HLA class II-restricted T cell receptor targeting mutant RAS

Abstract

Disclosed are isolated or purified T cell receptors (TCRs), wherein the TCRs have antigenic specificity for a mutated Kirsten rat sarcoma viral oncogene homolog (KRAS) presented by a human leukocyte antigen (HLA) class II molecule. Also provided are related polypeptides and proteins, as well as related nucleic acids, recombinant expression vectors, host cells, cell populations, and pharmaceutical compositions. Also disclosed are methods for detecting the presence of cancer in mammals and methods for treating or preventing cancer in mammals.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] 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.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This invention was made with government support through the National Cancer Institute of the National Institutes of Health under Grant No. BC010984. The government has certain rights in this invention.

[0005] Electronically Submitted Materials Incorporated by Reference

[0006] Incorporated herein by reference in its entirety is a computer-readable nucleotide / amino acid sequence listing filed concurrently with this document and identified as follows: a 59,753-byte ASCII (text) file named "739664_ST25.txt" and dated September 5, 2018. Background of the Invention

[0008] Some cancers may have very limited treatment options, especially when the cancer becomes metastatic and unresectable. Despite advances in treatments such as surgery, chemotherapy, and radiation therapy, the prognosis for many cancers (e.g., pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer, and prostate cancer) may be poor. Therefore, there is an unmet need for additional treatments for cancer.

[0009] Brief description of the invention

[0010] Embodiments of the present invention provide an isolated or purified T cell receptor (TCR), wherein the TCR has antigenic specificity for a mutated human Ras amino acid sequence presented by a human leukocyte antigen (HLA) class II molecule, wherein the mutated human RAS amino acid sequence is a mutated human Kirsten rat sarcoma viral oncogene homolog (KRAS) amino acid sequence, a mutated human Harvey rat sarcoma viral oncogene homolog (HRAS) amino acid sequence, or a mutated human neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence.

[0011] Another embodiment of the present invention provides an isolated or purified polypeptide comprising a functional portion of the TCR of the present invention, wherein the functional portion comprises the following amino acid sequence: (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.

[0012] Yet another embodiment of the present invention provides an isolated or purified protein comprising at least one polypeptide of the present invention.

[0013] 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.

[0014] Embodiments of the invention also provide methods of detecting the presence of cancer in a mammal and methods of treating or preventing cancer in a mammal.

[0015] Brief description of several views in the accompanying drawings

[0016] Figure 1 Depicts experimental data (dot plots) of cells stained for isotype (control) or for PD-1 and / or OX40 expression by flow cytometry. Values ​​in the histogram represent the percentage of cells expressing PD-1.

[0017] Figure 2 Graph showing the number of interferon gamma (IFNg) positive spots / well detected when pooled cultures of effector autologous T cells (culture numbers W1-W16) were co-cultured with target DC pulsed with pools of indicated 25-mer peptides (PP) or pools of peptides encoded by 25-mer tandem minigenes (TMG) covering various tumor-specific mutations. Autologous T cells cultured alone, with dimethyl sulfoxide (DMSO) or with OKT3 antibody were used as controls. Boxed symbols (▼) indicate pooled cultures of isolated TCRs (7 and 8).

[0018] Figure 3 is the number of IFNγ positive spots / 2×10 4 (2E4) Graph of cells detected when autologous T cells of culture number 7 (W7) were co-cultured with autologous DC pulsed with each of 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.

[0019] Figure 4: is a graph showing the percentage of effector T cells transduced by the TCR expressing 4-1BB in Example 2 after co-culture with target autologous APCs 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, with DMSO or phorbol myristate acetate (PMA) were used as controls. Effector cells transduced with empty vector (mock) co-cultured with target autologous APCs (pulsed with 1 ng / mL KRAS G12V peptide) were used as another control.

[0020] Figure 5 is a graph showing the following results: (i) IFN-γ counts measured by ELISPOT / 2×10 4 cells; and (ii) the percentage of mTCRβ+CD8+4-1BB+ cells measured by flow cytometry, both of which were obtained by combining T cells transduced with the TCR of Example 2 with autologous APCs (4148MB) or T cells from donors with DRB1 01:01 or DRB1 07:01 haplotypes and mutated with KRAS G12V Effector cells were co-cultured with APCs from HLA-DRB1 positive donors ("DRB mismatch") as controls. Effector cells cultured alone, with DMSO or with phorbol myristate acetate-ionomycin (PMA:Iono) were used as additional controls.

[0021] Figure 6 is a graph showing the following results: (i) IFN-γ counts measured by ELISPOT / 2×10 4 Cells (hatched bars); and (ii) the percentage of cells expressing 4-1BB and / or OX40 measured by flow cytometry (black bars), all of which were measured when T cells transduced by the TCR of Example 2 were co-cultured 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.

[0022] Figure 7is a graph showing the percentage of mTCRβ+CD8+4-1BB+ cells measured by flow cytometry after T cells transduced with the TCR of Example 2 were co-cultured with KRAS at the indicated concentrations. G12V Measured during overnight co-culture of autologous DCs pulsed with either peptide (triangles) or WT KRAS peptide (squares).

[0023] Figure 8 The number of IFN-γ measured by ELISPOT / 2×10 4 Figure 2 is a graph of cells measured when T cells transduced with the TCR of Example 2 were co-cultured with autologous DC pulsed with the indicated concentrations of the peptides in Table 9.

[0024] Fig. 9 Experimental data (dot plot) depicting the percentage of cells expressing the murine TCRβ chain and 4-1BB in the presence of a KRAS-encoding G12C MSGV-1-retrovirally transduced cells of TCR were treated with DMSO (control) or loaded with the indicated WT KRAS or KRAS at the indicated concentrations. G12C Peptide DC co-culture was measured. 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 is shown as follows (percentages in brackets): 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 10μg / ml: Q1 (13.6), Q2 (51.7), Q3 (1.61), Q4 (33.0). G12C 1μg / ml: Q1 (19.7), Q2 (46.9), Q3 (1.67), Q4 (31.7).

[0025] Fig.10 is a graph showing the percentage of cells expressing CD3 and 4-1BB after blocking membrane MHC-II molecules using antibodies against HLA-DQ, DR, DP or antibodies against all HLA-DQ, DR and DP. G12C TCR-transduced T cells were matched with a single HLA-DRB15:01 or HLA-DRB11:01 allele and expressed with KRAS G12CThe assay was performed after co-culture with autologous DC or allogeneic DC pulsed with 24-mer peptide. Transduced cells co-cultured with DC pulsed with WT KRAS peptide served as a control. Transduced cells co-cultured with PMA / ion served as another control. DETAILED DESCRIPTION OF THE INVENTION

[0027] RAS family proteins belong to a large family of small GTPases. Without being limited to a particular theory or mechanism, it is believed that, when mutated, RAS proteins can participate in the early signal transduction of tumor formation in many human cancers. Single amino acid substitutions can activate proteins. Mutated RAS protein products can be constitutively activated. Mutated RAS proteins can be expressed in any of various human cancers (e.g., pancreatic cancer (pancreatic cancer) (e.g., pancreatic cancer (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).

[0028] 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, reference to "KRAS" (mutated or unmutated (WT)) refers to variant A and variant B unless otherwise specified. Upon activation, mutant KRAS binds guanosine-5'-triphosphate (GTP) and converts GTP to guanosine-5'-diphosphate (GDP).

[0029] HRAS is another member of the RAS protein family. HRAS is also known as Harvey rat sarcoma viral oncoprotein, V-Ha-Ras Harvey rat sarcoma viral oncogene homolog or Ras family small GTP binding protein H-Ras. WT HRAS has the amino acid sequence of SEQ ID NO: 19.

[0030] NRAS is 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.

[0031] Embodiments of the present invention provide an isolated or purified TCR that has antigenic specificity for a mutant human RAS amino acid sequence (hereinafter "mutated RAS") presented by a human leukocyte antigen (HLA) class II molecule, wherein the mutant human RAS amino acid sequence is a mutant human KRAS, a mutant human HRAS or a mutant human NRAS amino acid sequence. In the following, unless otherwise specified, the "TCR" referred to also refers to the functional portion and functional variant of the TCR.

[0032] The TCR of the present invention may have antigenic specificity for any mutated human RAS protein, polypeptide or peptide amino acid sequence. In an embodiment of the present invention, the mutated human RAS amino acid sequence is a mutated human KRAS amino acid sequence, a mutated human HRAS amino acid sequence or a mutated 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 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 WT human KRAS protein. The amino acid residues 1-94 of WT human HRAS protein and WT human KRAS protein are 100% identical. In the following, unless otherwise specified, the "RAS" (mutated or unmutated (WT)) mentioned are collectively referred to as KRAS, HRAS and NRAS.

[0033] In an embodiment of the present invention, the mutant human RAS amino acid sequence comprises a WT RAS amino acid sequence in which glycine at position 12 is substituted, wherein position 12 is defined by reference to a WT RAS protein, respectively. The WT RAS protein may be any one of a WT KRAS protein (SEQ ID NO: 17 or 18), a WT HRAS protein (SEQ ID NO: 19), or a WT NRAS protein (SEQ ID NO: 20), because as explained above, amino acid residues 1-86 of the WT human NRAS protein and the WT human KRAS protein are 100% identical, and amino acid residues 1-94 of the WT human HRAS protein and the WT human KRAS protein are 100% identical. Therefore, the amino acid residue at position 12 of each of the WT KRAS, WT HRAS, and WT NRAS proteins is the same, i.e., glycine.

[0034] The glycine at position 12 of the WT RAS amino acid sequence can be substituted with any amino acid residue other than glycine. In an embodiment of the present invention, the substitution is to replace 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 with antigenic specificity for any WT RAS protein, polypeptide or peptide amino acid sequence having a G12V mutation or a G12C mutation.

[0035] In this article, the mutation and substitution of RAS are defined by reference to the amino acid sequence of WT RAS protein. Therefore, in this article, the mutation and substitution of RAS are described by reference to the amino acid residue present in a specific position in the WT RAS protein, followed by the position number, followed by the amino acid residue replacing the residue with the specific mutation or substitution discussed. A RAS amino acid sequence (such as a RAS peptide) may include amino acid residues less than all amino acid residues of the full-length WT RAS protein. Therefore, in this article, position 12 is defined by reference to the WT full-length RAS protein (i.e., any one of SEQ ID NO: 17-20), and it should be understood that the actual position of the corresponding residue in the specific example of the RAS amino acid sequence may be different. When the position is as defined by any one of SEQ ID NO: 17-20, the term "G12" refers to the glycine at position 12 of any one of SEQ ID NO: 17-20, and "G12V" indicates that the glycine at position 12 of any one of SEQ ID NO: 17-20 is replaced by valine. For example, in a specific example of a RAS amino acid sequence, for example, TEYKLVVVGA G In the case of 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" means that the underlined glycine in SEQ ID NO:29 is replaced by valine, even though the actual position of the underlined glycine in SEQ ID NO:29 is 11.

[0036] Examples of full-length RAS proteins with G12V or G12C mutations are shown in Table 1 below.

[0037] Table 1

[0038] 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

[0039] In an embodiment of the present invention, TCR has antigenic specificity to a RAS peptide having the above-mentioned G12V mutation or G12C mutation, wherein the mutated RAS peptide has any length. In an embodiment of the present invention, the mutated RAS peptide has any length suitable for binding to any HLA class II molecule described herein. For example, TCR may have antigenic specificity to a RAS peptide having a G12V mutation or a G12C mutation, and the RAS peptide has a length of about 11 to about 30 amino acid residues, about 12 to about 24 amino acid residues, or about 18 to about 20 amino acid residues. The mutated RAS peptide may include any continuous amino acid residues of a mutated RAS protein comprising a G12V or G12C mutation. In embodiments of the invention, the TCR may have antigenic specificity for a RAS peptide having a G12V mutation or a G12C mutation having a length of about 30 amino acid residues, about 29 amino acid residues, about 28 amino acid residues, about 27 amino acid residues, about 26 amino acid residues, about 25 amino acid residues, about 24 amino acid residues, about 23 amino acid residues, about 22 amino acid residues, about 21 amino acid residues, about 20 amino acid residues, about 19 amino acid residues, about 18 amino acid residues, about 17 amino acid residues, about 16 amino acid residues, about 15 amino acid residues, about 14 amino acid residues, about 13 amino acid residues, about 12 amino acid residues, about 11 amino acid residues, or a range of any two of the foregoing values. Examples of specific peptides each having a G12V mutation that can be recognized by the G12V TCR of the invention are shown in Table 9.

[0040] In an embodiment of the present invention, the TCR of the present invention is capable of recognizing a mutated RAS presented by an HLA class II molecule. In this regard, the TCR can elicit an immune response when binding to a mutated RAS within the context of an HLA class II molecule. The TCR of the present invention is capable of recognizing a mutated RAS presented by an HLA class II molecule and can bind to an HLA class II molecule in addition to the mutated RAS.

[0041] 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-DR α chain can be encoded by the HLA-DRA gene. The HLA-DR β 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 HLA-DR molecules may include, but are not limited to, HLA-DR1, HLA-DR2, HLA-DR3, HLA-DR4, HLA-DR5, HLA-DR6, HLA-DR7, HLA-DR8, HLA-DR9, HLA-DR10, HLA-DR11, HLA-DR12, HLA-DR13, HLA-DR14, HLA-DR15, and HLA-DR16. Preferably, the HLA-DR molecule is HLA-DR7 or HLA-DR11.

[0042] In an embodiment of the present invention, the HLA class II molecule is an HLA-DRB1 molecule. The HLA-DRB1 molecule may be any HLA-DRB1 molecule. Examples of HLA-DRB1 molecules may 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, HL A-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.

[0043] The TCRs of the present invention may provide any one or more of a number of advantages, including use for adoptive cell transfer when expressed by cells. Mutated RAS is expressed by cancer cells and is not expressed 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). In addition, the TCRs of the present invention may advantageously and successfully treat or prevent mutated 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 pancreatic cancer and colorectal cancer patients, respectively, and the KRAS G12C mutation is expressed in approximately 15% of lung cancer patients. In addition, the TCR of the present invention can provide high affinity recognition of mutant RAS, which can provide the ability to recognize untreated tumor cells (e.g., tumor cells that have not been treated with interferon (IFN)-γ, transfected with vectors encoding one or both of mutant RAS and HLA-DRB1*07:01, one or both of 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). In addition, in the United States, HLA-DRB1*07:01 and HLA-DRB1*11:01 alleles are expressed in approximately 25% and approximately 10.5% of individuals of Caucasian ethnicity, respectively. Thus, the TCRs of the 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 amenable to immunotherapy using TCRs that recognize RAS presented by other MHC molecules.

