion can be any portion that comprises contiguous amino acids of the TCR and is part thereof, provided that the functional portion specifically binds mutant RAS. The term "functional portion", when referring to the use of a TCR, refers to any portion or fragment of a TCR of the present invention that retains the biological activity of the TCR (parental TCR) from which the portion is derived. Functional portions encompass, for example, those portions of the TCR that retain the ability to: specifically bind mutant RAS (e.g., within the context of an HLA-DRB1*07:01 molecule or an HLA-DRB1*11:01 molecule) or detect, treat or prevent cancer to a similar, the same or a higher degree compared to the parental TCR. When referring to the parental TCR, the functional portion can comprise, for example, about 10%, about 25%, about 30%, about 50%, about 68%, about 80%, about 90%, about 95% or more of the parental TCR.

[0077] The functional portion can include additional amino acids at the amino or carboxyl terminus of the portion or at both termini, which additional amino acids are not found in the amino acid sequence of the parental TCR. Desirably, the additional amino acids do not interfere with the biological function of the functional portion, such as specifically binding mutant RAS and / or having the ability to detect cancer, treat or prevent cancer, etc. More desirably, the additional amino acids enhance the biological activity compared to the biological activity of the parental TCR.

[0078] The polypeptide can include a functional portion of any one or both of the α and β chains of a TCR of the present invention, such as a functional portion that includes one or more of CDR1, CDR2, and CDR3 of the variable region in the α and / or β chain of a TCR of the present invention. In an embodiment of the present invention, the polypeptide can 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 can 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.

[0079] In this regard, the polypeptides of the present invention may include any one or more of the amino acid sequences selected from SEQ ID NOs: 1-12. In an 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 preferred embodiment, the polypeptide includes the following amino acid sequences: (i) all of SEQ ID NOs: 1-6 or (ii) all of SEQ ID NOs: 7-12.

[0080] In an embodiment of the present invention, the polypeptides of the present invention may include, for example, the variable regions of the TCRs of the present invention that include combinations of the above CDR regions. In this regard, the polypeptide may include the following amino acid sequences: (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 NO: 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), or (vi) both SEQ ID NO: 15 and 16. Preferably, the polypeptide includes the following amino acid sequences: (i) both SEQ ID NO: 13 and 14 or (ii) both SEQ ID NO: 15 and 16.

[0081] In an embodiment of the present invention, the polypeptides of the present invention may further include the above constant regions of the TCRs of the present invention. In this regard, the polypeptide may further include the following amino acid sequences: SEQ ID NO: 32 (WT murine constant region of the α chain), SEQ ID NO: 33 (WT murine constant region of the β chain), SEQ ID NO: 30 (substituted murine constant region of the α chain), SEQ ID NO: 31 (substituted murine constant region of the β chain), both SEQ ID NO: 32 and 33, or both SEQ ID NO: 30 and 31. Preferably, the polypeptide further includes a combination of the amino acid sequences of SEQ ID NO: 30 and 31 or SEQ ID NO: 32 and 33 with any of the CDR regions or variable regions described herein for other aspects of the present invention. In an embodiment of the present invention, one or both of SEQ ID NOs: 30 and 31 of the polypeptide are defined as any one of Tables 2-4.

[0082] In an embodiment of the present invention, the polypeptide of the present invention may comprise the entire length of the α or β chain of the TCR described herein. In this regard, the polypeptide of the present invention may comprise the amino acid sequences of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37. Optionally, the polypeptide of the present invention may comprise both chains of the TCR described herein.

[0083] For example, the polypeptide of the present invention may comprise: (a) the amino acid sequence of SEQ ID NO:34, wherein: (i) X at position 179 of SEQ ID NO:34 is Thr or Cys; (ii) X at position 243 of SEQ ID NO:34 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 245 of SEQ ID NO:34 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 246 of SEQ ID NO:34 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) the amino acid sequence of SEQ ID NO:35, wherein X at position 189 of SEQ ID NO:35 is Ser or Cys; (c) the amino acid sequence of SEQ ID NO:36, wherein: (i) X at position 180 of SEQ ID NO:36 is Thr or Cys; (ii) X at position 244 of SEQ ID NO:36 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 246 of SEQ ID NO:36 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 247 of SEQ ID NO:36 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (d) the amino acid sequence of SEQ ID NO:37, wherein X at position 194 of SEQ ID NO:37 is Ser or Cys; (e) both (a) and (b); or (f) both (c) and (d). In an embodiment of the present invention, any one or more of SEQ ID NOs: 34-37 of the polypeptide are defined as any one of Tables 2-4.

[0084] The present invention also provides a protein comprising at least one polypeptide described herein. "Protein" means a molecule comprising one or more polypeptide chains.

[0085] In an embodiment, the protein of the present invention may comprise (a) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 1-3 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 4-6; or (b) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 7-9 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 10-12.

[0086] In another embodiment of the present invention, the protein may comprise (i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 13 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 14; or (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.

[0087] The protein of the present invention may further comprise any constant region described herein for other aspects of the present invention. In this regard, in an embodiment of the present invention, the first polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 32 and the second polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 31 or SEQ ID NO: 33. In an embodiment of the present invention, one or both of SEQ ID NO: 30 and 31 of the protein are defined as any one in Tables 2-4.

[0088] Optionally or alternatively, the protein of an embodiment of the present invention may comprise (a) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 34, wherein: (i) X at position 179 of SEQ ID NO: 34 is Thr or Cys; (ii) X at position 243 of SEQ ID NO: 34 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 245 of SEQ ID NO: 34 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 246 of SEQ ID NO: 34 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: 35, wherein X at position 189 of SEQ ID NO: 35 is Ser or Cys; (c) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 36, wherein: (i) X at position 180 of SEQ ID NO: 36 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 36 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 246 of SEQ ID NO: 36 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 247 of SEQ ID NO: 36 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: 37, wherein X at position 194 of SEQ ID NO: 37 is Ser or Cys; (e) both (a) and (b); or (f) both (c) and (d). In an embodiment of the present invention, one or more of SEQ ID NOs: 34-37 are defined as any one of Tables 2-4.

[0089] The protein of the present invention can be a TCR. Optionally, for example, if the protein comprises a single polypeptide chain containing the amino acid sequences of both SEQ ID NO: 34 and 35, and both SEQ ID NO: 36 and 37, or if the first and / or second polypeptide chain of the protein further comprises other amino acid sequences (such as amino acid sequences encoding an immunoglobulin or a portion thereof), then the protein of the present invention can be a fusion protein. In this regard, the present invention also provides a fusion protein comprising at least one polypeptide of the present invention described herein and at least one other polypeptide. The other polypeptide can exist as a separate polypeptide of the fusion protein or can exist in the form of a polypeptide expressed in-frame (in tandem) with one of the polypeptides of the present invention described herein. The other polypeptide can encode any peptide or protein molecule or a portion thereof, including but not limited to: immunoglobulins, CD3, CD4, CD8, MHC molecules, CD1 molecules (such as CD1a, CD1b, CD1c, CD1d), etc.

[0090] The fusion protein can comprise one or more copies of the polypeptide of the present invention and / or one or more copies of the other polypeptide. For example, the fusion protein can comprise 1, 2, 3, 4, 5 or more copies of the polypeptide of the present invention and / or the other polypeptide. Suitable methods for preparing the fusion protein are known in the art and include, for example, recombinant methods.

[0091] In some embodiments of the present invention, the TCR, polypeptide and protein of the present invention can be expressed as a single protein comprising a linker peptide connecting the α-chain and the β-chain. In this regard, the TCR, polypeptide and protein of the present invention can further comprise a linker peptide. The linker peptide can advantageously facilitate the expression of the recombinant TCR, polypeptide and / or protein in a host cell. The linker peptide can comprise any suitable amino acid sequence. For example, the linker peptide can be a furin-SGSG-P2A linker comprising the amino acid sequence of SEQ ID NO: 54. When the construct comprising the linker peptide is expressed by a host cell, the linker peptide can be cleaved, thereby generating separate α-chain and β-chain. In an embodiment of the present invention, the TCR, polypeptide or protein can comprise the amino acid sequence containing the full-length α-chain, the full-length β-chain and the linker peptide located between the α-chain and the β-chain.