[0044] As used herein, the phrase "antigen specificity" means that the TCR is able to specifically bind to and immunologically recognize a mutant RAS with high affinity. For example, if about 1×10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 4 About 1×10 5If a T cell expressing a TCR secretes at least about 200 pg / mL or more (e.g., 200 pg / mL or more, 300 pg / mL or more, 400 pg / mL or more, 500 pg / mL or more, 600 pg / mL or more, 700 pg / mL or more, 1000 pg / mL or more, 5,000 pg / mL or more, 7,000 pg / mL or more, 10,000 pg / mL or more, 20,000 pg / mL or more, or a range defined by any two of the foregoing values) of IFN-γ, the TCR can be considered to be "antigen-specific" for mutant RAS. Cells expressing the TCR of the invention can also secrete IFN-γ when co-cultured with antigen-negative, HLA class II-positive target cells pulsed with higher concentrations of mutant RAS peptides. The HLA class II molecule can be any HLA class II molecule described herein (eg, an HLA-DRB1*07:01 molecule or an HLA-DRB1*11:01 molecule).

[0045] Alternatively or additionally, a TCR can be considered to be "antigen-specific" for mutant RAS if T cells expressing the TCR secrete at least twice the amount of IFN-γ expressed by a negative control when co-cultured 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 a nucleotide sequence encoding mutant RAS has been introduced so that the target cells express mutant RAS. Negative controls can be, for example, (i) T cells expressing TCR co-cultured with the following cells: (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 mutated RAS peptide), or (b) antigen-negative, HLA class II molecule-positive target cells into which a nucleotide sequence encoding an irrelevant peptide has been introduced so that the target cells express the irrelevant peptide; or (ii) untransduced T cells (e.g., derived from PBMCs that do not express TCR) co-cultured with the following cells: (a) antigen-negative, HLA class II molecule-positive target cells pulsed with the same concentration of a mutated RAS peptide, or (b) antigen-negative, HLA class II molecule-positive target cells into which a nucleotide sequence encoding a mutated RAS has been introduced so that the target cells express the mutated RAS. The HLA class II molecules expressed by the target cells of the negative control are the same as the HLA class II molecules expressed by the target cells co-cultured with the T cells being tested. The HLA class II molecule may be any HLA class II molecule described herein (eg, an HLA-DRB1*07:01 molecule or an HLA-DRB1*11:01 molecule). IFN-γ secretion may be measured by methods known in the art (eg, enzyme-linked immunosorbent assay, ELISA).

[0046] Alternatively or additionally, a TCR may be considered to be "antigen specific" for a mutant RAS if at least twice the number of T cells expressing the TCR secrete IFN-γ when co-cultured with (a) antigen-negative, HLA class II-positive target cells pulsed with a low concentration of a mutant RAS peptide or (b) an antigen-negative, HLA class II-positive target cell into which a nucleotide sequence encoding a mutant RAS has been introduced so that the target cell expresses the mutant RAS, compared to the number of negative control T cells that secrete IFN-γ. The HLA class II molecule, peptide concentration, and negative control may be as described herein for other aspects of the invention. The number of cells secreting IFN-γ may be measured by methods known in the art (e.g., ELISPOT).

[0047] Alternatively or additionally, if the T cell expressing the TCR upregulates the expression of one or more T cell activation markers, the TCR can be considered to be "antigen-specific" for the mutated RAS, as measured by, for example, flow cytometry after stimulation with target cells expressing the mutated RAS. Examples of T cell activation markers include 4-1BB, OX40, CD107a, CD69, and cytokines that are upregulated upon antigen stimulation (e.g., tumor necrosis factor (TNF), interleukin (IL) -2, etc.).

[0048] Embodiments of the present invention provide a TCR comprising two polypeptides (i.e., polypeptide chains, such as an α chain of a TCR, a β chain of a TCR, a γ chain of a TCR, a δ chain of a TCR, or a combination thereof). The polypeptide of the TCR of the present invention may comprise any amino acid sequence, provided that the TCR has antigen specificity for a mutated RAS.

[0049] In an embodiment of the present invention, the TCR includes two polypeptide chains, each of which includes a variable region containing a complementary determining region (CDR) 1, CDR2 and CDR3 of the TCR. In an embodiment of the present invention, the TCR includes a first polypeptide chain and a second polypeptide chain, the first polypeptide chain includes a CDR1 (CDR1 of an α chain) containing an amino acid sequence of SEQ ID NO: 1, a CDR2 (CDR2 of an α chain) containing an amino acid sequence of SEQ ID NO: 2, and a CDR3 (CDR3 of an α chain) containing an amino acid sequence of SEQ ID NO: 3, and the second polypeptide chain includes a CDR1 (CDR1 of a β chain) containing an amino acid sequence of SEQ ID NO: 4, a CDR2 (CDR2 of a β chain) containing an amino acid sequence of SEQ ID NO: 5, and a CDR3 (CDR3 of a β chain) containing an amino acid sequence of SEQ ID NO: 6.

[0050] In another embodiment of the present invention, the TCR comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprises CDR1 (CDR1 of the α chain) comprising the amino acid sequence of SEQ ID NO:7, CDR2 (CDR2 of the α chain) comprising the amino acid sequence of SEQ ID NO:8, and CDR3 (CDR3 of the α chain) comprising the amino acid sequence of SEQ ID NO:9, and the second polypeptide chain comprises CDR1 (CDR1 of the β chain) comprising the amino acid sequence of SEQ ID NO:10, CDR2 (CDR2 of the β chain) comprising the amino acid sequence of SEQ ID NO:11, and CDR3 (CDR3 of the β chain) comprising the amino acid sequence of SEQ ID NO:12.

[0051] In this regard, the TCR of the present invention may include any one or more of the amino acid sequences selected from SEQ ID NO: 1-12. In embodiments of the present invention, the TCR includes 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 particularly preferred embodiments, the TCR includes the following amino acid sequences: (i) all of SEQ ID NO: 1-6 or (ii) all of SEQ ID NO: 7-12.

[0052] In an embodiment of the present invention, the TCR comprises an amino acid sequence of a variable region of a TCR comprising the above CDRs. In this regard, the TCR may comprise the following amino acid sequence: SEQ ID NO: 13 (variable region of α chain); SEQ ID NO: 14 (variable region of β chain); SEQ ID NO: 15 (variable region of α chain); SEQ ID NO: 16 (variable region of 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 sequence: (i) both SEQ ID NO: 13 and 14, or (ii) both SEQ ID NO: 15 and 16.

[0053] The TCR of the present invention may also include an α chain constant region and a β chain constant region. The constant region may be derived from any suitable species (e.g., humans or mice). In embodiments of the present invention, TCR also includes murine α chain and β chain constant regions or human α chain and β chain constant regions. As used herein, when referring to any component of TCR or TCR described herein (e.g., complementary determining region (CDR), variable region, constant region, α chain and / or β chain), the term "mouse" or "human" means that TCR (or its components) are derived from mice or humans, i.e., TCR (or its components) are derived from mouse T cells or human T cells, respectively, or are expressed once.

[0054] Embodiments of the present invention provide chimeric TCRs comprising human variable regions and murine constant regions, wherein the TCR has antigenic specificity for mutated human RAS amino acid sequences presented by HLA class II molecules. The murine constant region may provide any one or more advantages. For example, the murine constant region may reduce the mispairing of the TCR of the present invention with the endogenous TCR of the host cell into which the TCR of the present invention is introduced. Alternatively or additionally, the murine constant region may increase the expression of the TCR of the present invention compared to the same TCR with a human constant region. The chimeric TCR may include SEQ ID NO: 32 (wild-type (WT) murine α chain constant region), SEQ ID NO: 33 (WT murine β chain constant region) or SEQ ID NO: 32 and 33 both amino acid sequences. Preferably, the TCR of the present invention includes SEQ ID NO: 32 and 33 both amino acid sequences. The chimeric TCR may include any murine constant region described herein in combination with 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 NOs: 1-3 and 32; (b) all of SEQ ID NOs: 4-6 and 33; (c) all of SEQ ID NOs: 7-9 and 32; (d) all of SEQ ID NOs: 10-12 and 33; (e) all of SEQ ID NOs: 1-6 and 32-33; or (f) all of SEQ ID NOs: 7-12 and 32-33. In another embodiment of the invention, a chimeric TCR may include any of the murine constant regions described herein in combination with any of the variable regions described herein for other aspects of the invention. In this regard, the TCR may include the following amino acid sequences: (i) both SEQ ID NOs: 13 and 32; (ii) both SEQ ID NOs: 14 and 33; (iii) both SEQ ID NOs: 15 and 32; (iv) both SEQ ID NOs: 16 and 33; (v) all of SEQ ID NOs: 13-14 and 32-33; or (vi) all of SEQ ID NOs: 15-16 and 32-33.

[0055] In another embodiment of the present invention, the TCR comprises the following amino acid sequence: SEQ ID NO:38 (α chain with WT mouse constant region), SEQ ID NO:39 (β chain with WT mouse constant region), SEQ ID NO:40 (α chain with WT mouse constant region), SEQ ID NO:41 (β chain with WT mouse constant region), both SEQ ID NOs:38-39 or both SEQ ID NOs:40-41.

[0056] In an embodiment of the invention, the TCR comprises an alpha chain comprising a variable region and a constant region and a beta chain comprising a variable region and a constant region. In this regard, the TCR may include (a) an alpha 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 beta 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 alpha chain comprising the amino acid sequence of SEQ ID NO: 36, 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. (ii) X at position 180 of SEQ ID NO:36 is Thr or Cys; (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 beta strand 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 of (a) and (b); or (f) both of (c) and (d).

[0057] In an embodiment of the present invention, the TCR includes a substituted constant region. In this regard, the TCR may include any TCR described herein having one, two, three or four amino acid substitutions in the constant region of one or both of the α chain and the β chain. Preferably, the TCR includes 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 chain and the β chain. In a particularly preferred embodiment, the TCR includes 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 parent TCR including an unsubstituted (wild-type) constant region, a TCR including a substituted constant region advantageously provides one or more of the following: an increase in mutated RAS + Target recognition, increased host cell expression, reduced mispairing with endogenous TCRs, and increased anti-tumor activity. In general, the substituted amino acid sequences of the murine constant regions of the TCR α chain and β chain, SEQ ID NOs: 30 and 31, correspond to all or part of the unsubstituted murine constant region amino acid sequences, SEQ ID NOs: 32 and 33, respectively, 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 invention provide TCRs 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 NOs: 30 and 31. In embodiments of the invention, the TCR comprising SEQ ID NO: 30 does not include SEQ ID NO: 32 (unsubstituted murine constant region of the α chain). In an embodiment of the invention, the TCR comprising SEQ ID NO:31 does not include SEQ ID NO:33 (the unsubstituted murine constant region of the beta chain).

[0058] In an embodiment 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 relative cysteines in the α chain and the β chain provide a disulfide bond that connects the constant regions of the α chain and the β chain of the substituted TCR to each other and is not present in a TCR including an unsubstituted murine constant region. In this regard, the TCR may be a cysteine-substituted TCR, wherein one or both of the natural Thr (Thr48) at position 48 of SEQ ID NO:32 and the natural Ser (Ser57) at position 57 of SEQ ID NO:33 may be substituted with Cys. Preferably, the natural Thr48 of SEQ ID NO:32 and the natural Ser57 of SEQ ID NO:33 are both substituted with Cys. Examples of TCR constant region sequences substituted with cysteine ​​are shown in Table 2. In embodiments of the 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, wherein both SEQ ID NO: 30 and 31 are as defined in Table 2. In addition to any CDR or variable region described herein, the cysteine-substituted TCR of the invention may comprise a substituted constant region.

[0059] In an embodiment of the present invention, the chimeric TCR substituted with cysteine ​​includes a full-length α chain and a full-length β chain. Examples of chimeric TCR α chain and β chain sequences substituted with cysteine ​​are shown in Table 2. In an embodiment of the present invention, the TCR includes (i) SEQ ID NO: 34, (ii) SEQ ID NO: 35, (iii) SEQ ID NO: 36, (iv) SEQ ID NO: 37, (v) SEQ ID NO: 34 and 35 or (vi) SEQ ID NO: 36 and 37, wherein all SEQ ID NO: 34-37 are as defined in Table 2.

[0060] Table 2

[0061]

[0062] In an embodiment of the present invention, the substituted amino acid sequence comprises a substitution 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 with hydrophobic amino acids, thereby providing a TCR substituted with hydrophobic amino acids (also referred to herein as "LVL-modified TCR"). Compared with a TCR lacking a hydrophobic amino acid substitution in the TM domain, a 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 natural 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 with Leu, Ile or Val. In an embodiment of the 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 as defined in Table 3. In addition to any CDR or variable region described herein, the LVL-modified TCR of the invention may comprise a substituted constant region.

[0063] In an embodiment of the present invention, the LVL modified TCR comprises a full-length α chain and a full-length β chain. Examples of LVL modified TCR α chain and β chain sequences are shown in Table 3. In an embodiment 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) SEQ ID NO: 34 and 35 or (vi) SEQ ID NO: 36 and 37, wherein all SEQ ID NO: 34-37 are as defined in Table 3.

[0064] Table 3

[0065]

[0066]

[0067]

[0068] In an embodiment of the present invention, the substituted amino acid sequence comprises a cysteine ​​substitution combination in the constant region of one or both of the α chain and the β chain, and comprises a substitution 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 with 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 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 as defined in Table 4. In addition to any CDR or variable region described herein, the cysteine-substituted, LVL-modified TCR of the invention may comprise a substituted constant region.

[0069] 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 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 SEQ ID NO: 34-37 are as defined in Table 4.

[0070] Table 4

[0071]

[0072]

[0073]

[0074] The invention also provides polypeptides comprising a functional portion of any TCR described herein.The term "polypeptide" as used herein includes oligopeptides and refers to a single chain of amino acids linked by one or more peptide bonds.

[0075] For the polypeptide of the present invention, the functional part may be any part including the continuous amino acids of TCR and being a part thereof, provided that the functional part specifically binds to the mutated RAS. The term "functional part" refers to any part or fragment of the TCR of the present invention when referring to the use of TCR, and the part or fragment retains the biological activity of the TCR (parental TCR) that produces the part. The functional part covers, for example, those parts of TCR that retain the following ability: specifically bind to the mutated RAS (for example, in the context of HLA-DRB1*07:01 molecules or HLA-DRB1*11:01 molecules) or detect, treat or prevent cancer to a similar degree, the same degree or a higher degree compared to the parental TCR. Referring to the parental TCR, the functional part may include, for example, about 10%, about 25%, about 30%, about 50%, about 68%, about 80%, about 90%, about 95% or more of the parental TCR.

[0076] The functional portion may include additional amino acids at the amino or carboxyl terminus or at both termini of the portion that are not found in the amino acid sequence of the parent TCR. Desirably, the additional amino acids do not interfere with the biological function of the functional portion, such as specific binding to mutated RAS and / or the ability to detect, treat or prevent cancer, etc. More desirably, the additional amino acids enhance the biological activity compared to the biological activity of the parent TCR.

[0077] The polypeptide may include any one or two functional parts of the α chain and β chain of the TCR of the present invention, for example, one or more functional parts of CDR1, CDR2 and CDR3 of the variable region in the α chain and / or β chain of the TCR of the present invention. In an embodiment of the present invention, the polypeptide may include SEQ ID NO: 1 (CDR1 of α chain), SEQ ID NO: 2 (CDR2 of α chain), SEQ ID NO: 3 (CDR3 of α chain), SEQ ID NO: 4 (CDR1 of β chain), SEQ ID NO: 5 (CDR2 of β chain), SEQ ID NO: 6 (CDR3 of β chain) amino acid sequence or a combination thereof. In another embodiment of the present invention, the polypeptide may include SEQ ID NO: 7 (CDR1 of α chain), SEQ ID NO: 8 (CDR2 of α chain), SEQ ID NO: 9 (CDR3 of α chain), SEQ ID NO: 10 (CDR1 of β chain), SEQ ID NO: 11 (CDR2 of β chain), SEQ ID NO: 12 (CDR3 of β chain) amino acid sequence or a combination thereof.

[0078] In this regard, the polypeptide of the present invention may include any one or more of the amino acid sequences selected from SEQ ID NO: 1-12. In embodiments 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 preferred embodiments, the polypeptide comprises the following amino acid sequences: (i) all of SEQ ID NO: 1-6 or (ii) all of SEQ ID NO: 7-12.

[0079] In an embodiment of the present invention, the polypeptide of the present invention may include, for example, a variable region comprising a combination of the above-mentioned CDR regions in a TCR of the present invention. In this regard, the polypeptide may include the following amino acid sequence: (i) SEQ ID NO: 13 (variable region of the α chain), (ii) SEQ ID NO: 14 (variable region of the β chain), (iii) both SEQ ID 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 sequence: (i) both SEQ ID NO: 13 and 14 or (ii) both SEQ ID NO: 15 and 16.

[0080] In an embodiment of the present invention, the polypeptide of the present invention may also include the above-mentioned constant region of the TCR of the present invention. In this regard, the polypeptide may also include the following amino acid sequence: SEQ ID NO: 32 (WT murine constant region of α chain), SEQ ID NO: 33 (WT murine constant region of β chain), SEQ ID NO: 30 (substituted murine constant region of α chain), SEQ ID NO: 31 (substituted murine constant region of β chain), SEQ ID NO: 32 and 33 or SEQ ID NO: 30 and 31. Preferably, the polypeptide also includes the amino acid sequence of SEQ ID NO: 30 and 31 or SEQ ID NO: 32 and 33 in combination with any CDR region or variable region described herein for other aspects of the present invention. In an embodiment of the present invention, one or both of SEQ ID NO: 30 and 31 of the polypeptide are as defined in any one of Tables 2-4.

[0081] In embodiments of the invention, the polypeptide of the invention may include the entire length of the α or β chain of the TCR described herein. In this regard, the polypeptide of the invention may include the amino acid sequence of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37. Alternatively, the polypeptide of the invention may include two chains of the TCR described herein.

[0082] For example, a polypeptide of the invention may include: (a) an 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) an amino acid sequence of SEQ ID NO: 35, wherein X at position 189 of SEQ ID NO: 35 is Ser or Cys; (c) an 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 243 of SEQ ID NO: 34 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; NO:36 position 244 X is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) SEQ ID NO:36 position 246 X is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) SEQ ID NO:36 position 247 X is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (d) SEQ ID NO:37 amino acid sequence, wherein SEQ ID NO:37 position 194 X is Ser or Cys; (e) (a) and (b) both; or (f) (c) and (d) both. In an embodiment of the invention, any one or more of SEQ ID NO:34-37 of the polypeptide is as defined in any one of Tables 2-4.

[0083] The present invention also provides proteins comprising at least one polypeptide described herein. "Protein" means a molecule comprising one or more polypeptide chains.

[0084] In an embodiment, a protein of the invention may include (a) a first polypeptide chain including an amino acid sequence of SEQ ID NOs: 1-3 and a second polypeptide chain including an amino acid sequence of SEQ ID NOs: 4-6; or (b) a first polypeptide chain including an amino acid sequence of SEQ ID NOs: 7-9 and a second polypeptide chain including an amino acid sequence of SEQ ID NOs: 10-12.

[0085] In another embodiment of the present invention, the protein may include (i) a first polypeptide chain including the amino acid sequence of SEQ ID NO:13 and a second polypeptide chain including the amino acid sequence of SEQ ID NO:14; or (ii) a first polypeptide chain including the amino acid sequence of SEQ ID NO:15 and a second polypeptide chain including the amino acid sequence of SEQ ID NO:16.

[0086] The protein of the present invention may also include any constant region described herein for other aspects of the present invention. In this regard, in embodiments of the present invention, the first polypeptide chain may also include the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 32 and the second polypeptide chain may also include the amino acid sequence of SEQ ID NO: 31 or SEQ ID NO: 33. In embodiments of the present invention, one or both of SEQ ID NOs: 30 and 31 of the protein are as defined in any one of Tables 2-4.

[0087] Alternatively or additionally, a protein of an embodiment of the invention may include (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 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. NO:36 position 180 X is Thr or Cys; (ii) SEQ ID NO:36 position 244 X is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) SEQ ID NO:36 position 246 X is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) SEQ ID NO:36 position 247 X 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 SEQ ID NO:37 position 194 X is Ser or Cys; (e) (a) and (b) both; or (f) (c) and (d) both. In an embodiment of the invention, one or more of SEQ ID NO:34-37 is as defined in any one of Tables 2-4.

[0088] The protein of the present invention may be a TCR. Alternatively, for example, if the protein includes a single polypeptide chain containing the amino acid sequences of both SEQ ID NOs: 34 and 35, SEQ ID NOs: 36 and 37, or if the first and / or second polypeptide chains of the protein further include other amino acid sequences (e.g., amino acid sequences encoding immunoglobulins or portions thereof), the protein of the present invention may be a fusion protein. In this regard, the present invention also provides a fusion protein comprising at least one polypeptide of the present invention described herein and at least one other polypeptide. The other polypeptide may exist as a separate polypeptide of the fusion protein, or may exist in the form of a polypeptide expressed in the same frame (in series) as one of the polypeptides of the present invention described herein. The other polypeptide may encode any peptide or protein molecule or a portion thereof, including but not limited to: immunoglobulins, CD3, CD4, CD8, MHC molecules, CD1 molecules (e.g., CD1a, CD1b, CD1c, CD1d), etc.

[0089] The fusion protein may include one or more copies of a polypeptide of the present invention and / or one or more copies of other polypeptides. For example, the fusion protein may include 1, 2, 3, 4, 5 or more copies of a polypeptide of the present invention and / or other polypeptides. Suitable methods for preparing fusion proteins are known in the art and include, for example, recombinant methods.

[0090] In some embodiments of the present invention, the TCR, polypeptide and protein of the present invention may be expressed as a single protein including a linker peptide connecting an α chain and a β chain. In this regard, the TCR, polypeptide and protein of the present invention may also include a linker peptide. The linker peptide may advantageously promote the expression of recombinant TCR, polypeptide and / or protein in a host cell. The linker peptide may include any suitable amino acid sequence. For example, the linker peptide may be a furin-SGSG-P2A linker including the amino acid sequence of SEQ ID NO:54. When a construct comprising a linker peptide is expressed by a host cell, the linker peptide may be cut to produce separated α chains and β chains. In embodiments of the present invention, TCR, polypeptide or protein may include an amino acid sequence containing a full-length α chain, a full-length β chain and a linker peptide positioned between the α chain and the β chain.

[0091] The protein of the present invention may be a recombinant antibody or antigen binding portion thereof comprising at least one polypeptide of the present invention described herein. As used herein, "recombinant antibody" refers to a recombinant (e.g., genetically modified) protein comprising at least one of the polypeptides of the present invention and the polypeptide chains of the antibody or its antigen binding portion. The polypeptide of the antibody or its antigen binding portion may be a heavy chain, light chain, variable or constant region of a heavy chain or light chain, a single chain variable fragment (scFv) or Fc, Fab or F(ab)2' fragment, etc. of the antibody. The polypeptide chain of the antibody or its antigen binding portion may exist as a separate polypeptide of the recombinant antibody. Alternatively, the polypeptide chain of the antibody or its antigen binding portion may exist in the form of a polypeptide expressed in the same frame (in series) as the polypeptide of the present invention. The polypeptide of the antibody or its antigen binding portion may be a polypeptide of any antibody or any antibody fragment (including any antibody and antibody fragment described herein).

[0092] The scope of the present invention includes functional variants of the TCR, polypeptide or protein of the present invention described herein. The term "functional variant" as used herein refers to a TCR, polypeptide or protein having a large amount or significant sequence identity or similarity with a parent TCR, polypeptide or protein, and the functional variant retains the biological activity of the TCR, polypeptide or protein that produces the variant. Functional variants encompass those variants of (for example) TCR, polypeptide or protein (parent TCR, polypeptide or protein) described herein, which retain the ability to specifically bind to mutated RAS (parent TCR has antigen specificity for it or parent polypeptide or protein specifically binds to it) to a similar degree, the same degree or a higher degree compared to the parent TCR, polypeptide or protein. With reference to the parent TCR, polypeptide or protein, the amino acid sequence of the functional variant and the parent TCR, polypeptide or protein can be respectively (for example) at least about 30%, about 50%, about 75%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or more identical.

[0093] Functional variants may, for example, include an amino acid sequence of a parent TCR, polypeptide or protein having at least one conservative amino acid substitution. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is exchanged for another amino acid having the same chemical or physical properties. For example, conservative amino acid substitutions may be substitutions of one acidic amino acid for another acidic amino acid (e.g., Asp or Glu), substitutions of an amino acid having a non-polar side chain for another amino acid having a non-polar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), substitutions of a basic amino acid for another basic amino acid (Lys, Arg, etc.), substitutions of an amino acid having a polar side chain for another amino acid having a polar side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), etc.

[0094] Alternatively or additionally, a functional variant may include an amino acid sequence of a parent 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 parent TCR, polypeptide or protein.

[0095] TCR, polypeptide or protein can be substantially composed of a specified amino acid sequence or a sequence described herein, so that other components of TCR, polypeptide or protein (e.g., other amino acids) do not substantially change the biological activity of TCR, polypeptide or protein. In this regard, the TCR, polypeptide or protein of the present invention can be (for example) substantially composed of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:34-35 or SEQ ID NO:36-37. Similarly, for example, the TCR, polypeptide or protein of the present invention can be substantially composed 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 or (vi) SEQ ID NO:15 and 16. In addition, the TCR, polypeptide or protein of the present invention may essentially consist of the following amino acid sequences: (a) any one or more of SEQ ID NOs: 1-12; (b) all of SEQ ID NOs: 1-3; (c) all of SEQ ID NOs: 4-6; (d) all of SEQ ID NOs: 7-9; (e) all of SEQ ID NOs: 10-12; (f) all of SEQ ID NOs: 1-6; or (g) all of SEQ ID NOs: 7-12.

[0096] The TCRs, polypeptides and proteins of the present invention may have any length, i.e., may include any number of amino acids, provided that the TCRs, polypeptides or proteins retain their biological activity (e.g., the ability to specifically bind to mutated RAS; detect cancer in a mammal; or treat or prevent cancer in a mammal, etc.). For example, the length of the polypeptide may be in the range of 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 length. In this regard, the polypeptides of the present invention also include oligopeptides.

[0097] The TCR, polypeptide and protein of the present invention may include synthetic amino acids to replace one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3-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.

[0098] The TCRs, polypeptides and proteins of the invention may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, or converted into acid addition salts and / or optionally dimerized or polymerized or conjugated, for example, via disulfide bridges.

[0099] The TCRs, polypeptides and / or proteins of the invention can be obtained by methods known in the art (e.g., de novo synthesis). Likewise, polypeptides and proteins can be produced recombinantly using the nucleic acids described herein and 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). Alternatively, the TCRs, polypeptides and / or proteins described herein may 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 invention may be synthetic, recombinant, isolated and / or purified.

[0100] The scope of the invention includes conjugates (e.g., bioconjugates) containing any TCR, polypeptide or protein of the invention (including any functional part or variant thereof), nucleic acid, recombinant expression vector, host cell, host cell population or antibody or antigen binding portion thereof. Conjugates and general methods for synthesizing conjugates are known in the art.

[0101] Embodiments of the present invention provide nucleic acids comprising nucleotide sequences encoding any TCR, polypeptide or protein described herein. "Nucleic acid" as used herein includes "polynucleotide", "oligonucleotide" and "nucleic acid molecule" and generally means a polymer of DNA or RNA, which may be single-stranded or double-stranded, which may contain natural, non-natural or changed nucleotides, and which may contain natural, non-natural or changed internucleotide linkages (e.g., phosphoramidate linkages or thiophosphate linkages) to replace the phosphodiester found between the nucleotides of unmodified oligonucleotides. In embodiments, nucleic acids include complementary DNA (cDNA). It is generally preferred that nucleic acids do not include any insertion, deletion, inversion and / or substitution. However, in some cases, as discussed herein, nucleic acids may be suitable for including one or more insertions, deletions, inversions and / or substitutions.

[0102] Preferably, the nucleic acids of the invention are recombinant. As used herein, the term "recombinant" means that (i) the molecule is constructed outside a living cell by joining a natural or synthetic nucleic acid segment to a nucleic acid molecule that can replicate in a living cell, or (ii) the molecule is derived from replication of those described in (i) above. For purposes herein, replication can be in vitro replication or in vivo replication.