[0092] The protein of the present invention can be a recombinant antibody or an antigen-binding portion thereof comprising at least one polypeptide of the present invention described herein. As used herein, a "recombinant antibody" refers to a recombinant (such as genetically engineered) protein comprising at least one of the polypeptide of the present invention and a polypeptide chain of an antibody or an antigen-binding portion thereof. The polypeptide of the antibody or an antigen-binding portion thereof can be a heavy chain, a light chain, a variable or constant region of a heavy chain or a light chain, a single-chain variable fragment (scFv) or Fc, Fab or F(ab) 2'Fragments, etc. The polypeptide chains of the antibody or its antigen-binding portion may exist as separate polypeptides of the recombinant antibody. Optionally, the polypeptide chains of the antibody or its antigen-binding portion may exist in the form of polypeptides expressed in-frame (in tandem) with the polypeptides of the present invention. The polypeptide of the antibody or its antigen-binding portion may be the polypeptide of any antibody or any antibody fragment (including any antibody and antibody fragment described herein).

[0093] The scope of the present invention includes functional variants of the TCR, polypeptide or protein of the present invention described herein. As used herein, the term "functional variant" refers to a TCR, polypeptide or protein that has substantial or significant sequence identity or similarity to the parental TCR, polypeptide or protein, and that retains the biological activity of the TCR, polypeptide or protein from which the variant is derived. Functional variants encompass, for example, those variants of the TCR, polypeptide or protein (parental TCR, polypeptide or protein) described herein that retain the ability to bind specifically to mutant RAS (to which the parental TCR has antigenic specificity or with which the parental polypeptide or protein binds specifically) to a similar degree, the same degree or a higher degree compared to the parental TCR, polypeptide or protein. With reference to the parental TCR, polypeptide or protein, the amino acid sequence of the functional variant may be, respectively, at least about 30%, about 50%, about 75%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or higher identical to the parental TCR, polypeptide or protein.

[0094] Functional variants may, for example, include the amino acid sequence of the parental TCR, polypeptide or protein having at least one conservative amino acid substitution. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is exchanged for another amino acid having the same chemical or physical properties. For example, a conservative amino acid substitution may be the substitution of one acidic amino acid for another acidic amino acid (e.g., Asp or Glu), the substitution of an amino acid with a nonpolar side chain for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), the substitution of a basic amino acid for another basic amino acid (Lys, Arg, etc.), the substitution of an amino acid with a polar side chain for another amino acid with a polar side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), etc.

[0095] Optionally or additionally, functional variants may include the amino acid sequence of the parental TCR, polypeptide or protein having at least one non-conservative amino acid substitution. 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 increased compared to the parental TCR, polypeptide or protein.

[0096] The TCR, polypeptide or protein may consist essentially of a specified amino acid sequence or the sequences described herein, such that other components of the TCR, polypeptide or protein (e.g., other amino acids) do not substantially alter the biological activity of the TCR, polypeptide or protein. In this regard, the TCR, polypeptide or protein of the present invention may, for example, consist essentially of the amino acid sequences of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:34-35 both, or SEQ ID NO:36-37 both. Similarly, for example, the TCR, polypeptide or protein of the present invention may consist essentially of (i) SEQ ID NO:13, (ii) SEQ ID NO:14, (iii) SEQ ID NO:15, (iv) SEQ ID NO:16, (v) SEQ ID NO:13 and 14 both, or (vi) SEQ ID NO:15 and 16 both. Additionally, the TCR, polypeptide or protein of the present invention may consist essentially of the following amino acid sequences: (a) any one or more of SEQ ID NO:1-12; (b) all of SEQ ID NO:1-3; (c) all of SEQ ID NO:4-6; (d) all of SEQ ID NO:7-9; (e) all of SEQ ID NO:10-12; (f) all of SEQ ID NO:1-6; or (g) all of SEQ ID NO:7-12.

[0097] The TCRs, polypeptides and proteins of the present invention can have any length, i.e., can include any number of amino acids, provided that the TCR, polypeptide or protein retains its biological activity (e.g., being able to specifically bind to mutant RAS; detecting cancer in a mammal; or treating or preventing cancer in a mammal, etc.). For example, the length of the polypeptide can range from about 50 to about 5000 amino acids, such as about 50, about 70, about 75, about 100, about 125, about 150, about 175, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000 or more amino acids. In this regard, the polypeptides of the present invention also include oligopeptides.

[0098] 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-aminodecanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine-β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0099] The TCRs, polypeptides, and proteins of the present invention may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, or converted to acid addition salts and / or optionally dimerized or polymerized or conjugated, for example, via disulfide bridges.

[0100] The TCRs, polypeptides, and / or proteins of the present invention can be obtained by methods known in the art, such as resynthesis. Similarly, the nucleic acids described herein can be used and polypeptides and proteins can be produced recombinantly using standard recombinant methods. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual , 4th Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Optionally, 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.

[0101] The scope of the present invention includes conjugates (e.g., bioconjugates) containing any TCR, polypeptide, or protein of the present invention, including any functional portion or variant thereof, nucleic acid, recombinant expression vector, host cell, host cell population, or antibody or antigen-binding portion thereof. Conjugates and general methods of synthesizing conjugates are known in the art.

[0102] Embodiments of the present invention provide nucleic acids comprising nucleotide sequences encoding any of the TCRs, polypeptides, or proteins described herein. As used herein, "nucleic acid" encompasses "polynucleotide", "oligonucleotide", and "nucleic acid molecule" and generally refers to a polymer of DNA or RNA, which can be single-stranded or double-stranded, which can contain natural, non-natural, or altered nucleotides, and which can contain natural, non-natural, or altered internucleotide linkages (e.g., phosphoramidate linkages or thiophosphate linkages) in place of the phosphodiester found between nucleotides in an unmodified oligonucleotide. In embodiments, the nucleic acid comprises complementary DNA (cDNA). Generally preferably, the nucleic acid does not include any insertions, deletions, inversions, and / or substitutions. However, in some cases, as discussed herein, the nucleic acid may be adapted to include one or more insertions, deletions, inversions, and / or substitutions.

[0103] Preferably, the nucleic acids of the present invention are recombinant. As used herein, the term "recombinant" means: (i) a molecule is constructed outside of a living cell by joining natural or synthetic nucleic acid segments to a nucleic acid molecule that can replicate in a living cell, or (ii) a molecule is derived from the replication of those described in (i) above. For purposes herein, replication can be in vitro replication or in vivo replication.

[0104] Nucleic acids can be constructed using procedures known in the art based on chemical synthesis and / or enzymatic ligation reactions. See, e.g., Green and Sambrook et al. (supra). For example, nucleic acids can be synthesized chemically using naturally occurring nucleotides or nucleotides modified in various ways, the modified nucleotides being designed to increase the biological stability of the molecule or the physical stability of the duplex formed upon hybridization (e.g., thiophosphate 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-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine, inosine, N 6 -isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6 -substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6-Isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl uracil-5-oxyacetate, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Optionally, one or more of the nucleic acids of the present invention may be purchased from companies such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0105] The nucleic acid may include any nucleotide sequence encoding any TCR, polypeptide, or protein described herein. In an embodiment of the present invention, the nucleic acid may include the nucleotide sequence of any one of SEQ ID NOs: 42-45 (Table 5). In an embodiment of the present invention, the nucleic acid includes the nucleotide sequences of both SEQ ID NOs: 42-43 or both SEQ ID NOs: 44-45.

[0106] Table 5

[0107]

[0108] In an 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 can increase the translation efficiency of the mRNA transcript. Codon optimization of the nucleotide sequence may involve replacing a native codon with another codon that encodes the same amino acid but can be translated by a tRNA that is more readily available within the cell, thereby increasing the translation efficiency. Optimization of the nucleotide sequence may also reduce secondary mRNA structures that interfere with translation, thereby increasing the translation efficiency.

[0109] The present invention also provides a nucleic acid comprising a nucleotide sequence complementary to the nucleotide sequence of any nucleic acid described herein or a nucleotide sequence that hybridizes to the nucleotide sequence of any nucleic acid described herein under stringent conditions.

[0110] Nucleotide sequences hybridized under stringent conditions are preferably hybridized under high-stringency conditions. "High-stringency conditions" means that a nucleotide sequence hybridizes specifically to a 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 a polynucleotide containing an exactly complementary sequence or a polynucleotide containing only a few scattered mismatches from a random sequence having a few small regions (e.g., 3-10 bases) of exactly matching nucleotide sequences. Such small complementary regions are more readily melted than full-length complements of 14-17 bases or more, and high-stringency hybridization allows them to be readily distinguished. Relatively high-stringency conditions will 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 little mismatch, if any, between the nucleotide sequence and the template or target strand, and are particularly suitable for detecting the expression of any TCR of the present invention. It is generally believed that more stringent conditions can be achieved by adding increasing amounts of formamide.