[0103] Nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, for example, Green and Sambrook et al. (supra). For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or nucleotides modified in various ways designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides). Examples of modified nucleotides that can be used to generate nucleic acids include, but are not limited to: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylquenoside, inosine, N- 6 -Isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6 Substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosyl quercetin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6-isopentenyl adenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl) uracil and 2,6-diaminopurine. Alternatively, one or more nucleic acids of the invention can be purchased from companies such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0104] Nucleic acid may include any nucleotide sequence encoding any TCR, polypeptide or protein described herein. In embodiments of the present invention, nucleic acid may include a nucleotide sequence of any one of SEQ ID NO:42-45 (Table 5). In embodiments of the present invention, nucleic acid includes a nucleotide sequence of both SEQ ID NO:42-43 or both SEQ ID NO:44-45.

[0105] Table 5

[0106]

[0107] In an embodiment of the present invention, 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 nucleotide sequences can increase the translation efficiency of mRNA transcripts. Codon optimization of nucleotide sequences can involve replacing natural codons to obtain another codon, which encodes the same amino acid, but can be translated by tRNA that is more easily available in the cell, thereby increasing translation efficiency. Optimization of nucleotide sequences can also reduce secondary mRNA structures that interfere with translation, thereby increasing translation efficiency.

[0108] The present invention also provides nucleic acids comprising a nucleotide sequence that is complementary to the nucleotide sequence of any nucleic acid described herein or a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of any nucleic acid described herein.

[0109] The nucleotide sequence that hybridizes under stringent conditions preferably hybridizes under high stringency conditions. "High stringency conditions" means that the nucleotide sequence specifically hybridizes with the target sequence (nucleotide sequence of any nucleic acid described herein) with an amount that is detectably stronger than non-specific hybridization. High stringency conditions include conditions that distinguish the polynucleotide containing the accurate complementary sequence or the polynucleotide containing only several dispersed mispairings from the random sequence of several small regions (e.g., 3-10 bases) that happen to have a matching nucleotide sequence. Such small complementary regions are more fusible than the full-length complement of 14-17 or more bases, and high stringency hybridization makes it easy to distinguish. Relatively high stringency conditions will include, for example, low salt and / or high temperature conditions, such as provided by about 0.02-0.1M NaCl or equivalents at a temperature of about 50-70°C. Such high stringency conditions tolerate (if present) very few mispairings between the nucleotide sequence and the template or target chain, and are particularly suitable for detecting the expression of any TCR of the present invention. It is generally believed that more stringent conditions can be caused by adding an increased amount of formamide.

[0110] The 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 of the nucleic acids described herein. In this regard, the nucleic acid may consist essentially of any of the nucleotide sequences described herein.

[0111] The nucleic acid of the present invention can be incorporated into a recombinant expression vector. In this regard, the present invention provides a recombinant expression vector comprising any nucleic acid of the present invention. In an embodiment of the present invention, the recombinant expression vector comprises a nucleotide sequence encoding an α chain, a β chain and a linker peptide.

[0112] For the purposes of this article, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that allows a host cell to express an mRNA, protein, polypeptide or peptide when the construct includes a nucleotide sequence encoding the mRNA, protein, polypeptide or peptide and the vector is contacted with the cell under conditions sufficient to allow the mRNA, protein, polypeptide or peptide to be expressed in the cell. The vector of the present invention is not entirely naturally occurring. However, portions of the vector may be naturally occurring. The recombinant expression vector of the present invention may include any type of nucleotide, including but not limited to: DNA and RNA, may be single-stranded or double-stranded, synthesized or partially obtained from a natural source, and may contain natural, non-natural or altered nucleotides. The recombinant expression vector may include naturally occurring, non-naturally occurring internucleotide linkages, or both types of linkages. Preferably, non-naturally occurring or altered nucleotides or internucleotide linkages do not hinder transcription or replication of the vector.

[0113] The recombinant expression vector of the present invention may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host cell. Suitable vectors include vectors designed for reproduction and amplification or for expression or for both, such as plasmids and viruses. The vector may be selected from: pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX series (Clontech, Palo Alto, CA). Phage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4 and λNM1149 may also be used. Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). Preferably, the recombinant expression vector is a viral vector (eg, a retroviral vector). In a particularly preferred embodiment, the recombinant expression vector is a MSGV1 vector.

[0114] The recombinant expression vectors of the present invention can be prepared using standard recombinant DNA techniques described, for example, in Green and Sambrook et al. (supra). Circular or linear constructs of the expression vector can be prepared to contain a functional replication system in a prokaryotic or eukaryotic host cell. The replication system can be derived, for example, from ColE1, 2μ plasmid, lambda, SV40, bovine papilloma virus, and the like.

[0115] Desirably, recombinant expression vectors contain regulatory sequences, such as transcription and translation start and stop codons, which are specific to the type of host cell (e.g., bacteria, fungi, plants, or animals) into which the vector is to be introduced, as appropriate and taking into account whether the vector is DNA or RNA based.

[0116] The recombinant expression vector may comprise one or more marker genes, which allow selection of transformed or transfected host cells. Marker genes include biocide resistance (e.g., resistance to antibiotics, heavy metals, etc.), complementation in auxotrophic host cells to provide prototrophy, etc. Suitable marker genes for expression vectors of the present invention include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histamine resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0117] The recombinant expression vector may include a natural or non-natural promoter, which may be operably linked to a nucleotide sequence encoding a TCR, a polypeptide or a protein or to a nucleotide sequence complementary to or hybridizing with a nucleotide sequence encoding a TCR, a polypeptide or a protein. The selection of promoters, such as strong, weak, inducible, tissue-specific and development-specific, is within the capabilities of those of ordinary skill in the art. Similarly, the combination of nucleotide sequence and promoter is also within the capabilities of the technician. Promoters may be non-viral promoters or viral promoters, such as cytomegalovirus (CMV) promoters, SV40 promoters, RSV promoters, and promoters found in the long terminal repeats of murine stem cell viruses.

[0118] The recombinant expression vector of the present invention can be designed for transient expression, stable expression or for both. Likewise, the recombinant expression vector can be prepared for constitutive expression or for inducible expression.

[0119] In addition, the recombinant expression vector can be prepared to include a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the cell expressing the suicide gene to die. A suicide gene can be a gene that imparts sensitivity to an agent (e.g., a drug) to the cell in which the gene is expressed, and causes cell death when the cell contacts or is exposed to the agent. Suicide genes are known in the art and include, for example, herpes simplex virus (HSV) thymidine kinase (TK) genes, cytosine deaminase, purine nucleoside phosphorylase, nitroreductase, and inducible caspase 9 gene systems.

[0120] Another embodiment of the present invention also provides a host cell comprising any recombinant expression vector described herein. As used herein, the term "host cell" refers to any type of cell that may contain a recombinant expression vector of the present invention. The host cell may be a eukaryotic cell (e.g., a plant, an animal, a fungus, or an algae), or may be a prokaryotic cell (e.g., a bacterium or a protozoan). The host cell may be a cultured cell or a primary cell (i.e., directly isolated from an organism such as a human). The host cell may be an adherent cell or a suspension cell, i.e., a cell grown in a 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 a recombinant expression vector, the host cell is preferably a prokaryotic cell, such as a DH5α cell. For the purpose of producing a recombinant TCR, a polypeptide, or a protein, the host cell is preferably a mammalian cell. More preferably, the host cell is a human cell. Although the host cell may be any cell type, may be derived from any type of tissue, and may 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.

[0121] For the purposes of this article, T cells can be any T cells, such as cultured T cells (e.g., primary T cells, or T cells from cultured T cell lines (e.g., Jurkat, SupT1, etc.), or T cells obtained from mammals. If obtained from mammals, T cells can be obtained from a variety of sources, including but not limited to: blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells can also be enriched or purified. Preferably, T cells are human T cells. T cells can be any type of T cells and can be at any developmental stage, including but not limited to: CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells (such as Th1 and Th2 cells), CD4 + T cells, CD8 + T cells (such as cytotoxic T cells), tumor infiltrating lymphocytes (TIL), memory T cells (such as central memory T cells and effector memory T cells), naive T cells, etc.

[0122] The present invention also provides a cell colony including at least one host cell described herein. The cell colony can be a heterogeneous population of host cells containing any recombinant expression vector in addition to at least one other cell, and the other cells are, for example, host cells (such as T cells) that do not contain any recombinant expression vectors or cells other than T cells, such as B cells, macrophages, neutrophils, erythrocytes, hepatocytes, endothelial cells, epithelial cells, muscle cells, brain cells, etc. Alternatively, the cell colony can be a substantially homogeneous population, wherein the colony mainly comprises host cells containing recombinant expression vectors (such as substantially composed of host cells containing recombinant expression vectors). The colony can also be a clonal cell colony, wherein all cells of the colony are clones of a single host cell containing a recombinant expression vector, so that all cells of the colony include the recombinant expression vector. In one embodiment of the present invention, the cell colony is a clonal population of host cells including a recombinant expression vector as described herein.

[0123] In embodiments of the present invention, the number of cells in the population can be rapidly expanded. The expansion of the number of T cells can be achieved by any of the many methods known in the art as described in, for example, U.S. Patent 8,034,334; U.S. Patent 8,383,099; U.S. Patent Application Publication No. 2012 / 0244133; Dudley et al., J. Immunother., 26:332-42 (2003); and Riddell et al., J. Immunol. Methods, 128:189-201 (1990). . In embodiments, the number of T cells is expanded by culturing T cells with OKT3 antibody, IL-2, and feeder PBMCs (e.g., irradiated allogeneic PBMCs).

[0124] The TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors and host cells (including populations thereof) of the present invention may be isolated and / or purified. The term "isolated" as used herein means taken out from its natural environment. The term "purified" as used herein means having an increased purity, wherein "purity" is a relative term and need not be interpreted as absolute purity. For example, the purity may be at least about 50%, may be greater than about 60%, about 70%, about 80%, about 90%, about 95%, or may be about 100%.

[0125] The TCR, polypeptide, protein, nucleic acid, recombinant expression vector and host cell (including its population) of the present invention (hereinafter referred to as "TCR material of the present invention") can be formulated into a composition (e.g., a pharmaceutical composition). In this regard, the present invention provides a pharmaceutical composition comprising any of the TCR, polypeptide, protein, nucleic acid, expression vector and host cell (including its population) described herein and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention containing any TCR material of the present invention may include more than one TCR material of the present invention (e.g., polypeptide and nucleic acid) or two or more different TCRs. Optionally, a pharmaceutical composition may include a TCR material of the invention in combination with another pharmaceutically active agent or drug, which is, for example, a chemotherapeutic agent, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, and the like.

[0126] Preferably, the carrier is a pharmaceutically acceptable carrier. For pharmaceutical compositions, the carrier may be any of those conventionally used for the specific TCR material of the present invention under consideration. Methods for preparing compositions that can be administered are known to or apparent to those skilled in the art and are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, 22nd edition, Pharmaceutical Press (2012). Preferably, a pharmaceutically acceptable carrier is a carrier that has no harmful side effects or toxicity under the conditions of use.

[0127] The choice of carrier depends in part on the specific TCR material of the present invention and the specific method for administering the TCR material of the present invention. Therefore, there are various suitable formulations of the pharmaceutical composition of the present invention. Suitable formulations may include any of those for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, intratumoral or intraperitoneal administration. More than one approach can be used to administer the TCR material of the present invention, and in some cases, a specific approach can provide a more direct and more effective response than another approach.

[0128] Preferably, the TCR material of the invention is administered by injection (e.g., intravenously). When the TCR material of the invention is a host cell (or a population thereof) expressing the TCR of the invention, the pharmaceutically acceptable carrier for the cells for injection may comprise any isotonic carrier, such as physiological 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.

[0129] For the purposes of the present invention, the amount or dosage 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 cause, for example, a therapeutic or preventive response in an individual or animal within a reasonable time frame. For example, the dosage of the TCR material of the present invention should be sufficient to bind to a cancer antigen (e.g., a mutated 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, the time period may even be longer. The dosage will be determined by the efficacy of the specific TCR material of the present invention and the condition of the animal (e.g., human) and the weight of the animal (e.g., human) to be treated.

[0130] 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 after administering a given dose of T cells expressing the TCR, polypeptide or protein of the present invention to a mammal in a group of mammals to which different doses of T cells are each administered. The extent of target cell lysis or IFN-γ secretion by T cells can be analyzed by methods known in the art when a certain dose is administered.

[0131] The dosage of the TCR material of the 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 invention. Typically, the attending physician will determine the dosage of the TCR material of the invention to be used to treat each individual patient, taking into account various factors, such as age, weight, general health, diet, sex, the TCR material of the invention to be administered, the route of administration, and the severity of the cancer being treated. In embodiments where the TCR material of the invention is a cell population, the number of cells administered per infusion may vary, for example, from about 1×10 6 About 1×10 12 In some embodiments, less than 1×10 6 cells.

[0132] It will be readily understood by those of ordinary skill in the art that the inventive TCR materials of the present invention can be modified in any number of ways, thereby increasing the therapeutic or preventive efficacy of the TCR materials of the present invention via modification. For example, the TCR materials of the present invention may be conjugated to a chemotherapeutic agent directly or indirectly via a bridge. The practice of conjugating compounds to chemotherapeutic agents is known in the art. Those of ordinary skill in the art recognize that sites on the TCR materials of the present invention that are not required for the function of the TCR materials of the present invention are suitable sites for attaching bridges and / or chemotherapeutic agents, provided that the bridges and / or chemotherapeutic agents do not interfere with the function of the TCR materials of the present invention after being attached to the TCR materials of the present invention (i.e., capable of binding to mutated RAS or capable of detecting, treating or preventing cancer).

[0133] 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 TCR of the present invention specifically binds to mutated RAS, so that the TCR (or related polypeptides or proteins of the present invention) can mediate an immune response to target cells expressing mutated RAS when expressed by cells. In this regard, the present invention provides a method for treating or preventing cancer in a mammal, comprising administering the following to the mammal in an amount effective for treating or preventing cancer in the mammal: any pharmaceutical composition, TCR, polypeptide, or protein described herein; any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any TCR, polypeptide, or protein described herein; or any host cell or cell population comprising a recombinant vector encoding any TCR, polypeptide, or protein described herein.

[0134] Embodiments of the present invention provide the following substances for treating or preventing cancer in mammals: any pharmaceutical composition, TCR, polypeptide or protein described herein; any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any TCR, polypeptide, protein described herein; or any host cell or cell population comprising a recombinant vector encoding any TCR, polypeptide or protein described herein.