[0111] The present invention also provides nucleic acids comprising a nucleotide sequence that is at least about 70% or more (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 nucleic acid described herein. In this regard, the nucleic acid can consist essentially of any nucleotide sequence described herein.

[0112] 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 nucleic acid of the present invention. In an embodiment of the present invention, the recombinant expression vector comprises nucleotide sequences encoding an α-chain, a β-chain, and a linker peptide.

[0113] For the purposes herein, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that, when the construct comprises a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide and the vector is contacted with a cell under conditions sufficient to permit expression of the mRNA, protein, polypeptide, or peptide within the cell, allows a host cell to express the mRNA, protein, polypeptide, or peptide. The vectors of the present invention are not entirely naturally occurring. However, portions of the vector can be naturally occurring. The recombinant expression vectors of the present invention can comprise any type of nucleotide, including but not limited to: DNA and RNA, which can be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and can contain natural, non-natural, or altered nucleotides. The recombinant expression vectors can comprise naturally occurring, non-naturally occurring nucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or altered nucleotides or nucleotide linkages do not impede transcription or replication of the vector.

[0114] 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 vectors designed for propagation and amplification or for expression or for both, such as plasmids and viruses. The 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 can also be used, such as λGT10, λGT11, λZapII (Stratagene), λEMBL4 and λNM1149. 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 the MSGV1 vector.

[0115] The recombinant expression vector of the present invention can be prepared using standard recombinant DNA techniques described, for example, by Green and Sambrook et al. (see above). Circular or linear constructs of the expression vector can be prepared to contain a functional replication system in prokaryotic or eukaryotic host cells. The replication system can be derived from, for example, ColE1, 2μ plasmid, λ, SV40, bovine papillomavirus, etc.

[0116] Desirably, the recombinant expression vector contains regulatory sequences such as transcription and translation start codons and stop codons, which are specific for the type of host cell to be introduced into the vector (such as bacteria, fungi, plants or animals), depending on the situation and considering whether the vector is based on DNA or RNA.

[0117] The recombinant expression vector can contain one or more marker genes, which allows the selection of transformed or transfected host cells. Marker genes include biocide resistance (such as resistance to antibiotics, heavy metals, etc.), complementation in auxotrophic host cells to provide prototrophy, etc. Suitable marker genes for the expression vectors of the present invention include, for example, neomycin / G418 resistance gene, hygromycin resistance gene, histidinol resistance gene, tetracycline resistance gene and ampicillin resistance gene.

[0118] The recombinant expression vector may include a natural or non-natural promoter that is operably linked to a nucleotide sequence encoding a TCR, polypeptide, or protein or to a nucleotide sequence complementary or hybridizable to the nucleotide sequence encoding a TCR, polypeptide, or protein. The selection of a promoter, such as strong, weak, inducible, tissue-specific, and developmentally specific, is within the ability of one of ordinary skill in the art. Similarly, the combination of a nucleotide sequence with a promoter is also within the ability of the skilled person. The promoter may be a non-viral promoter or a viral promoter, such as the cytomegalovirus (CMV) promoter, SV40 promoter, RSV promoter, and the promoter found in the long terminal repeat of murine stem cell virus.

[0119] The recombinant expression vector of the present invention can be designed for transient expression, stable expression, or both. Similarly, the recombinant expression vector can be prepared for constitutive expression or for inducible expression.

[0120] In addition, the recombinant expression vector can be prepared to contain a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the death of a cell expressing the suicide gene. A suicide gene can be a gene that confers sensitivity of the cell in which it is expressed to a reagent (such as a drug), and causes cell death when the cell comes into contact with or is exposed to the reagent. Suicide genes are known in the art and include, for example, the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, nitroreductase, and the inducible caspase 9 gene system.

[0121] Another embodiment of the present invention also 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 the recombinant expression vector of the present invention. The host cell can be a eukaryotic cell (such as a plant, animal, fungus, or alga), or can be a prokaryotic cell (such as a bacterium or protozoan). 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 adherent cell or a suspension cell, i.e., a cell that grows in suspension. Suitable host cells are known in the art and include, for example, DH5α Escherichia coli (E. coli) cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. For the purpose of amplifying or replicating the recombinant expression vector, the host cell is preferably a prokaryotic cell, such as DH5α cells. For the purpose of producing a recombinant TCR, polypeptide, or protein, the host cell is preferably a mammalian cell. More preferably, the host cell is a human cell. Although the host cell can be of any cell type, can be derived from any type of tissue, and can be at 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 is a T cell.

[0122] For the purposes of the present disclosure, the T cell can be any T cell, such as a cultured T cell (e.g., a primary T cell, or a T cell from a cultured T cell line (e.g., Jurkat, SupT1, etc.)), or a T cell obtained from a mammal. If obtained from a mammal, the T cell can be obtained from a variety of sources, including but not limited to: blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The T cell can also be enriched or purified. Preferably, the T cell is a human T cell. The T cell can be any type of T cell and can be at any stage of development, including but not limited to: CD4 + / CD8 + double positive T cells, CD4 + helper T cells (e.g., Th 1 and Th 2 cells), CD4 + T cells, CD8 + T cells (e.g., cytotoxic T cells), tumor infiltrating lymphocytes (TILs), memory T cells (e.g., central memory T cells and effector memory T cells), naive T cells, etc.

[0123] The present disclosure also provides a cell population comprising at least one host cell described herein. The cell population can be a heterogeneous population comprising, in addition to at least one other cell, a host cell containing any of the recombinant expression vectors described herein, the other cells being, for example, host cells that do not contain any recombinant expression vectors (e.g., T cells) or cells other than T cells, such as B cells, macrophages, neutrophils, red blood cells, hepatocytes, endothelial cells, epithelial cells, muscle cells, brain cells, etc. Optionally, the cell population can be a substantially homogeneous population, wherein the population mainly comprises host cells containing the recombinant expression vector (e.g., consisting essentially of host cells containing the recombinant expression vector). The population can also be a clonal cell population, wherein all the cells of the population are clones of a single host cell containing the recombinant expression vector such that all the cells of the population contain the recombinant expression vector. In one embodiment of the present disclosure, the cell population is a clonal population comprising host cells containing the recombinant expression vector as described herein.

[0124] In an embodiment of the present invention, the number of cells in a population can be rapidly expanded. Expansion of the number of T cells can be achieved by any of a number of methods known in the art, such as, 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. Immunol. Ther., 26:332-42 (2003); and Riddell et al., J. Immunol. Methods, 128:189-201 (1990). In an embodiment, the number of T cells is expanded by culturing the T cells together with OKT3 antibody, IL-2, and feeder PBMCs (e.g., irradiated allogeneic PBMCs).

[0125] 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. As used herein, the term "isolated" means removed from its natural environment. As used herein, the term "purified" means having increased purity, where "purity" is a relative term and need not be construed 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%.

[0126] The TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, and host cells (including populations thereof) of the present invention (hereinafter collectively referred to as "the TCR materials of the present invention") can be formulated into compositions (e.g., pharmaceutical compositions). In this regard, the present invention provides pharmaceutical compositions comprising any one of the TCRs, polypeptides, proteins, nucleic acids, expression vectors, and host cells (including populations thereof) described herein and a pharmaceutically acceptable carrier. The pharmaceutical compositions of the present invention containing any of the TCR materials of the present invention can include more than one of the TCR materials of the present invention (e.g., polypeptides and nucleic acids) or two or more different TCRs. Optionally, the pharmaceutical composition can include a combination of a TCR material of the present invention with another pharmaceutically active agent or drug, which other pharmaceutically active agent or drug is, for example, a chemotherapeutic agent such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.

[0127] Preferably, the carrier is a pharmaceutically acceptable carrier. In the case of pharmaceutical compositions, the carrier can be any of those carriers conventionally used for the specific TCR materials of the present invention under consideration. Methods for preparing administrable compositions are known to those skilled in the art or will be apparent to those skilled in the art and are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, 22nd Edition, Pharmaceutical Press (2012). Preferably, a pharmaceutically acceptable carrier is a carrier that is free of adverse side effects or toxicity under the conditions of use.

[0128] The choice of carrier depends in part on the particular TCR material of the present invention and the particular method for administering the TCR material of the present invention. Accordingly, there are various suitable formulations of the pharmaceutical compositions of the present invention. Suitable formulations can include any of those 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 certain instances, a particular route may provide a more direct and more effective response than another route.

[0129] 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 a population thereof) expressing the TCR of the present invention, a pharmaceutically acceptable carrier for the cells for injection can include any isotonic carrier, such as normal saline (about 0.90% w / v NaCl in water, about 300 mOsm / L NaCl in water, or about 9.0 g NaCl / liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water, or Ringer's lactate. In an embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumin.