[0135] The terms "treatment" and "prevention" used herein and words derived therefrom do not necessarily imply 100% or complete treatment or prevention. On the contrary, there are different 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 mammals at any level in any amount. In addition, the treatment or prevention provided by the methods of the present invention may include treatment or prevention of one or more conditions or symptoms of the cancer being treated or prevented. For example, treatment or prevention may include promoting tumor regression. Similarly, for the purposes of this article, "prevention" may cover delaying the onset of cancer or its symptoms or conditions. Alternatively or additionally, "prevention" may cover preventing or delaying the recurrence of cancer or its symptoms or conditions.

[0136] Also provided is a method for detecting the presence of cancer in a mammal. The method comprises: (i) contacting a sample comprising one or more cells from a mammal with any of the TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, cell populations, or pharmaceutical compositions of the invention described herein, thereby forming a complex; and detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

[0137] In the present method for detecting cancer in a mammal, the cell sample may be a sample comprising whole cells, lysates thereof, or fractions of whole cell lysates (eg, nuclear or cytoplasmic fractions, total protein fractions, or nucleic acid fractions).

[0138] For the purposes of the methods of the present invention for detecting cancer, contacting can occur in vitro or in vivo in a mammal. Preferably, contacting is in vitro.

[0139] Likewise, the complex can be detected by any number of means known in the art. For example, the TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, or cell populations of the invention described herein can be labeled with a detectable marker, such as a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

[0140] For the purposes of the methods of the invention in which a host cell or cell population is administered, the cell may be allogeneic or autologous to the mammal. Preferably, the cell is autologous to the mammal.

[0141] For the purposes of the present invention, the cancer may 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 rectal anal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer or pleural cancer, nasal cancer, nasal cancer or middle ear cancer, oral cancer, vaginal cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumors, glioma, Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, Preferably, the cancer is pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer, or prostate cancer. Preferably, the lung cancer is lung adenocarcinoma, the ovarian cancer is epithelial ovarian cancer, and the pancreatic cancer is pancreatic adenocarcinoma. In an embodiment of the present invention, the cancer expresses a mutated human RAS amino acid sequence, wherein the mutated human RAS amino acid sequence is a mutated human KRAS, a mutated human HRAS, or a mutated human NRAS amino acid sequence. The mutated human KRAS, mutated human HRAS, and mutated human NRAS expressed by the cancer may be as described herein for other aspects of the present invention.

[0142] The mammal mentioned in the method of the present invention can be any mammal. As used herein, the term "mammal" refers to any mammal, including but not limited to: rodent mammals (e.g., mice and hamsters) and lagomorpha mammals (e.g., rabbits). Preferably, the mammal is from the order Carnivora, including felines (cats) and canines (dogs). More preferably, the mammal is from the order Artiodactyla (including cattle (cows) and swine (pigs)) or the order Perssodactyla (including horses (horses)). Most preferably, the mammal is a primate, a ceboid, a simian (monkey), or anthropoid (human and ape). A particularly preferred mammal is a human.

[0143] The following examples further illustrate the present invention but, of course, should not be construed as limiting the scope of the invention in any way.

[0144] Example 1

[0145] This example demonstrates the isolation of a TCR with antigenic specificity for human KRAS with the G12V mutation presented by the HLA-DRB1*07:01 molecule.

[0146] TCRs with antigen specificity for human KRAS with G12V mutation presented by HLA-DRB1*07:01 molecules were isolated from endometrial tumor samples from patients. Briefly, tumor samples were minced, digested, and frozen. Tumor digests were thawed and placed overnight in the absence of cytokines before cell sorting. TCRs were selected based on PD-1 and / or OX40 expression (gated on PI - T cells (live cells) > CD3+) were sorted from tumor digests using FACS. FACS results are shown in Figure 1 Isotype-stained cells were used as controls.

[0147] The sorted cell numbers were expanded for 3.5 weeks according to the rapid expansion protocol (REP).For REP, T cells were cultured in microtiter 96-well plates (3 cells / well) in the presence of OKT3 antibody, IL-2 and irradiated allogeneic PBMCs.

[0148] Amplified numbers of cells were pooled and tested for reactivity against autologous dendritic cells (DCs) pulsed with pooled 25-mer peptides or peptides encoded by 25-mer tandem minigenes (TMGs) covering various tumor-specific mutations detected in patient tumors. Each pool contained 17-21 peptides or TMGs, respectively. Interferon-γ (IFN-γ) secretion was measured by enzyme-linked immunospot (ELISPOT). Results are shown in Figure 2 In. Figure 2 As shown in , the pooled effector autologous T cells in culture numbers 7 and 8 recognized target DCs pulsed with peptide pool 1 (PP1) and peptide pool 2 (PP2).

[0149] Mutation-reactive T cell cultures were tested against autologous DC pulsed with each single peptide from the relevant peptide pool. Figure 3 Shown are the results obtained after co-culturing autologous T cell culture No. 7 (W7) with autologous DC pulsed with each of peptides 1-17 (P1-P17) from peptide pool 1 (PP1). Figure 3 As shown in Figure 1, T cells from culture number 7 showed high specificity for peptide P17. Peptide 17 (P17) encodes KRAS G12V mutation.

[0150] Total RNA was isolated from cells of autologous T cell culture number (W7). The total RNA was then subjected to rapid amplification of the 5' complementary DNA end (5' RACE) using constant primers for the TCR-α and -β chains. The TCR PCR products were then separated by standard agarose gel electrophoresis and gel extraction. The products were sequenced directly. The nucleotide sequences of the TCR α chain and β chain variable regions are SEQ ID NOs: 42 and 43, respectively. The amino acid sequences of the TCR α chain and β chain variable regions are shown in Table 6. The complementary determining regions (CDRs) are underlined.

[0151] Table 6

[0152]

[0153]

[0154] Example 2

[0155] This example demonstrates that the TCR isolated in Example 1 recognizes the KRAS G12V peptide antigen presented in the context of HLA-DR molecules.

[0156] 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 comprising a cysteine-substituted, LVL-modified murine constant region is cloned into a retroviral expression vector. The α chain murine constant region 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 β chain constant region 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 are transduced using a retroviral expression vector.

[0157] The transduced cells (effector cells) were then cultured with KRAS G12V Target autologous antigen presenting cells (APCs) pulsed with 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) were co-cultured. Effector transduced cells cultured alone, with DMSO or phorbol myristate acetate (PMA) were used as controls. Effector cells transduced with empty vector (mock) co-cultured with target autologous APCs (pulsed with 1 ng / mL KRAS G12V peptide (SEQ ID NO: 53)) were used as another control.

[0158] The responsiveness of effector cells to target cells was measured by 4-1BB expression detected by flow cytometry (gated on CD3+mTCRβ chain+ cells). The results are shown in Figure 4 In. Figure 4 As shown in , IVA12 and HB55 antibodies blocked the reactivity of effector cells against target cells, indicating that the transduced effector cells recognized the KRASG12V peptide antigen presented in the context of HLA-DR molecules.

[0159] Example 3

[0160] This example demonstrates that the TCR of Example 2 recognizes the KRAS G12V peptide antigen presented in the context of the HLA-DRB1*07:01 molecule.

[0161] 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 a donor with the DRB1 01:01 or DRB1 07:01 haplotype (target cells). G12VTarget cells were pulsed with either the WT KRAS 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 a control) (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 Table 7, "TNTC" stands for "too many to count". The percentage of mTCR expressing cells expressing 4-1BB was also measured by flow cytometry. The results are shown in Figure 5 The results showed that the TCR was specifically reactive against mutant KRAS presented by HLA-DRB*07:01.

[0162] Table 7

[0163]

[0164] Example 4

[0165] This example demonstrates the isolation of a TCR with antigenic specificity for human KRAS with the G12C mutation presented by the HLA-DRB 1*11:01 molecule.

[0166] Use of patients with KRAS G12C - Repeated in vitro sensitization (IVS) of peripheral blood T cell subsets from ovarian cancer patients expressing KRAS G12C Reactive TCR.

[0167] Autologous DCs were pulsed with the G12C mutated peptide (SEQ ID NO: 56) and co-cultured with sorted T cell subsets for 10 days and then tested for reactivity as described in Example 1.

[0168] To further enrich for reactive cells, the reactive fraction against KRAS mutant peptides was sorted based on 4-1BB / OX40 expression and restimulated with the mutant peptides. Reactive T cells were sorted based on 4-1BB / OX40 expression and sequenced.

[0169] Total RNA was isolated from cells. The total RNA was then subjected to rapid amplification of the 5' complementary DNA end (5' RACE) using constant primers for the TCR-α and -β chains. The TCR PCR products were then separated by standard agarose gel electrophoresis and gel extraction. The products were sequenced directly. The nucleotide sequences of the TCR α chain and β chain variable regions are SEQ ID NOs: 44 and 45, respectively. The amino acid sequences of the TCR α chain and β chain variable regions are shown in Table 8. The complementary determining regions (CDRs) are underlined.

[0170] Table 8

[0171]

[0172] Example 5

[0173] This example demonstrates that the TCR isolated in Example 4 recognizes the KRAS G12C peptide antigen presented in the context of HLA-DR molecules.

[0174] The nucleic acid sequence encoding the isolated G12C reactive TCR of Example 4 (comprising the nucleotide sequences of SEQ ID NO:44 and SEQ ID NO:45) and comprising a cysteine-substituted, LVL-modified murine constant region is cloned into a retroviral expression vector. The α chain murine constant region 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 β chain constant region 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 are transduced using a retroviral expression vector.

[0175] After blocking membrane MHC class II molecules using antibodies against HLA-DQ, HLA-DR, or HLA-DP or pan-specific antibodies against all of HLA-DP, HLA-DR, and HLA-DQ, transduced cells (effector cells) were inoculated with antibodies matched to a single HLA-DRB15:01 or HLA-DRB11:01 allele and pulsed with KRAS G12C Target autologous DCs or allogeneic DCs were co-cultured with 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.

[0176] The responsiveness of effector cells to target cells was measured by 4-1BB expression detected by flow cytometry (gated on CD3+mTCRβ chain+ cells). The results are shown in Fig.10 In. Fig.10 As shown in Figure 2, TCR targets KRAS presented by HLA-DRB*11:01. G12C Has specific reactivity.

[0177] Example 6

[0178] This example demonstrates that the KRAS G12C TCR-transduced PBMCs recognize KRAS G12C Peptide-pulsed autologous DCs.

[0179] Using KRAS encoding Example 5 G12C Allogeneic T cells were genetically modified by MSGV-1-retrovirus with TCR. Autologous DC were loaded with WT KRAS (SEQ ID NO: 55) or KRAS G12C Peptide (SEQ ID NO: 56) was co-cultured with TCR-transduced cells for 18 hours, followed by flow cytometry analysis of 4-1BB upregulation. The results are shown in Fig. 9 In. Fig. 9 As shown in Example 5, KRAS G12C TCR-transduced PBMCs recognize KRAS G12C Peptide-pulsed autologous DCs.

[0180] Example 7

[0181] This example demonstrates that KRAS G12V mutant protein is processed and presented by DCs and recognized by the TCR of Example 2.

[0182] 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 that upregulated 4-1BB and / or OX40 was measured by flow cytometry. The number of cells expressing IFNγ was measured by ELISPOT (spots / 2×10 4 cells). The results are shown in Figure 6 In. Figure 6 As shown in , the KRAS G12V mutant protein is processed and presented by DCs and recognized by the TCR of Example 2.

[0183] Example 8

[0184] This example demonstrates that cells transduced with the G12V-DRB1*07:01 TCR of Example 2 specifically recognize KRAS G12V Peptide.

[0185] Allogeneic T cells transduced with the G12V-DRB1*07:01 TCR of Example 2 were incubated with various concentrations of the 24-mer peptide KRAS G12V (SEQ ID NO: 53) or WT KRAS (SEQ ID NO: 55) pulsed autologous DCs were co-cultured overnight. The percentage of mTCRβ+CD8+4-1BB+ cells was measured by flow cytometry. The results are shown in Figure 7 In. Figure 7 As shown in Example 2, cells transduced with G12V-DRB1*07:01TCR specifically recognize KRAS G12V Peptide.

[0186] Example 9

[0187] Cells transduced with G12V-DRB1*07:01TCR of Example 2 specifically recognize various KRAS G12V Peptide.

[0188] Allogeneic T cells transduced with the G12V-DRB1*07:01 TCR of Example 2 were co-cultured overnight with autologous DC 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 In. Figure 8 As shown in Example 2, although cells transduced with G12V-DRB1*07:01 TCR specifically recognized all KRAS G12V Peptides, but SEQ ID NO:52 is the most preferred.

[0189] Table 9

[0190]

[0191]

[0192] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0193] Unless otherwise indicated herein or clearly contradicted by the context, in the context of describing the present invention (especially in the context of the following claims), the use of the terms "a" and "an" and "the" and "at least one" and similar indicators should be interpreted as covering both the singular and the plural. Unless otherwise indicated herein or clearly contradicted by the context, the term "at least one" used immediately following a list of one or more items (e.g., "at least one of A and B") should be interpreted as meaning one item (A or B) or any combination of two or more listed items (A and B) selected from the listed items. Unless otherwise noted, the terms "including", "having", "including" and "containing" should be interpreted as open terms (i.e., meaning "including but not limited to"). Unless otherwise indicated herein, the description of numerical ranges herein is intended only as a shorthand method of individually referring to each individual value falling within this range, and each individual value is generally incorporated into the specification as if it were individually listed herein. Unless otherwise indicated herein or clearly contradicted by the context, all methods described herein can be implemented in any suitable order. Unless otherwise claimed, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present invention and does not impose limitations on the scope of the present invention. Any language in this specification should not be construed as indicating that any unclaimed element is essential to the practice of the present invention.