[0130] 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 elicit, for example, a therapeutic or prophylactic response in an individual or animal within a reasonable 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., mutant RAS) or detect, treat, or prevent cancer within a period of about 2 hours or longer (e.g., 12 to 24 or more hours) from the time of administration. In certain embodiments, this period may even be longer. The dose will be determined by the efficacy of the particular TCR material of the present invention and the condition of the animal (e.g., human) as well as the weight of the animal (e.g., human) to be treated.

[0131] Many assays for determining the administered dose are known in the art. For the purposes of the present invention, an assay can be used to determine the starting dose to be administered to a mammal, the assay comprising comparing the extent of target cell lysis or IFN-γ secretion by T cells in a group of mammals each given a different dose of T cells, after administration to the mammal of a given dose of T cells expressing the TCR, polypeptide or protein of the present invention. The extent of target cell lysis or IFN-γ secretion at a given dose can be assayed by methods known in the art.

[0132] The dose 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 will consider a variety of factors to determine the dose of the TCR material of the present invention to be used to treat each individual patient, the factors being (e.g.) age, weight, general 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, e.g., from about 1×10 6 to about 1×10 12 cells or more. In certain embodiments, fewer than 1×10 6 cells can be administered.

[0133] One of ordinary skill in the art will readily appreciate that the inventive TCR material of the present invention can be modified in any number of ways to increase the therapeutic or prophylactic efficacy of the TCR material of the present invention via the modification. For example, the TCR material of the present invention can be conjugated directly or via a bridge to a chemotherapeutic agent. The practice of conjugating compounds to chemotherapeutic agents is known in the art. One of ordinary skill in the art recognizes that sites on the TCR material of the present invention that are not required for the function of the TCR material of the present invention are suitable sites for attaching the bridge and / or chemotherapeutic agent, provided that the bridge and / or chemotherapeutic agent do not interfere with the function of the TCR material of the present invention (i.e., are capable of binding mutant RAS or are capable of detecting, treating or preventing cancer) after attachment to the TCR material of the present invention.

[0134] The pharmaceutical compositions, TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells and cell populations of the present invention are expected to be used 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 mutant 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 mutant RAS when expressed by cells. In this regard, the present invention provides methods for treating or preventing cancer in a mammal, which comprise administering to the mammal an amount effective to treat or prevent cancer in the mammal any of the following substances: any of the pharmaceutical compositions, TCRs, polypeptides or proteins described herein; any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCRs, polypeptides, proteins described herein; or any host cell or cell population comprising a recombinant vector encoding any of the TCRs, polypeptides or proteins described herein.

[0135] Embodiments of the present invention provide the following substances for treating or preventing cancer in a mammal: any of the pharmaceutical compositions, TCRs, polypeptides or proteins described herein; any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCRs, polypeptides, proteins described herein; or any host cell or cell population comprising a recombinant vector encoding any of the TCRs, polypeptides or proteins described herein.

[0136] As used herein, the terms “treatment” and “prevention” and words derived therefrom do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention, which are considered by those of ordinary skill in the art to have potential benefits or therapeutic effects. In this regard, the methods of the present invention can provide treatment or prevention of cancer in a mammal at any amount and at any level. Additionally, 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, the treatment or prevention can include promoting tumor regression. Similarly, for the purposes herein, “prevention” can encompass delaying the onset of cancer or its symptoms or conditions. Optionally or additionally, “prevention” can encompass preventing or delaying the recurrence of cancer or its symptoms or conditions.

[0137] Also provided are methods for detecting the presence of cancer in a mammal. The methods include: (i) contacting a sample comprising one or more cells from the mammal with any of the present invention TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, cell populations or pharmaceutical compositions described herein, thereby forming a complex; and detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

[0138] For the methods of the present invention for detecting cancer in a mammal, the cell sample can be a sample comprising whole cells, lysates thereof or whole cell lysate fractions (such as nuclear or cytoplasmic fractions, whole protein fractions or nucleic acid fractions).

[0139] For the purposes of the methods of the invention for detecting cancer, the contacting can occur in vitro or in vivo in a mammal. Preferably, the contacting is in vitro.

[0140] Similarly, the complex can be detected by any number of ways 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 label, which can be (for example) a radioisotope, a fluorophore (such as fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (such as alkaline phosphatase, horseradish peroxidase) and elemental particles (such as gold particles).

[0141] For the purposes of the methods of the invention for administering a host cell or cell population, the cells can be allogeneic or autologous cells of a mammal. Preferably, the cells are autologous to the mammal.

[0142] For the methods of the invention, the cancer can be any cancer, including any of the following cancers: 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 tumor, glioma, Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, oropharyngeal cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal cancer, omental cancer and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, ureteral cancer and bladder cancer. Preferred cancers are pancreatic 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 an embodiment of the invention, the cancer expresses a mutant human RAS amino acid sequence, wherein the mutant human RAS amino acid sequence is a mutant human KRAS, mutant human HRAS or mutant human NRAS amino acid sequence. 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 invention.

[0143] The mammalian species referred to in the method of the present invention can be any mammalian species. As used herein, the term "mammalian species" refers to any mammalian species, including but not limited to: mammalian species of the order Rodentia (e.g., mice and hamsters) and mammalian species of the order Lagomorpha (e.g., rabbits). Preferably, the mammalian species is from the order Carnivora, including felines (cats) and canines (dogs). More preferably, the mammalian species is from the order Artiodactyla (including bovines (cows) and porcines (pigs)) or the order Perissodactyla (including equines (horses)). Most preferably, the mammalian species is of the order Primate, suborder Ceboidea or suborder Simiiformes (monkeys) or suborder Hominoidea (humans and apes). A particularly preferred mammalian species is the human species.

[0144] The following examples further illustrate the present invention, but should of course not be construed as limiting the scope of the present invention in any way.

[0145] Example 1

[0146] This example demonstrates the isolation of a TCR with antigen specificity for human KRAS with the G12V mutation presented by the HLA-DRB1*07:01 molecule.

[0147] A TCR with antigen specificity for human KRAS with the G12V mutation presented by the HLA-DRB1*07:01 molecule was isolated from an endometrial tumor sample of a patient. Briefly, the tumor sample was minced, digested, and frozen. Prior to cell sorting, the tumor digest was thawed and left overnight in the absence of cytokines. T cells were sorted from the tumor digest by FACS based on PD-1 and / or OX40 expression (gated on PI - (live cells)>CD3+). The FACS results are shown in Figure 1 . Isotype-stained cells were used as controls.

[0148] Sorted cells were expanded for 3.5 weeks according to the rapid expansion protocol (REP). For the REP, T cells were cultured in microtiter 96-well plates (3 cells / well) in the presence of OKT3 antibody, IL-2, and irradiated allogeneic PBMC.

[0149] Pooled expanded cells were tested for reactivity against autologous dendritic cells (DC) pulsed with pooled 25-mer peptides or peptides encoded by 25-mer tandem minigenes (TMGs) covering various tumor-specific mutations detected in the patient's tumor. Each pool contained 17 - 21 peptides or TMGs. Interferon-γ (IFN-γ) secretion was measured by enzyme-linked immunospot (ELISPOT). The results are shown in Figure 2 . AsFigure 2 As shown, the pooled effector autologous T cells in cultures 7 and 8 recognize target DCs pulsed with peptide pool 1 (PP1) and peptide pool 2 (PP2).

[0150] The mutant-reactive T cell cultures were tested against autologous DCs pulsed with each single peptide from the relevant peptide pools. Figure 3 Results obtained after co-culturing autologous T cell culture 7 (W7) with autologous DCs pulsed with each of peptides 1-17 (P1-P17) from peptide pool 1 (PP1) are shown. As Figure 3 shown, the T cells of culture 7 exhibit high specificity for peptide P17. Peptide 17 (P17) encodes a KRAS G12V mutation.

[0151] Total RNA was isolated from the cells of autologous T cell culture number (W7). Then, using TCR-α and -β chain constant primers, the total RNA was subjected to rapid amplification of the 5' complementary DNA end (5’RACE). The TCR PCR products were then separated by standard agarose gel electrophoresis and gel extraction. The products were directly sequenced. The nucleotide sequences of the variable regions of the TCRα and β chains are SEQ ID NO:42 and 43, respectively. The amino acid sequences of the variable regions of the TCRα and β chains are shown in Table 6. The complementary determining regions (CDRs) are underlined.

[0152] Table 6

[0153]

[0154]

[0155] Example 2

[0156] This example confirms that the TCR isolated in Example 1 recognizes the KRAS G12V peptide antigen presented in the context of HLA-DR molecules.