[0194] Preferred embodiments of the present invention are described herein, including the best mode known to the inventor for implementing the present invention. By reading the foregoing description, the changes of these preferred embodiments are obvious to those of ordinary skill in the art. The inventor expects that the technician will appropriately apply such modifications, and the inventor intends to implement the present invention in a manner different from that specifically described herein. Therefore, as permitted by applicable law, the present invention includes all modifications and equivalents of the subject matter recited in the appended claims. In addition, the present invention includes any combination of the above-mentioned elements with all possible variant forms thereof, unless otherwise indicated herein or clearly contradictory to the context. Sequence Listing <110> The United States Department of Health and Human Services SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES) <120> HLA class II-restricted T cell receptor for mutant RAS <130> 739664 <150> US 62 / 560,930 <151> 2017-09-20 <160> 56 <170> PatentIn version 3.5 <210> 1 <211> 6 <212> PRT <213> Homo sapiens <400> 1 Asp Ser Ala Ser Asn Tyr 1 5 <210> 2 <211> 7 <212> PRT <213> Homo sapiens <400> 2 Ile Arg Ser Asn Val Gly Glu 1 5 <210> 3 <211> 13 <212> PRT <213> Homo sapiens <400> 3 Cys Ala Ala Ser Thr Gly Gly Gly Asn Lys Leu Thr Phe 1 5 10 <210> 4 <211> 6 <212> PRT <213> Homo sapiens <400> 4 Asp Phe Gln Ala Thr Thr 1 5 <210> 5 <211> 7 <212> PRT <213> Homo sapiens <400> 5 Ser Asn Glu Gly Ser Lys Ala 1 5 <210> 6 <211> 15 <212> PRT <213> Homo sapiens <400> 6 Cys Ser Ala Arg Glu Gly Ala Gly Gly Met Gly Thr Gln Tyr Phe 1 5 10 15 <210> 7 <211> 6 <212> PRT <213> Homo sapiens <400> 7 Ser Ser Asn Phe Tyr Ala 1 5 <210> 8 <211> 7 <212> PRT <213> Homo sapiens <400> 8 Met Thr Leu Asn Gly Asp Glu 1 5 <210> 9 <211> 11 <212> PRT <213> Homo sapiens <400> 9 Cys Ala Phe Thr Thr Gly Asn Gln Phe Tyr Phe 1 5 10 <210> 10 <211> 6 <212> PRT <213> Homo sapiens <400> 10 Ser Gly His Asp Tyr Leu 1 5 <210> 11 <211> 6 <212> PRT <213> Homo sapiens <400> 11 Phe Asn Asn Asn Val Pro 1 5 <210> 12 <211> 16 <212> PRT <213> Homo sapiens <400> 12 Cys Ala Ser Ser Ser Tyr Gly Gly Tyr Ser Asn Gln Pro Gln His Phe 1 5 10 15 <210> 13 <211> 131 <212> PRT <213> Homo sapiens <400> 13 Met Thr Ser Ile Arg Ala Val Phe Ile Phe Leu Trp Leu Gln Leu Asp 1 5 10 15 Leu Val Asn Gly Glu Asn Val Glu Gln His Pro Ser Thr Leu Ser Val 20 25 30 Gln Glu Gly Asp Ser Ala Val Ile Lys Cys Thr Tyr Ser Asp Ser Ala 35 40 45 Ser Asn Tyr Phe Pro Trp Tyr Lys Gln Glu Leu Gly Lys Gly Pro Gln 50 55 60 Leu Ile Ile Asp Ile Arg Ser Asn Val Gly Glu Lys Lys Asp Gln Arg 65 70 75 80 Ile Ala Val Thr Leu Asn Lys Thr Ala Lys His Phe Ser Leu His Ile 85 90 95 Thr Glu Thr Gln Pro Glu Asp Ser Ala Val Tyr Phe Cys Ala Ala Ser 100 105 110 Thr Gly Gly Gly Asn Lys Leu Thr Phe Gly Thr Gly Thr Gln Leu Lys 115 120 125 Val Glu Leu 130 <210> 14 <211> 132 <212> PRT <213> Homo sapiens <400> 14 Met Leu Leu Leu Leu Leu Leu Leu Gly Pro Ala Gly Ser Gly Leu Gly 1 5 10 15 Ala Val Val Ser Gln His Pro Ser Arg Val Ile Cys Lys Ser Gly Thr 20 25 30 Ser Val Lys Ile Glu Cys Arg Ser Leu Asp Phe Gln Ala Thr Thr Met 35 40 45 Phe Trp Tyr Arg Gln Phe Pro Lys Gln Ser Leu Met Leu Met Ala Thr 50 55 60 Ser Asn Glu Gly Ser Lys Ala Thr Tyr Glu Gln Gly Val Glu Lys Asp 65 70 75 80 Lys Phe Leu Ile Asn His Ala Ser Leu Thr Leu Ser Thr Leu Thr Val 85 90 95 Thr Ser Ala His Pro Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg 100 105 110 Glu Gly Ala Gly Gly Met Gly Thr Gln Tyr Phe Gly Pro Gly Thr Arg 115 120 125 Leu Leu Val Leu 130 <210> 15 <211> 132 <212> PRT <213> Homo sapiens <400> 15 Met Glu Lys Asn Pro Leu Ala Ala Pro Leu Leu Ile Leu Trp Phe His 1 5 10 15 Leu Asp Cys Val Ser Ser Ile Leu Asn Val Glu Gln Ser Pro Gln Ser 20 25 30 Leu His Val Gln Glu Gly Asp Ser Thr Asn Phe Thr Cys Ser Phe Pro 35 40 45 Ser Ser Asn Phe Tyr Ala Leu His Trp Tyr Arg Trp Glu Thr Ala Lys 50 55 60 Ser Pro Glu Ala Leu Phe Val Met Thr Leu Asn Gly Asp Glu Lys Lys 65 70 75 80 Lys Gly Arg Ile Ser Ala Thr Leu Asn Thr Lys Glu Gly Tyr Ser Tyr 85 90 95 Leu Tyr Ile Lys Gly Ser Gln Pro Glu Asp Ser Ala Thr Tyr Leu Cys 100 105 110 Ala Phe Thr Thr Gly Asn Gln Phe Tyr Phe Gly Thr Gly Thr Ser Leu 115 120 125 Thr Val Ile Pro 130 <210> 16 <211> 137 <212> PRT <213> Homo sapiens <400> 16 Met Gly Ser Trp Thr Leu Cys Cys Val Ser Leu Cys Ile Leu Val Ala 1 5 10 15 Lys His Thr Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr 20 25 30 Glu Met Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His 35 40 45 Asp Tyr Leu Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu 50 55 60 Leu Ile Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro 65 70 75 80 Glu Asp Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu 85 90 95 Lys Ile Gln Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala 100 105 110 Ser Ser Ser Tyr Gly Gly Tyr Ser Asn Gln Pro Gln His Phe Gly Asp 115 120 125 Gly Thr Arg Leu Ser Ile Leu Glu Asp 130 135 <210> 17 <211> 189 <212> PRT <213> Homo sapiens <400> 17 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Gly Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr 20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly 35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr 50 55 60 Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys 65 70 75 80 Val Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile His His Tyr 85 90 95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Glu Asp Val Pro Met Val 100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Pro Ser Arg Thr Val Asp Thr Lys 115 120 125 Gln Ala Gln Asp Leu Ala Arg Ser Tyr Gly Ile Pro Phe Ile Glu Thr 130 135 140 Ser Ala Lys Thr Arg Gln Arg Val Glu Asp Ala Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Gln Tyr Arg Leu Lys Lys Ile Ser Lys Glu Glu Lys 165 170 175 Thr Pro Gly Cys Val Lys Ile Lys Lys Cys Ile Ile Met 180 185 <210> 18 <211> 188 <212> PRT <213> Homo sapiens <400> 18 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Gly Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr 20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly 35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr 50 55 60 Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys 65 70 75 80 Val Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile His His Tyr 85 90 95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Glu Asp Val Pro Met Val 100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Pro Ser Arg Thr Val Asp Thr Lys 115 120 125 Gln Ala Gln Asp Leu Ala Arg Ser Tyr Gly Ile Pro Phe Ile Glu Thr 130 135 140 Ser Ala Lys Thr Arg Gln Gly Val Asp Asp Ala Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Lys His Lys Glu Lys Met Ser Lys Asp Gly Lys Lys 165 170 175 Lys Lys Lys Lys Ser Lys Thr Lys Cys Val Ile Met 180 185 <210> 19 <211> 189 <212> PRT <213> Homo sapiens <400> 19 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Gly Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr 20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly 35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr 50 55 60 Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys 65 70 75 80 Val Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile His Gln Tyr 85 90 95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Asp Asp Val Pro Met Val 100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Ala Ala Arg Thr Val Glu Ser Arg 115 120 125 Gln Ala Gln Asp Leu Ala Arg Ser Tyr Gly Ile Pro Tyr Ile Glu Thr 130 135 140 Ser Ala Lys Thr Arg Gln Gly Val Glu Asp Ala Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Gln His Lys Leu Arg Lys Leu Asn Pro Pro Asp Glu 165 170 175 Ser Gly Pro Gly Cys Met Ser Cys Lys Cys Val Leu Ser 180 185 <210> 20 <211> 189 <212> PRT <213> Homo sapiens <400> 20 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Gly Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr 20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly 35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr 50 55 60 Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys 65 70 75 80 Val Phe Ala Ile Asn Asn Ser Lys Ser Phe Ala Asp Ile Asn Leu Tyr 85 90 95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Asp Asp Val Pro Met Val 100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Pro Thr Arg Thr Val Asp Thr Lys 115 120 125 Gln Ala His Glu Leu Ala Lys Ser Tyr Gly Ile Pro Phe Ile Glu Thr 130 135 140 Ser Ala Lys Thr Arg Gln Gly Val Glu Asp Ala Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Gln Tyr Arg Met Lys Lys Leu Asn Ser Ser Asp Asp 165 170 175 Gly Thr Gln Gly Cys Met Gly Leu Pro Cys Val Val Met 180 185 <210> 21 <211> 189 <212> PRT <213> Homo sapiens <400> 21 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Val Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr 20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly 35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr 50 55 60 Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys 65 70 75 80 Val Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile His His Tyr 85 90 95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Glu Asp Val Pro Met Val 100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Pro Ser Arg Thr Val Asp Thr Lys 115 120 125 Gln Ala Gln Asp Leu Ala Arg Ser Tyr Gly Ile Pro Phe Ile Glu Thr 130 135 140 Ser Ala Lys Thr Arg Gln Arg Val Glu Asp Ala Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Gln Tyr Arg Leu Lys Lys Ile Ser Lys Glu Glu Lys 165 170 175 Thr Pro Gly Cys Val Lys Ile Lys Lys Cys Ile Ile Met 180 185 <210> 22 <211> 188 <212> PRT <213> Homo sapiens <400> 22 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Val Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr 20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly 35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr 50 55 60 Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys 65 70 75 80 Val Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile His His Tyr 85 90 95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Glu Asp Val Pro Met Val 100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Pro Ser Arg Thr Val Asp Thr Lys 115 120 125 Gln Ala Gln Asp Leu Ala Arg Ser Tyr Gly Ile Pro Phe Ile Glu Thr 130 135 140 Ser Ala Lys Thr Arg Gln Gly Val Asp Asp Ala Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Lys His Lys Glu Lys Met Ser Lys Asp Gly Lys Lys 165 170 175 Lys Lys Lys Lys Ser Lys Thr Lys Cys Val Ile Met 180 185 <210> 23 <211> 189 <212> PRT <213> Homo sapiens <400> 23 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Val Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr 20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly 35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr 50 55 60 Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys 65 70 75 80 Val Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile His Gln Tyr 85 90 95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Asp Asp Val Pro Met Val 100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Ala Ala Arg Thr Val Glu Ser Arg 115 120 125 Gln Ala Gln Asp Leu Ala Arg Ser Tyr Gly Ile Pro Tyr Ile Glu Thr 130 135 140 Ser Ala Lys Thr Arg Gln Gly Val Glu Asp Ala Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Gln His Lys Leu Arg Lys Leu Asn Pro Pro Asp Glu 165 170 175 Ser Gly Pro Gly Cys Met Ser Cys Lys Cys Val Leu Ser 180 185 <210> 24 <211> 189 <212> PRT <213> Homo sapiens <400> 24 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Val Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr 20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly 35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr 50 55 60 Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys 65 70 75 80 Val Phe Ala Ile Asn Asn Ser Lys Ser Phe Ala Asp Ile Asn Leu Tyr 85 90 95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Asp Asp Val Pro Met Val 100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Pro Thr Arg Thr Val Asp Thr Lys 115 120 125 Gln Ala His Glu Leu Ala Lys Ser Tyr Gly Ile Pro Phe Ile Glu Thr 130 135 140 Ser Ala Lys Thr Arg Gln Gly Val Glu Asp Ala Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Gln Tyr Arg Met Lys Lys Leu Asn Ser Ser Asp Asp 165 170 175 Gly Thr Gln Gly Cys Met Gly Leu Pro Cys Val Val Met 180 185 <210> 25 <211> 189 <212> PRT <213> Homo sapiens <400> 25 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Cys Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr 20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly 35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr 50 55 60 Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys 65 70 75 80 Val Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile His His Tyr 85 90 95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Glu Asp Val Pro Met Val 100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Pro Ser Arg Thr Val Asp Thr Lys 115 120 125 Gln Ala Gln Asp Leu Ala Arg Ser Tyr Gly Ile Pro Phe Ile Glu Thr 130 135 140 Ser Ala Lys Thr Arg Gln Arg Val Glu Asp Ala Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Gln Tyr Arg Leu Lys Lys Ile Ser Lys Glu Glu Lys 165 170 175 Thr Pro Gly Cys Val Lys Ile Lys Lys Cys Ile Ile Met 180 185 <210> 26 <211> 188 <212> PRT <213> Homo sapiens <400> 26 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Cys Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr 20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly 35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr 50 55 60 Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys 65 70 75 80 Val Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile His His Tyr 85 90 95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Glu Asp Val Pro Met Val 100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Pro Ser Arg Thr Val Asp Thr Lys 115 120 125 Gln Ala Gln Asp Leu Ala Arg Ser Tyr Gly Ile Pro Phe Ile Glu Thr 130 135 140 Ser Ala Lys Thr Arg Gln Gly Val Asp Asp Ala Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Lys His Lys Glu Lys Met Ser Lys Asp Gly Lys Lys 165 170 175 Lys Lys Lys Lys Ser Lys Thr Lys Cys Val Ile Met 180 185 <210> 27 <211> 189 <212> PRT <213> Homo sapiens <400> 27 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Cys Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr 20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly 35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr 50 55 60 Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys 65 70 75 80 Val Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile His Gln Tyr 85 90 95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Asp Asp Val Pro Met Val 100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Ala Ala Arg Thr Val Glu Ser Arg 115 120 125 Gln Ala Gln Asp Leu Ala Arg Ser Tyr Gly Ile Pro Tyr Ile Glu Thr 130 135 140 Ser Ala Lys Thr Arg Gln Gly Val Glu Asp Ala Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Gln His Lys Leu Arg Lys Leu Asn Pro Pro Asp Glu 165 170 175 Ser Gly Pro Gly Cys Met Ser Cys Lys Cys Val Leu Ser 180 185 <210> 28 <211> 189 <212> PRT <213> Homo sapiens <400> 28 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Cys Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr 20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly 35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr 50 55 60 Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys 65 70 75 80 Val Phe Ala Ile Asn Asn Ser Lys Ser Phe Ala Asp Ile Asn Leu Tyr 85 90 95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Asp Asp Val Pro Met Val 100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Pro Thr Arg Thr Val Asp Thr Lys 115 120 125 Gln Ala His Glu Leu Ala Lys Ser Tyr Gly Ile Pro Phe Ile Glu Thr 130 135 140 Ser Ala Lys Thr Arg Gln Gly Val Glu Asp Ala Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Gln Tyr Arg Met Lys Lys Leu Asn Ser Ser Asp Asp 165 170 175 Gly Thr Gln Gly Cys Met Gly Leu Pro Cys Val Val Met 180 185 <210> 29 <211> 23 <212> PRT <213> Homo sapiens <400> 29 Thr Glu Tyr Lys Leu Val Val Val Gly Ala Gly Gly Val Gly Lys Ser 1 5 10 15 Ala Leu Thr Ile Gln Leu Ile 20 <210> 30 <211> 137 <212> PRT <213> Artificial Sequence <220> <223> synthesis <220> <221> MISC_FEATURE <222> (48)..(48) <223> X is Thr or Cys <220> <221> MISC_FEATURE <222> (112)..(112) <223> X is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp <220> <221> MISC_FEATURE <222> (114)..(114) <223> X is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp <220> <221> MISC_FEATURE <222> (115)..(115) <223> X is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp <400> 30 Asp Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys Asp Pro Arg 1 5 10 15 Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser Gln Ile 20 25 30 Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile Thr Asp Lys Xaa 35 40 45 Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala Ile Ala 50 55 60 Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile Phe Lys Glu Thr 65 70 75 80 Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala Thr Leu Thr 85 90 95 Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gln Asn Leu Xaa 100 105 110 Val Xaa Xaa Leu Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu 115 120 125 Leu Met Thr Leu Arg Leu Trp Ser Ser 130 135 <210> 31 <211> 173 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> MISC_FEATURE <222> (57)..