[0157] The nucleic acid sequence encoding the isolated G12V-reactive TCR of Example 1 (comprising the nucleotide sequences of SEQ ID NO:42 and SEQ ID NO:43) and containing a cysteine-substituted, LVL-modified murine constant region was cloned into a retroviral expression vector. The murine constant region of the α-chain comprises the amino acid sequence of SEQ ID NO:30, 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 constant region of the β-chain comprises the amino acid sequence of SEQ ID NO:31, wherein X at position 57 is Cys. A linker comprising the amino acid sequence of SEQ ID NO:54 is positioned between the α-chain constant region and the β-chain variable region. Allogeneic T cells were transduced with the retroviral expression vector.

[0158] The transduced cells (effector cells) were co-cultured with target autologous antigen-presenting cells (APCs) pulsed with KRAS G12V peptide (1 ng / mL) and HLA-blocking antibodies W6 / 32 (anti-HLA-A, -B, -C), IVA12 (pan-specific, anti-HLA class II), B7 / 21 (anti-HLA-DP), HB55 (anti-HLA-DR), or SPV-L3 (HLA-DQ) (target cells). Effector transduced cells cultured alone or in the presence of DMSO or phorbol myristate acetate (PMA) were used as controls. Effector cells transduced with an empty vector (mock) and co-cultured with target autologous APCs pulsed with 1 ng / mL KRAS G12V peptide (SEQ ID NO:53) were used as another control.

[0159] The reactivity of the effector cells against the target cells was measured by 4-1BB expression detected using flow cytometry (gating on CD3+mTCRβ-chain+ cells). The results are shown in Figure 4 as Figure 4 shown, the IVA12 and HB55 antibodies blocked the reactivity of the effector cells against the target cells, indicating that the transduced effector cells recognize the KRAS G12V peptide antigen presented in the context of HLA-DR molecules.

[0160] Example 3

[0161] This example confirmed that the TCR of Example 2 recognizes the KRAS G12V peptide antigen presented in the context of HLA-DRB1*07:01 molecules.

[0162] Allogeneic T cells transduced with the TCR of Example 2 (effector cells) were co-cultured with autologous APCs from the patient of Example 1 or APCs from donors with the DRB101:01 or DRB107:01 haplotype (target cells). With KRAS G12VTarget cells were pulsed with the peptide (SEQ ID NO:53) or the WT KRAS peptide (SEQ ID NO:55). Effector cells were co-cultured with APCs from HLA-DRB1 positive donors (as controls), where one but not both donor alleles were DRB1*07:01 ("DRB mismatch"). Effector cells cultured alone, with DMSO, or with PMA-ionomycin were used as additional controls. IFN-γ secretion was measured by ELISPOT. The number of positive wells was counted. The results are shown in Table 7 and Figure 5 in. In Table 7, "TNTC" stands for "too numerous to count". The percentage of mTCR-expressing cells expressing 4-1BB was also measured by flow cytometry. The results are shown in Figure 5 in. The results show that the TCR has specific reactivity against mutant KRAS presented by HLA-DRB*07:01.

[0163] Table 7

[0164]

[0165] Example 4

[0166] This example confirmed the isolation of TCRs antigen-specific for human KRAS with a G12C mutation presented by HLA-DRB1*11:01 molecules.

[0167] Repeated in vitro sensitization (IVS) of a subset of peripheral blood T cells from an ovarian cancer patient with KRAS G12C -expressing tumors was used to identify KRAS G12C reactive TCRs.

[0168] Autologous DCs were pulsed with the G12C mutant peptide (SEQ ID NO:56) and co-cultured with the sorted T cell subset for 10 days, and then reactivity was tested as described in Example 1.

[0169] To further enrich for reactive cells, the reactive fraction against the KRAS mutant peptide was sorted based on 4-1BB / OX40 expression and re-stimulated with the mutant peptide. Reactive T cells were sorted based on 4-1BB / OX40 expression and sequenced.

[0170] Total RNA was isolated from leukocytes. Then, using TCR-α and -β chain constant primers, rapid amplification of the 5' complementary DNA end (5’RACE) of the total RNA was performed. The TCR PCR products were then separated by standard agarose gel electrophoresis and gel extraction. The products were directly sequenced. The nucleotide sequences of the variable regions of the TCRα and β chains are SEQ ID NO:44 and 45, respectively. The amino acid sequences of the variable regions of the TCRα and β chains are shown in Table 8. The complementarity-determining regions (CDRs) are underlined.

[0171] Table 8

[0172]

[0173] Example 5

[0174] This example confirmed that the TCR isolated in Example 4 recognizes the KRAS G12C peptide antigen presented in the context of HLA-DR molecules.

[0175] The nucleic acid sequence encoding the isolated G12C-reactive TCR of Example 4 (including the nucleotide sequences of SEQ ID NO:44 and SEQ ID NO:45) and containing cysteine substitutions and an LVL-modified murine constant region was cloned into a retroviral expression vector. The murine constant region of the α chain includes the amino acid sequence of SEQ ID NO:30, where X at position 48 is Cys, X at position 112 is Leu, X at position 114 is Ile, and X at position 115 is Val. The constant region of the β chain includes the amino acid sequence of SEQ ID NO:31, where X at position 57 is Cys. A linker including the amino acid sequence of SEQ ID NO:54 was positioned between the constant region of the α chain and the variable region of the β chain. Allogeneic T cells were transduced using the retroviral expression vector.

[0176] After blocking membrane MHC class II molecules using an antibody against HLA-DQ, HLA-DR, or HLA-DP or a pan-specific antibody against all of HLA-DP, HLA-DR, and HLA-DQ, the transduced cells (effector cells) were co-cultured with target autologous DCs or allogeneic DCs that were matched to a single HLA-DRB15:01 or HLA-DRB11:01 allele and pulsed with the KRAS G12C 24-mer peptide (SEQ ID NO:56). Effector transduced cells cultured with phorbol myristate acetate (PMA) or WT KRAS (SEQ ID NO:55) were used as controls.

[0177] The reactivity of the effector cells against the target cells was measured by 4-1BB expression detected using flow cytometry (gating on CD3+mTCRβ chain+ cells). The results are shown inFigure 10 In. As Figure 10 shown, the TCR has specific reactivity against KRAS presented by HLA-DRB*11:01. G12C

[0178] Example 6

[0179] This example confirmed that the KRAS G12C TCR-transduced PBMCs from Example 5 recognized autologous DCs pulsed with KRAS G12C peptides.

[0180] Allogeneic T cells were genetically modified with the MSGV-1-retrovirus encoding the KRAS G12C TCR of Example 5. Autologous DCs were loaded with WT KRAS (SEQ ID NO:55) or KRAS G12C peptides (SEQ ID NO:56) and co-cultured with the TCR-transduced cells for 18 hours, followed by analysis of 4-1BB upregulation using flow cytometry. The results are shown in Figure 9 In. As Figure 9 shown, the KRAS G12C TCR-transduced PBMCs from Example 5 recognized autologous DCs pulsed with KRAS G12C peptides.

[0181] Example 7

[0182] This example confirmed that the protein of the KRAS G12V mutation was processed and presented by DCs and recognized by the TCR of Example 2.

[0183] Allogeneic T cells transduced with G12V-DRB1*07:01 in Example 2 were co-cultured overnight with autologous DCs pulsed with cell lysates of tumor cell lines expressing one of the following KRAS G12 mutations: G12R, G12C, G12D, or G12V. Transduced cells co-cultured with autologous DCs pulsed with cell lysates of tumor cell lines expressing WT KRAS were used as controls. Transduced cells cultured alone or with PMA or DMSO were used as other controls. The percentage of cells upregulating 4-1BB and / or OX40 was measured by flow cytometry. The number of cells expressing IFNγ (spots / 2×10 4 cells) was measured by ELISPOT. The results are shown in Figure 6 In. As Figure 6 shown, the protein of the KRAS G12V mutation was processed and presented by DCs and recognized by the TCR of Example 2.

[0184] Example 8

[0185] ​This example confirmed that the cells transduced with the G12V-DRB1*07:01 TCR of Example 2 specifically recognized KRAS G12V peptide.

[0186] Allogeneic T cells transduced with the G12V-DRB1*07:01 TCR of Example 2 were co-cultured overnight with autologous DCs pulsed with various concentrations of the 24-mer peptide KRAS G12V (SEQ ID NO:53) or WT KRAS (SEQ ID NO:55). The percentage of mTCRβ+CD8+4-1BB+ cells was measured by flow cytometry. The results are shown in Figure 7 . As Figure 7 shown therein, the cells transduced with the G12V-DRB1*07:01 TCR of Example 2 specifically recognized the KRAS G12V peptide.

[0187] Example 9

[0188] The cells transduced with the G12V-DRB1*07:01 TCR of Example 2 specifically recognized various KRAS G12V peptides.

[0189] Allogeneic T cells transduced with the G12V-DRB1*07:01 TCR of Example 2 were co-cultured overnight with autologous DCs pulsed with various concentrations of the peptides listed in Table 9 below. IFNγ secretion was measured by ELISPOT. The results are shown in Figure 8 . As Figure 8 shown therein, although the cells transduced with the G12V-DRB1*07:01 TCR of Example 2 specifically recognized all KRAS G12V peptides, SEQ ID NO:52 was the best.

[0190] Table 9

[0191]

[0192]

[0193] All references cited herein (including publications, patent applications, and patents) are hereby incorporated by reference as if each reference were individually and specifically indicated to be incorporated by reference herein and were set forth in its entirety herein.

[0194] Unless the context clearly dictates otherwise or there is an obvious contradiction, in the context of describing the present invention (especially in the context of the following claims), the use of the terms "a", "an", "the", "at least one", and similar referents shall be construed to cover both the singular and the plural. Unless the context clearly dictates otherwise or there is an obvious contradiction, the term "at least one" (e.g., "at least one of A and B") used immediately following a list of one or more items shall be construed to mean either one of the listed items (A or B) or any combination of two or more of the listed items (A and B). Unless otherwise noted, the terms "comprising", "having", "including", and "containing" shall be construed as open-ended terms (i.e., meaning "including but not limited to"). Unless otherwise indicated herein, the recitation of numerical ranges herein is merely intended to be 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 the context clearly dictates otherwise or there is an obvious contradiction, all methods described herein may be performed in any suitable order. Unless otherwise claimed, the use of any and all examples or exemplary language (e.g., "such as") provided herein is merely intended 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 as essential to the practice of the invention.

[0195] Preferred embodiments of the invention are described herein, including the best mode known to the inventors for practicing the invention. Variations of these preferred embodiments will be apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to appropriately apply such variations, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, as permitted by applicable law, the invention includes all modifications and equivalent subjects of the subject matter recited in the appended claims. In addition, the invention includes any combination of the above elements in all possible variant forms, unless otherwise indicated herein or clearly contradicted by the context.

Claims

1. A method for preparing a cell or cell population expressing a T cell receptor (TCR), the method comprising: introducing a recombinant expression vector into a separated or purified human cell or separated or purified human cell population, wherein the recombinant expression vector comprises a nucleotide sequence encoding a TCR, wherein the TCR is antigen - specific for a mutant human RAS amino acid sequence presented by a human leukocyte antigen (HLA) class II molecule, wherein the mutant human RAS amino acid sequence is the amino acid sequence of mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS), the amino acid sequence of mutant human Harvey rat sarcoma viral oncogene homolog (HRAS), or the amino acid sequence of mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS), and wherein the TCR comprises: (a) the α - chain complementarity - determining region (CDR) 1 amino acid sequence of SEQ ID NO:1, the α - chain CDR2 amino acid sequence of SEQ ID NO:2, the α - chain CDR3 amino acid sequence of SEQ ID NO:3, the β - chain CDR1 amino acid sequence of SEQ ID NO:4, the β - chain CDR2 amino acid sequence of SEQ ID NO:5, and the β - chain CDR3 amino acid sequence of SEQ ID NO:6; or (b) the α - chain CDR1 amino acid sequence of SEQ ID NO:7, the α - chain CDR2 amino acid sequence of SEQ ID NO:8, the α - chain CDR3 amino acid sequence of SEQ ID NO:9, the amino acid sequence of the β - chain CDR1 of SEQ ID NO:10, the β - chain CDR2 amino acid sequence of SEQ ID NO:11, and the β - chain CDR3 amino acid sequence of SEQ ID NO:

12.

2. The method according to claim 1, wherein the HLA class II molecule is an HLA - DR molecule.

3. The method according to claim 1, wherein the HLA class II molecule is an HLA - DRB1 molecule.

4. The method according to claim 1, wherein the HLA class II molecule is an HLA - DRB1*07:01 molecule or an HLA - DRB1*11:01 molecule.

5. The method according to claim 1, wherein the mutant human RAS amino acid sequence comprises a wild - type human KRAS, wild - type human HRAS, or wild - type human NRAS amino acid sequence with glycine substituted at position 12, wherein position 12 is defined by reference to the wild - type human KRAS, wild - type human HRAS, or wild - type human NRAS protein, respectively.

6. The method according to claim 5, wherein the substitution is substituting glycine at position 12 with valine or cysteine.

7. The method according to any one of claims 1 to 6, wherein the TCR 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; or (v) both (i) and (ii); or both (iii) and (iv).

8. The method according to any one of claims 1 to 6, wherein the TCR further comprises: (a) an α-chain constant region comprising the amino acid sequence of SEQ ID NO: 30, wherein: (i) X at position 48 of SEQ ID NO: 30 is Thr or Cys; (ii) X at position 112 of SEQ ID NO: 30 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 114 of SEQ ID NO: 30 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 115 of SEQ ID NO: 30 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) a β-chain constant region comprising the amino acid sequence of SEQ ID NO: 31, wherein X at position 57 of SEQ ID NO: 31 is Ser or Cys; or (c) both (a) and (b).

9. The method according to any one of claims 1 to 6, wherein the TCR comprises: (a) an α-chain, the α-chain comprising the amino acid sequence of SEQ ID NO: 34, wherein: (i) X at position 179 of SEQ ID NO: 34 is Thr or Cys; (ii) X at position 243 of SEQ ID NO: 34 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 245 of SEQ ID NO: 34 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 246 of SEQ ID NO: 34 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) a β-chain, the β-chain comprising the amino acid sequence of SEQ ID NO: 35, wherein X at position 189 of SEQ ID NO: 35 is Ser or Cys; (c) an α-chain, the α-chain comprising the amino acid sequence of SEQ ID NO: 36, wherein: (i) X at position 180 of SEQ ID NO: 36 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 36 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 246 of SEQ ID NO: 36 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 247 of SEQ ID NO:36 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (d) a β-chain comprising the amino acid sequence of SEQ ID NO:37, wherein X at position 194 of SEQ ID NO:37 is Ser or Cys; or (e) both (a) and (b); or both (c) and (d).

10. A method for preparing a cell or cell population that expresses a polypeptide, the method comprising: introducing a recombinant expression vector into an isolated or purified human cell or an isolated or purified human cell population, wherein the recombinant expression vector comprises a nucleotide sequence encoding a polypeptide comprising a functional portion of a T cell receptor (TCR), wherein the functional portion is antigen-specific for a mutant human RAS amino acid sequence presented by a human leukocyte antigen (HLA) class II molecule, wherein the mutant human RAS amino acid sequence is the amino acid sequence of mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS), the amino acid sequence of mutant human Harvey rat sarcoma viral oncogene homolog (HRAS), or the amino acid sequence of mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS), and wherein the functional portion comprises: (a) a TCR α-chain sequence and a TCR β-chain sequence, the TCR α-chain sequence comprising the TCR α-chain complementarity determining region (CDR) 1 amino acid sequence of SEQ ID NO:1, the TCR α-chain CDR2 amino acid sequence of SEQ ID NO:2, and the TCR α-chain CDR3 amino acid sequence of SEQ ID NO:3; the TCR β-chain sequence comprising the TCR β-chain CDR1 amino acid sequence of SEQ ID NO:4, the TCR β-chain CDR2 amino acid sequence of SEQ ID NO:5, and the TCR β-chain CDR3 amino acid sequence of SEQ ID NO:6; or (b) a TCR α-chain sequence and a TCR β-chain sequence, the TCR α-chain sequence comprising the TCR α-chain CDR1 amino acid sequence of SEQ ID NO:7, the TCR α-chain CDR2 amino acid sequence of SEQ ID NO:8, and the TCR α-chain CDR3 amino acid sequence of SEQ ID NO:9; the TCR β-chain sequence comprising the amino acid sequence of TCR β-chain CDR1 of SEQ ID NO:10, the TCR β-chain CDR2 amino acid sequence of SEQ ID NO:11, and the TCR β-chain CDR3 amino acid sequence of SEQ ID NO:

12.

11. The method according to claim 10, wherein the functional portion 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; or (v) both (i) and (ii); or both (iii) and (iv).