(57) <223> X is Ser or Cys <400> 31 Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser Leu Phe Glu Pro 1 5 10 15 Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr Leu Val Cys Leu 20 25 30 Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn 35 40 45 Gly Lys Glu Val His Ser Gly Val Xaa Thr Asp Pro Gln Ala Tyr Lys 50 55 60 Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala 65 70 75 80 Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe 85 90 95 His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser Pro Lys Pro 100 105 110 Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly 115 120 125 Ile Thr Ser Ala Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu 130 135 140 Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser 145 150 155 160 Thr Leu Val Val Met Ala Met Val Lys Arg Lys Asn Ser 165 170 <210> 32 <211> 137 <212> PRT <213> Mus musculus <400> 32 Asp Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys Asp Pro Arg 1 5 10 15 Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser Gln Ile 20 25 30 Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile Thr Asp Lys Thr 35 40 45 Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala Ile Ala 50 55 60 Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile Phe Lys Glu Thr 65 70 75 80 Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala Thr Leu Thr 85 90 95 Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gln Asn Leu Ser 100 105 110 Val Met Gly Leu Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu 115 120 125 Leu Met Thr Leu Arg Leu Trp Ser Ser 130 135 <210> 33 <211> 173 <212> PRT <213> Mus musculus <400> 33 Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser Leu Phe Glu Pro 1 5 10 15 Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr Leu Val Cys Leu 20 25 30 Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn 35 40 45 Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Ala Tyr Lys 50 55 60 Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala 65 70 75 80 Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe 85 90 95 His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser Pro Lys Pro 100 105 110 Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly 115 120 125 Ile Thr Ser Ala Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu 130 135 140 Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser 145 150 155 160 Thr Leu Val Val Met Ala Met Val Lys Arg Lys Asn Ser 165 170 <210> 34 <211> 268 <212> PRT <213> Artificial Sequence <220> <223> synthesis <220> <221> MISC_FEATURE <222> (179)..(179) <223> X is Thr or Cys <220> <221> MISC_FEATURE <222> (243)..(243) <223> X is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp <220> <221> MISC_FEATURE <222> (245)..(245) <223> X is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp <220> <221> MISC_FEATURE <222> (246)..(246) <223> X is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp <400> 34 Met Thr Ser Ile Arg Ala Val Phe Ile Phe Leu Trp Leu Gln Leu Asp 1 5 10 15 Leu Val Asn Gly Glu Asn Val Glu Gln His Pro Ser Thr Leu Ser Val 20 25 30 Gln Glu Gly Asp Ser Ala Val Ile Lys Cys Thr Tyr Ser Asp Ser Ala 35 40 45 Ser Asn Tyr Phe Pro Trp Tyr Lys Gln Glu Leu Gly Lys Gly Pro Gln 50 55 60 Leu Ile Ile Asp Ile Arg Ser Asn Val Gly Glu Lys Lys Asp Gln Arg 65 70 75 80 Ile Ala Val Thr Leu Asn Lys Thr Ala Lys His Phe Ser Leu His Ile 85 90 95 Thr Glu Thr Gln Pro Glu Asp Ser Ala Val Tyr Phe Cys Ala Ala Ser 100 105 110 Thr Gly Gly Gly Asn Lys Leu Thr Phe Gly Thr Gly Thr Gln Leu Lys 115 120 125 Val Glu Leu Asp Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys 130 135 140 Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp 145 150 155 160 Ser Gln Ile Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile Thr 165 170 175 Asp Lys Xaa Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly 180 185 190 Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile Phe 195 200 205 Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala 210 215 220 Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gln 225 230 235 240 Asn Leu Xaa Val Xaa Xaa Leu Arg Ile Leu Leu Leu Lys Val Ala Gly 245 250 255 Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 <210> 35 <211> 305 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <220> <221> MISC_FEATURE <222> (189)..(189) <223> X is Ser or Cys <400> 35 Met Leu Leu Leu Leu Leu Leu Leu Gly Pro Ala Gly Ser Gly Leu Gly 1 5 10 15 Ala Val Val Ser Gln His Pro Ser Arg Val Ile Cys Lys Ser Gly Thr 20 25 30 Ser Val Lys Ile Glu Cys Arg Ser Leu Asp Phe Gln Ala Thr Thr Met 35 40 45 Phe Trp Tyr Arg Gln Phe Pro Lys Gln Ser Leu Met Leu Met Ala Thr 50 55 60 Ser Asn Glu Gly Ser Lys Ala Thr Tyr Glu Gln Gly Val Glu Lys Asp 65 70 75 80 Lys Phe Leu Ile Asn His Ala Ser Leu Thr Leu Ser Thr Leu Thr Val 85 90 95 Thr Ser Ala His Pro Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg 100 105 110 Glu Gly Ala Gly Gly Met Gly Thr Gln Tyr Phe Gly Pro Gly Thr Arg 115 120 125 Leu Leu Val Leu Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser 130 135 140 Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Xaa Thr Asp Pro 180 185 190 Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu 195 200 205 Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys 210 215 220 Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly 225 230 235 240 Ser Pro Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg 245 250 255 Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr Gln Gln Gly Val Leu Ser 260 265 270 Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala 275 280 285 Val Leu Val Ser Thr Leu Val Val Met Ala Met Val Lys Arg Lys Asn 290 295 300 Ser 305 <210> 36 <211> 269 <212> PRT <213> Artificial Sequence <220> <223> synthesis <220> <221> MISC_FEATURE <222> (180)..(180) <223> X is Thr or Cys <220> <221> MISC_FEATURE <222> (244)..(244) <223> X is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp <220> <221> MISC_FEATURE <222> (246)..(246) <223> X is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp <220> <221> MISC_FEATURE <222> (247)..(247) <223> X is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp <400> 36 Met Glu Lys Asn Pro Leu Ala Ala Pro Leu Leu Ile Leu Trp Phe His 1 5 10 15 Leu Asp Cys Val Ser Ser Ile Leu Asn Val Glu Gln Ser Pro Gln Ser 20 25 30 Leu His Val Gln Glu Gly Asp Ser Thr Asn Phe Thr Cys Ser Phe Pro 35 40 45 Ser Ser Asn Phe Tyr Ala Leu His Trp Tyr Arg Trp Glu Thr Ala Lys 50 55 60 Ser Pro Glu Ala Leu Phe Val Met Thr Leu Asn Gly Asp Glu Lys Lys 65 70 75 80 Lys Gly Arg Ile Ser Ala Thr Leu Asn Thr Lys Glu Gly Tyr Ser Tyr 85 90 95 Leu Tyr Ile Lys Gly Ser Gln Pro Glu Asp Ser Ala Thr Tyr Leu Cys 100 105 110 Ala Phe Thr Thr Gly Asn Gln Phe Tyr Phe Gly Thr Gly Thr Ser Leu 115 120 125 Thr Val Ile Pro Asp Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu 130 135 140 Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe 145 150 155 160 Asp Ser Gln Ile Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile 165 170 175 Thr Asp Lys Xaa Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn 180 185 190 Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile 195 200 205 Phe Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp 210 215 220 Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe 225 230 235 240 Gln Asn Leu Xaa Val Xaa Xaa Leu Arg Ile Leu Leu Leu Lys Val Ala 245 250 255 Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 <210> 37 <211> 310 <212> PRT <213> Artificial Sequence <220> <223> synthesis <220> <221> MISC_FEATURE <222> (194)..(194) <223> X is Ser or Cys <400> 37 Met Gly Ser Trp Thr Leu Cys Cys Val Ser Leu Cys Ile Leu Val Ala 1 5 10 15 Lys His Thr Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr 20 25 30 Glu Met Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His 35 40 45 Asp Tyr Leu Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu 50 55 60 Leu Ile Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro 65 70 75 80 Glu Asp Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu 85 90 95 Lys Ile Gln Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala 100 105 110 Ser Ser Ser Tyr Gly Gly Tyr Ser Asn Gln Pro Gln His Phe Gly Asp 115 120 125 Gly Thr Arg Leu Ser Ile Leu Glu Asp Glu Asp Leu Arg Asn Val Thr 130 135 140 Pro Pro Lys Val Ser Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn 145 150 155 160 Lys Gln Lys Ala Thr Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp 165 170 175 His Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly 180 185 190 Val Xaa Thr Asp Pro Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys 195 200 205 Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg 210 215 220 Asn His Phe Arg Cys Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp 225 230 235 240 Lys Trp Pro Glu Gly Ser Pro Lys Pro Val Thr Gln Asn Ile Ser Ala 245 250 255 Glu Ala Trp Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr Gln 260 265 270 Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys 275 280 285 Ala Thr Leu Tyr Ala Val Leu Val Ser Thr Leu Val Val Met Ala Met 290 295 300 Val Lys Arg Lys Asn Ser 305 310 <210> 38 <211> 268 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 38 Met Thr Ser Ile Arg Ala Val Phe Ile Phe Leu Trp Leu Gln Leu Asp 1 5 10 15 Leu Val Asn Gly Glu Asn Val Glu Gln His Pro Ser Thr Leu Ser Val 20 25 30 Gln Glu Gly Asp Ser Ala Val Ile Lys Cys Thr Tyr Ser Asp Ser Ala 35 40 45 Ser Asn Tyr Phe Pro Trp Tyr Lys Gln Glu Leu Gly Lys Gly Pro Gln 50 55 60 Leu Ile Ile Asp Ile Arg Ser Asn Val Gly Glu Lys Lys Asp Gln Arg 65 70 75 80 Ile Ala Val Thr Leu Asn Lys Thr Ala Lys His Phe Ser Leu His Ile 85 90 95 Thr Glu Thr Gln Pro Glu Asp Ser Ala Val Tyr Phe Cys Ala Ala Ser 100 105 110 Thr Gly Gly Gly Asn Lys Leu Thr Phe Gly Thr Gly Thr Gln Leu Lys 115 120 125 Val Glu Leu Asp Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys 130 135 140 Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp 145 150 155 160 Ser Gln Ile Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile Thr 165 170 175 Asp Lys Thr Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly 180 185 190 Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile Phe 195 200 205 Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp Ala 210 215 220 Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe Gln 225 230 235 240 Asn Leu Ser Val Met Gly Leu Arg Ile Leu Leu Leu Lys Val Ala Gly 245 250 255 Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 <210> 39 <211> 305 <212> PRT <213> Artificial Sequence <220> <223> synthesis <400> 39 Met Leu Leu Leu Leu Leu Leu Gly Pro Ala Gly Ser Gly Leu Gly 1 5 10 15 Ala Val Val Ser Gln His Pro Ser Arg Val Ile Cys Lys Ser Gly Thr 20 25 30 Ser Val Lys Ile Glu Cys Arg Ser Leu Asp Phe Gln Ala Thr Thr Met 35 40 45 Phe Trp Tyr Arg Gln Phe Pro Lys Gln Ser Leu Met Leu Met Ala Thr 50 55 60 Ser Asn Glu Gly Ser Lys Ala Thr Tyr Glu Gln Gly Val Glu Lys Asp 65 70 75 80 Lys Phe Leu Ile Asn His Ala Ser Leu Thr Leu Ser Thr Leu Thr Val 85 90 95 Thr Ser Ala His Pro Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg 100 105 110 Glu Gly Ala Gly Gly Met Gly Thr Gln Tyr Phe Gly Pro Gly Thr Arg 115 120 125 Leu Leu Val Leu Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser 130 135 140 Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro 180 185 190 Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu 195 200 205 Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys 210 215 220 Gln Val Gln Phe His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly 225 230 235 240 Ser Pro Lys Pro Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg 245 250 255 Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr Gln Gln Gly Val Leu Ser 260 265 270 Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala 275 280 285 Val Leu Val Ser Thr Leu Val Val Met Ala Met Val Lys Arg Lys Asn 290 295 300 Ser 305 <210> 40 <211> 269 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 40 Met Glu Lys Asn Pro Leu Ala Ala Pro Leu Leu Ile Leu Trp Phe His 1 5 10 15 Leu Asp Cys Val Ser Ser Ile Leu Asn Val Glu Gln Ser Pro Gln Ser 20 25 30 Leu His Val Gln Glu Gly Asp Ser Thr Asn Phe Thr Cys Ser Phe Pro 35 40 45 Ser Ser Asn Phe Tyr Ala Leu His Trp Tyr Arg Trp Glu Thr Ala Lys 50 55 60 Ser Pro Glu Ala Leu Phe Val Met Thr Leu Asn Gly Asp Glu Lys Lys 65 70 75 80 Lys Gly Arg Ile Ser Ala Thr Leu Asn Thr Lys Glu Gly Tyr Ser Tyr 85 90 95 Leu Tyr Ile Lys Gly Ser Gln Pro Glu Asp Ser Ala Thr Tyr Leu Cys 100 105 110 Ala Phe Thr Thr Gly Asn Gln Phe Tyr Phe Gly Thr Gly Thr Ser Leu 115 120 125 Thr Val Ile Pro Asp Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu 130 135 140 Lys Asp Pro Arg Ser Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe 145 150 155 160 Asp Ser Gln Ile Asn Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile 165 170 175 Thr Asp Lys Thr Val Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn 180 185 190 Gly Ala Ile Ala Trp Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile 195 200 205 Phe Lys Glu Thr Asn Ala Thr Tyr Pro Ser Ser Asp Val Pro Cys Asp 210 215 220 Ala Thr Leu Thr Glu Lys Ser Phe Glu Thr Asp Met Asn Leu Asn Phe 225 230 235 240 Gln Asn Leu Ser Val Met Gly Leu Arg Ile Leu Leu Leu Lys Val Ala 245 250 255 Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 <210> 41 <211> 310 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 41 Met Gly Ser Trp Thr Leu Cys Cys Val Ser Leu Cys Ile Leu Val Ala 1 5 10 15 Lys His Thr Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr 20 25 30 Glu Met Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His 35 40 45 Asp Tyr Leu Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu 50 55 60 Leu Ile Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro 65 70 75 80 Glu Asp Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu 85 90 95 Lys Ile Gln Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala 100 105 110 Ser Ser Ser Tyr Gly Gly Tyr Ser Asn Gln Pro Gln His Phe Gly Asp 115 120 125 Gly Thr Arg Leu Ser Ile Leu Glu Asp Glu Asp Leu Arg Asn Val Thr 130 135 140 Pro Pro Lys Val Ser Leu Phe Glu Pro Ser Lys Ala Glu Ile Ala Asn 145 150 155 160 Lys Gln Lys Ala Thr Leu Val Cys Leu Ala Arg Gly Phe Phe Pro Asp 165 170 175 His Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly 180 185 190 Val Ser Thr Asp Pro Gln Ala Tyr Lys Glu Ser Asn Tyr Ser Tyr Cys 195 200 205 Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp His Asn Pro Arg 210 215 220 The hybridization of Arg Cys Gln Val Gln was hybridized to Gly Leu Ser Glu Glu Asp 225 230 235 240 Lys Trp Pro Glu Gly Ser Pro Lys Pro Val Thr Gln Asn Ile Ser Ala 245 250 255 Glu Ala Trp Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr Gln 260 265 270 Gln Gly Val Can Be Thr and Tyr Glu and Gly Lys 275 280 285 Ala Thr Leu Tyr Ala Val Leu Val Ser Thr Leu Val Val Met Ala Met 290,295,300 Val Lys Arg Lys Asn Ser 305 310 <210> 42 <211> 393 <212> DNA <213> Homo sapiens <400> 42 atgaccagca tcagggccgt gttcatctc ctgtggctgc agctgacct ggtgaacggc 60 gagaacgtgg agcagcaccc cagcaccctg agcgtgcagg agggcgacag cgccgtgatc 120 aagtgcacct acagcgacag cgccagcaac tactccct ggtacaagca ggagctgggc 180 aagggccccc agctgatcat cgacatcagg agcaacgtgg gcgagaagaa ggaccagagg 240 atcgccgtga ccctgaacaa gaccgccaag cacttcagcc tgcacatcac cgagacccag 300 cccgaggaca gcgccgtgta cttctgcgcc gccagcaccg gcggcggcaa caagctgacc 360 ttcggcaccg gcacccagct gaaggtggag ctg 393 <210> 43 <211> 393 <212> DNA <213> Homo sapiens <400> 43 atgaccagca tcagggccgt gttcatcttc ctgtggctgc agctggacct ggtgaacggc 60 gagaacgtgg agcagcaccc cagcaccctg agcgtgcagg agggcgacag cgccgtgatc 120 aagtgcacct acagcgacag cgccagcaac tacttcccct ggtacaagca ggagctgggc 180 aagggccccc agctgatcat cgacatcagg agcaacgtgg gcgagaagaa ggaccagagg 240 atcgccgtga ccctgaacaa gaccgccaag cacttcagcc tgcacatcac cgagacccag 300 cccgaggaca gcgccgtgta cttctgcgcc gccagcaccg gcggcggcaa caagctgacc 360 ttcggcaccg gcacccagct gaaggtggag ctg 393 <210> 44 <211> 396 <212> DNA <213> Homo sapiens <400> 44 atggagaaga accccctggc cgcccccctg ctgatcctgt ggttccacct ggactgcgtg 60 agcagcatcc tgaacgtgga gcagagcccc cagagcctgc acgtgcagga gggcgacagc 120 accaacttca cctgcagctt ccccagcagc aacttctacg ccctgcactg gtacaggtgg 180 gagaccgcca agagccccga ggccctgttc gtgatgaccc tgaacggcga cgagaagaag 240 aagggcagga tcagcgccac cctgaacacc aaggagggct acagctacct gtacatcaag 300 ggcagccagc ccgaggacag cgccacctac ctgtgcgcct tcaccaccgg caaccagttc 360 tacttcggca ccggcaccag cctgaccgtg atcccc 396 <210> 45 <211> 411 <212> DNA <213> Homo sapiens <400> 45 atgggcagct ggaccctgtg ctgcgtgagc ctgtgcatcc tggtggccaa gcacaccgac 60 gccggcgtga tccagagccc caggcacgag gtgaccgaga tgggccagga ggtgaccctg 120 aggtgcaagc ccatcagcgg ccacgactac ctgttctggt acaggcagac catgatgagg 180 ggcctggagc tgctgatcta cttcaacaac aacgtgccca tcgacgacag cggcatgccc 240 gaggacaggt tcagcgccaa gatgcccac gccagcttca gcaccctgaa gatccagccc 300 agcgagccca gggacagcgc cgtgtacttc tgcgccagca gcagctacgg cggctacagc 360 aaccagcccc agcactcgg cgacggcacc aggctgagca tcctggagga c 411 <210> 46 <211> 11 <212> PRT <213> Homo sapiens <400> 46 Leu Val Val Val Gly Ala Val Gly Val Gly Lys 1 5 10 <210> 47 <211> 12 <212> PRT <213> Homo sapiens <400> 47 Lys Leu Val Val Val Gly Ala Val Gly Val Gly Lys 1 5 10 <210> 48 <211> 12 <212> PRT <213> Homo sapiens <400> 48 Leu Val Val Val Gly Ala Val Gly Val Gly Lys Ser 1 5 10 <210> 49 <211> 13 <212> PRT <213> Homo sapiens <400> 49 Tyr Lys Leu Val Val Val Gly Ala Val Gly Val Gly Lys 1 5 10 <210> 50 <211> 13 <212> PRT <213> Homo sapiens <400> 50 Lys Leu Val Val Val Gly Ala Val Gly Val Gly Lys Ser 1 5 10 <210> 51 <211> 13 <212> PRT <213> Homo sapiens <400> 51 Leu Val Val Val Gly Ala Val Gly Val Gly Lys Ser Ala 1 5 10 <210> 52 <211> 15 <212> PRT <213> Homo sapiens <400> 52 Glu Tyr Lys Leu Val Val Val Gly Ala Val Gly Val Gly Lys Ser 1 5 10 15 <210> 53 <211> 24 <212> PRT <213> Homo sapiens <400> 53 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Val Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile 20 <210> 54 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 54 Arg Ala Lys Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys 1 5 10 15 Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro 20 25 <210> 55 <211> 24 <212> PRT <213> Homo sapiens <400> 55 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Gly Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile 20 <210> 56 <211> 24 <212> PRT <213> Homo sapiens <400> 56 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Cys Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile 20