12. The method according to claim 11, wherein the functional portion further comprises: (a) the amino acid sequence of SEQ ID NO:30, wherein: (i) X at position 48 of SEQ ID NO:30 is Thr or Cys; (ii) X at position 112 of SEQ ID NO:30 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 114 of SEQ ID NO:30 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 115 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 57 of SEQ ID NO:31 is Ser or Cys; or (c) both (a) and (b).

13. The method according to claim 11, wherein the polypeptide comprises: (a) the amino acid sequence of SEQ ID NO:34, wherein: (i) X at position 179 of SEQ ID NO:34 is Thr or Cys; (ii) X at position 243 of SEQ ID NO:34 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 245 of SEQ ID NO:34 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 246 of SEQ ID NO:34 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) the amino acid sequence of SEQ ID NO:35, wherein X at position 189 of SEQ ID NO:35 is Ser or Cys; (c) the amino acid sequence of SEQ ID NO:36, wherein: (i) X at position 180 of SEQ ID NO:36 is Thr or Cys; (ii) X at position 244 of SEQ ID NO:36 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 246 of SEQ ID NO:36 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 247 of SEQ ID NO:36 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (d) the amino acid sequence of SEQ ID NO:37, wherein X at position 194 of SEQ ID NO:37 is Ser or Cys; or (e) both (a) and (b); or both (c) and (d).

14. A method for preparing a cell or cell population expressing a protein, the method comprising: introducing a recombinant expression vector into an isolated or purified human cell or an isolated or purified human cell population, wherein the recombinant expression vector comprises a nucleotide sequence encoding a protein, wherein the protein is antigen - specific for a mutant human RAS amino acid sequence presented by human leukocyte antigen (HLA) class II molecules, wherein the mutant human RAS amino acid sequence is the amino acid sequence of mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS), the amino acid sequence of mutant human Harvey rat sarcoma viral oncogene homolog (HRAS), or the amino acid sequence of mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS), and wherein the protein comprises: (a) a TCRα chain and a TCRβ chain, the TCRα chain comprising the TCRα - chain complementarity - determining region (CDR) 1 amino acid sequence of SEQ ID NO:1, the TCRα - chain CDR2 amino acid sequence of SEQ ID NO:2, and the TCRα - chain CDR3 amino acid sequence of SEQ ID NO:3; the TCRβ chain comprising the TCRβ - chain CDR1 amino acid sequence of SEQ ID NO:4, the TCRβ - chain CDR2 amino acid sequence of SEQ ID NO:5, and the TCRβ - chain CDR3 amino acid sequence of SEQ ID NO:6; or (b) a TCRα chain and a TCRβ chain, the TCRα chain comprising the TCRα - chain CDR1 amino acid sequence of SEQ ID NO:7, the TCRα - chain CDR2 amino acid sequence of SEQ ID NO:8, and the TCRα - chain CDR3 amino acid sequence of SEQ ID NO:9; the TCRβ chain comprising the TCRβ - chain CDR1 amino acid sequence of SEQ ID NO:10, the TCRβ - chain CDR2 amino acid sequence of SEQ ID NO:11, and the TCRβ - chain CDR3 amino acid sequence of SEQ ID NO:

12.

15. The method according to claim 14, wherein: (i) the TCRα chain comprises the amino acid sequence of SEQ ID NO:13, and the TCRβ chain comprises the amino acid sequence of SEQ ID NO:14; or (ii) the TCRα - chain sequence comprises the amino acid sequence of SEQ ID NO:15, and the TCRβ chain comprises the amino acid sequence of SEQ ID NO:

16.

16. The method according to claim 14, wherein: (a) the TCRα chain further comprises the amino acid sequence of SEQ ID NO:30, wherein: (i) X at position 48 of SEQ ID NO:30 is Thr or Cys; (ii) X at position 112 of SEQ ID NO:30 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii)X at position 114 of SEQ ID NO:30 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv)X at position 115 of SEQ ID NO:30 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) the TCR β chain further comprises the amino acid sequence of SEQ ID NO:31, wherein X at position 57 of SEQ ID NO:31 is Ser or Cys; or (c) both (a) and (b).

17. The method according to claim 14, wherein: (a) the TCR α chain comprises the amino acid sequence of SEQ ID NO:34, wherein: (i)X at position 179 of SEQ ID NO:34 is Thr or Cys; (ii)X at position 243 of SEQ ID NO:34 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii)X at position 245 of SEQ ID NO:34 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv)X at position 246 of SEQ ID NO:34 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) the TCR β chain comprises the amino acid sequence of SEQ ID NO:35, wherein X at position 189 of SEQ ID NO:35 is Ser or Cys; (c) the TCR α chain comprises the amino acid sequence of SEQ ID NO:36, wherein: (i)X at position 180 of SEQ ID NO:36 is Thr or Cys; (ii)X at position 244 of SEQ ID NO:36 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii)X at position 246 of SEQ ID NO:36 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv)X at position 247 of SEQ ID NO:36 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (d) the TCR β chain comprises the amino acid sequence of SEQ ID NO:37, wherein X at position 194 of SEQ ID NO:37 is Ser or Cys; or (e) both (a) and (b); or both (c) and (d).

18. The following uses in the preparation of a medicament for inducing an immune response against cancer in a mammal: (i) a T cell receptor (TCR), (ii) a nucleic acid comprising a nucleotide sequence encoding the TCR, (iii) a recombinant expression vector comprising the nucleic acid, (iv) a host cell comprising the recombinant expression vector, or (v) A cell population comprising the host cell, wherein the TCR is antigen - specific for a mutant human RAS amino acid sequence presented by a human leukocyte antigen (HLA) class II molecule, wherein the mutant human RAS amino acid sequence is the amino acid sequence of mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS), mutant human Harvey rat sarcoma viral oncogene homolog (HRAS), or mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS), and wherein the TCR comprises: (a) The α - chain complementarity - determining region (CDR) 1 amino acid sequence of SEQ ID NO:1, the α - chain CDR2 amino acid sequence of SEQ ID NO:2, the α - chain CDR3 amino acid sequence of SEQ ID NO:3, the β - chain CDR1 amino acid sequence of SEQ ID NO:4, the β - chain CDR2 amino acid sequence of SEQ ID NO:5, and the β - chain CDR3 amino acid sequence of SEQ ID NO:6; or (b) The α - chain CDR1 amino acid sequence of SEQ ID NO:7, the α - chain CDR2 amino acid sequence of SEQ ID NO:8, the α - chain CDR3 amino acid sequence of SEQ ID NO:9, the β - chain CDR1 amino acid sequence of SEQ ID NO:10, the β - chain CDR2 amino acid sequence of SEQ ID NO:11, and the β - chain CDR3 amino acid sequence of SEQ ID NO:

12.

19. The use according to claim 18, wherein the HLA class II molecule is an HLA - DR molecule.

20. The use according to claim 18, wherein the HLA class II molecule is an HLA - DRB1 molecule.

21. The use according to claim 18, wherein the HLA class II molecule is an HLA - DRB1*07:01 molecule or an HLA - DRB1*11:01 molecule.

22. The method according to claim 18, wherein the mutant human RAS amino acid sequence comprises a wild - type human KRAS, wild - type human HRAS, or wild - type human NRAS amino acid sequence with glycine substituted at position 12, where position 12 is defined by reference to the wild - type human KRAS, wild - type human HRAS, or wild - type human NRAS protein, respectively.

23. The use according to claim 22, wherein the substitution is the replacement of glycine at position 12 with valine or cysteine.

24. The use according to any one of claims 18 to 23, wherein the TCR 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; or (v) Both (i) and (ii); or both (iii) and (iv).

25. The use according to any one of claims 18 to 23, wherein the TCR further comprises: (a) an α-chain constant region comprising the amino acid sequence of SEQ ID NO: 30, wherein: (i) X at position 48 of SEQ ID NO: 30 is Thr or Cys; (ii) X at position 112 of SEQ ID NO: 30 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 114 of SEQ ID NO: 30 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 115 of SEQ ID NO: 30 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) a β-chain constant region comprising the amino acid sequence of SEQ ID NO: 31, wherein X at position 57 of SEQ ID NO: 31 is Ser or Cys; or (c) both (a) and (b).