Claims

1. An isolated or purified T cell receptor (TCR), wherein the TCR has antigenic specificity for a mutated human RAS amino acid sequence presented by a human leukocyte antigen (HLA) class II molecule, wherein the mutated human RAS amino acid sequence is a mutated human Kirsten rat sarcoma viral oncogene homolog (KRAS) amino acid sequence, a mutated human Harvey rat sarcoma viral oncogene homolog (HRAS) amino acid sequence, or a mutated human neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence, wherein the TCR comprises: (a) the α chain complementary 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 complementary determining region (CDR) 1 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.

2. The TCR of claim 1, wherein the HLA class II molecule is an HLA-DR molecule.

3. The TCR of claim 1, wherein the HLA class II molecule is an HLA-DRB1 molecule.

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

5. The TCR of claim 1, 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 replacing glycine 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 TCR of claim 5, wherein the substitution is substitution of glycine at position 12 with valine or cysteine.

7. The TCR according to any one of claims 1 to 6, comprising: (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 of (i) and (ii); or both of (iii) and (iv).

8. The TCR of any one of claims 1 to 6, further comprising: (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 beta 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 isolated or purified TCR according to any one of claims 1 to 6, comprising: (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, He, 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 strand 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, He, Pro, Phe, Met or Trp; (iii) X at position 246 of SEQ ID NO:36 is Met, Ala, Val, Leu, He, 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. An isolated or purified polypeptide comprising a functional portion of a T cell receptor (TCR), wherein the functional portion has antigenic specificity for a mutated human RAS amino acid sequence presented by a human leukocyte antigen (HLA) class II molecule, wherein the mutated human RAS amino acid sequence is a mutated human Kirsten rat sarcoma viral oncogene homolog (KRAS) amino acid sequence, a mutated human Harvey rat sarcoma viral oncogene homolog (HRAS) amino acid sequence, or a mutated human neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence, wherein the functional portion comprises: (a) a TCR α chain sequence and a TCR β chain sequence, wherein the TCR α chain sequence comprises the TCR α chain complementary 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 comprises 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, wherein the TCR α chain sequence comprises the TCR α chain complementary determining region (CDR) 1 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; and the TCR β chain sequence comprises the TCR β chain CDR1 amino acid sequence 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 isolated or purified polypeptide of 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 of (i) and (ii); or both of (iii) and (iv).

12. The isolated or purified polypeptide of claim 11, further comprising: (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, He, 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 isolated or purified polypeptide of claim 11, comprising: (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, He, 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, He, Pro, Phe, Met or Trp; (iii) X at position 246 of SEQ ID NO:36 is Met, Ala, Val, Leu, He, 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. An isolated or purified protein, wherein the protein has antigenic specificity for a mutated human RAS amino acid sequence presented by a human leukocyte antigen (HLA) class II molecule, wherein the mutated human RAS amino acid sequence is a mutated human Kirsten rat sarcoma viral oncogene homolog (KRAS) amino acid sequence, a mutated human Harvey rat sarcoma viral oncogene homolog (HRAS) amino acid sequence, or a mutated human neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence, wherein the protein comprises: (a) a TCR α chain sequence and a TCR β chain sequence, wherein the TCR α chain sequence comprises the TCR α chain complementary 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 comprises 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, wherein the TCR α chain sequence comprises the TCR α chain complementary determining region (CDR) 1 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; and the TCR β chain sequence comprises the TCR β chain CDR1 amino acid sequence 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.

15. The isolated or purified protein of claim 14, comprising: (i) a TCR α chain comprising the amino acid sequence of SEQ ID NO: 13 and a TCR β chain comprising the amino acid sequence of SEQ ID NO: 14; or (ii) a TCR α chain comprising the amino acid sequence of SEQ ID NO:15 and a TCR β chain comprising the amino acid sequence of SEQ ID NO:

16.

16. The isolated or purified protein of claim 14, further comprising: (a) a TCR alpha chain 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, He, 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 TCR β chain 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).

17. The isolated or purified protein of claim 14, comprising: (a) a TCR alpha 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, He, 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 TCR 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) a TCR alpha 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, He, 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 TCR 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).

18. An isolated or purified nucleic acid comprising a nucleotide sequence encoding the TCR of any one of claims 1 to 9, the polypeptide of any one of claims 10 to 13, or the protein of any one of claims 14 to 17.

19. A recombinant expression vector comprising the nucleic acid of claim 18.

20. An isolated or purified host cell comprising the recombinant expression vector of claim 19.

21. An isolated or purified cell population comprising the host cell of claim 20.

22. A pharmaceutical composition comprising (a) the TCR of any one of claims 1 to 9, the polypeptide of any one of claims 10 to 13, the protein of any one of claims 14 to 17, the nucleic acid of claim 18, the recombinant expression vector of claim 19, the host cell of claim 20 or the cell population of claim 21; and (b) a pharmaceutically acceptable carrier.

23. Use of the TCR of any one of claims 1 to 9, the polypeptide of any one of claims 10 to 13, the protein of any one of claims 14 to 17, the nucleic acid of claim 18, the recombinant expression vector of claim 19, the host cell of claim 20, the cell population of claim 21 or the pharmaceutical composition of claim 22 in the preparation of a medicament for detecting, treating or preventing cancer in a mammal, wherein the cancer expresses a mutated human RAS amino acid sequence, wherein the mutated human RAS amino acid sequence is a mutated human KRAS amino acid sequence, a mutated human HRAS amino acid sequence or a mutated human NRAS amino acid sequence.

24. The use of claim 23, 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 in which glycine at position 12 is substituted, wherein position 12 is defined by reference to the wild-type human KRAS amino acid sequence, the wild-type human HRAS amino acid sequence or the wild-type human NRAS amino acid sequence, respectively.

25. Use according to claim 24, wherein the substitution is substitution of glycine at position 12 with valine or cysteine.

26. The use according to claim 24 or 25, wherein the mutated human RAS amino acid sequence is a mutated human Kirsten rat sarcoma viral oncogene homolog (KRAS) amino acid sequence.

27. The use of claim 24 or 25, wherein the mutated human Ras amino acid sequence is a mutated human neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence.

28. The use according to claim 24 or 25, wherein the mutated human RAS amino acid sequence is a mutated human Harvey rat sarcoma viral oncogene homolog (HRAS) amino acid sequence.

29. The use of any one of claims 23 to 25, wherein the cancer is pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer or prostate cancer.

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