26. The use according to any one of claims 18 to 23, wherein the TCR comprises: (a) an α-chain, the α-chain comprising the amino acid sequence of SEQ ID NO: 34, wherein: (i) X at position 179 of SEQ ID NO: 34 is Thr or Cys; (ii) X at position 243 of SEQ ID NO: 34 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 245 of SEQ ID NO: 34 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 246 of SEQ ID NO: 34 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) a β-chain, the β-chain comprising the amino acid sequence of SEQ ID NO: 35, wherein X at position 189 of SEQ ID NO: 35 is Ser or Cys; (c) an α-chain, the α-chain comprising the amino acid sequence of SEQ ID NO: 36, wherein: (i) X at position 180 of SEQ ID NO: 36 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 36 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 246 of SEQ ID NO: 36 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 247 of SEQ ID NO: 36 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (d) a β-chain, said β-chain comprising the amino acid sequence of SEQ ID NO:37, wherein X at position 194 of SEQ ID NO:37 is Ser or Cys; or (e) both (a) and (b); or both (c) and (d).

27. Use in the preparation of a medicament for inducing an immune response against cancer in a mammal: (i) a polypeptide comprising a functional portion of a T cell receptor (TCR), (ii) a nucleic acid comprising a nucleotide sequence encoding said polypeptide, (iii) a recombinant expression vector comprising said nucleic acid, (iv) a host cell comprising said recombinant expression vector, or (v) a cell population comprising said host cell, wherein said functional portion is antigen-specific for a mutant human RAS amino acid sequence presented by a human leukocyte antigen (HLA) class II molecule, wherein said mutant human RAS amino acid sequence is the amino acid sequence of mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS), the amino acid sequence of mutant human Harvey rat sarcoma viral oncogene homolog (HRAS), or the amino acid sequence of mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS), and wherein said functional portion comprises: (a) a TCR α-chain sequence and a TCR β-chain sequence, said TCR α-chain sequence comprising the TCR α-chain complementarity determining region (CDR) 1 amino acid sequence of SEQ ID NO:1, the TCR α-chain CDR2 amino acid sequence of SEQ ID NO:2, and the TCR α-chain CDR3 amino acid sequence of SEQ ID NO:3; said TCR β-chain sequence comprising the TCR β-chain CDR1 amino acid sequence of SEQ ID NO:4, the TCR β-chain CDR2 amino acid sequence of SEQ ID NO:5, and the TCR β-chain CDR3 amino acid sequence of SEQ ID NO:6; or (b) a TCR α-chain sequence and a TCR β-chain sequence, said TCR α-chain sequence comprising the TCR α-chain CDR1 amino acid sequence of SEQ ID NO:7, the TCR α-chain CDR2 amino acid sequence of SEQ ID NO:8, and the TCR α-chain CDR3 amino acid sequence of SEQ ID NO:9; said TCR β-chain sequence comprising the amino acid sequence of TCR β-chain CDR1 of SEQ ID NO:10, the TCR β-chain CDR2 amino acid sequence of SEQ ID NO:11, and the TCR β-chain CDR3 amino acid sequence of SEQ ID NO:

12.

28. The use according to claim 27, wherein said functional portion 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; or (v) both (i) and (ii); or both (iii) and (iv).

29. The use according to claim 27, wherein the functional part further comprises: (a) The amino acid sequence of SEQ ID NO: 30, wherein: (i) X at position 48 of SEQ ID NO: 30 is Thr or Cys; (ii) X at position 112 of SEQ ID NO: 30 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 114 of SEQ ID NO: 30 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 115 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 57 of SEQ ID NO: 31 is Ser or Cys; or (c) Both (a) and (b).

30. The use according to claim 28, wherein the polypeptide comprises: (a) The amino acid sequence of SEQ ID NO: 34, wherein: (i) X at position 179 of SEQ ID NO: 34 is Thr or Cys; (ii) X at position 243 of SEQ ID NO: 34 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 245 of SEQ ID NO: 34 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 246 of SEQ ID NO: 34 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) The amino acid sequence of SEQ ID NO: 35, wherein X at position 189 of SEQ ID NO: 35 is Ser or Cys; (c) The amino acid sequence of SEQ ID NO: 36, wherein: (i) X at position 180 of SEQ ID NO: 36 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 36 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 246 of SEQ ID NO: 36 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 247 of SEQ ID NO: 36 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (d) The amino acid sequence of SEQ ID NO: 37, wherein X at position 194 of SEQ ID NO: 37 is Ser or Cys; or (e) Both (a) and (b); or both (c) and (d).

31. Use in the preparation of a medicament for inducing an immune response against cancer in a mammal: (i) a protein, (ii) a nucleic acid comprising a nucleotide sequence encoding said protein, (iii) a recombinant expression vector comprising said nucleic acid, (iv) a host cell comprising said recombinant expression vector, or (v) a cell population comprising said host cell, wherein said protein is antigen - specific for a mutant human RAS amino acid sequence presented by a human leukocyte antigen (HLA) class II molecule, wherein said mutant human RAS amino acid sequence is the amino acid sequence of mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS), the amino acid sequence of mutant human Harvey rat sarcoma viral oncogene homolog (HRAS), or the amino acid sequence of mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS), and wherein said protein comprises: (a) a TCRα chain and a TCRβ chain, wherein the TCRα chain comprises the TCRα chain complementarity - determining region (CDR) 1 amino acid sequence of SEQ ID NO:1, the TCRα chain CDR2 amino acid sequence of SEQ ID NO:2, and the TCRα chain CDR3 amino acid sequence of SEQ ID NO:3; the TCRβ chain comprises the TCRβ chain CDR1 amino acid sequence of SEQ ID NO:4, the TCRβ chain CDR2 amino acid sequence of SEQ ID NO:5, and the TCRβ chain CDR3 amino acid sequence of SEQ ID NO:6; or (b) a TCRα chain and a TCRβ chain, wherein the TCRα chain comprises the TCRα chain CDR1 amino acid sequence of SEQ ID NO:7, the TCRα chain CDR2 amino acid sequence of SEQ IDNO:8, and the TCRα chain CDR3 amino acid sequence of SEQ ID NO:9; the TCRβ chain comprises the amino acid sequence of TCRβ chain CDR1 of SEQ ID NO:10, the TCRβ chain CDR2 amino acid sequence of SEQ ID NO:11, and the TCRβ chain CDR3 amino acid sequence of SEQ ID NO:

12.

32. The use according to claim 31, wherein: (i) the TCRα chain comprises the amino acid sequence of SEQ ID NO:13, and the TCRβ chain comprises the amino acid sequence of SEQ ID NO:14; or (ii) the TCRα chain sequence comprises the amino acid sequence of SEQ ID NO:15, and the TCRβ chain comprises the amino acid sequence of SEQ IDNO:

16.

33. The use according to claim 31, wherein: (a) the TCRα chain further comprises the amino acid sequence of SEQ ID NO:30, wherein: (i) X at position 48 of SEQ ID NO:30 is Thr or Cys; (ii) The X at position 112 of SEQ ID NO:30 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) The X at position 114 of SEQ ID NO:30 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) The X at position 115 of SEQ ID NO:30 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) The TCRβ chain further comprises the amino acid sequence of SEQ ID NO:31, wherein the X at position 57 of SEQ ID NO:31 is Ser or Cys; or (c) Both (a) and (b).

34. The use according to claim 31, wherein: (a) The TCRα chain comprises the amino acid sequence of SEQ ID NO:34, wherein: (i) The X at position 179 of SEQ ID NO:34 is Thr or Cys; (ii) The X at position 243 of SEQ ID NO:34 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) The X at position 245 of SEQ ID NO:34 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) The X at position 246 of SEQ ID NO:34 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) The TCRβ chain comprises the amino acid sequence of SEQ ID NO:35, wherein the X at position 189 of SEQ ID NO:35 is Ser or Cys; (c) The TCRα chain comprises the amino acid sequence of SEQ ID NO:36, wherein: (i) The X at position 180 of SEQ ID NO:36 is Thr or Cys; (ii) The X at position 244 of SEQ ID NO:36 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) The X at position 246 of SEQ ID NO:36 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) The X at position 247 of SEQ ID NO:36 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (d) The TCRβ chain comprises the amino acid sequence of SEQ ID NO:37, wherein the X at position 194 of SEQ ID NO:37 is Ser or Cys; or (e) Both (a) and (b); or both (c) and (d).

35. The use according to any one of claims 18 - 34, wherein the mutated human RAS amino acid sequence comprises a wild - type human KRAS, wild - type human HRAS or wild - type human NRAS amino acid sequence with glycine substituted at position 12, wherein position 12 is defined by reference to the wild - type human KRAS, wild - type human HRAS or wild - type human NRAS amino acid sequence, respectively.

36. The use according to claim 35, wherein the substitution is substitution of glycine at position 12 with valine or cysteine.

37. The use according to any one of claims 18 - 36, wherein the cancer is pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer or prostate cancer.

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