HLA class II-restricted DRB T cell receptor for RAS with G12V mutation

Abstract

An isolated or purified T cell receptor (TCR) is disclosed that has antigen specificity for a mutant human RAS amino acid sequence in which glycine at position 12 is substituted with valine. The TCR can recognize G12V RAS presented by HLA-DR heterodimers. Related polypeptides and proteins, as well as related nucleic acids, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions are also provided. Also disclosed are methods for detecting the presence of cancer in a mammal and methods for treating or preventing cancer in a mammal.

Description

[Technical Field]

[0001] Cross-reference of related applications This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 052,502, filed July 16, 2020, which is incorporated herein by reference in its entirety.

[0002] Description of federally supported research or development This invention was made with government support from the National Institutes of Health and the National Cancer Institute under project number ZIABC010984. The government reserves certain rights in this invention.

[0003] References to electronically submitted literature The computer-readable nucleotide / amino acid sequence listing submitted concurrently with this specification and identified below is incorporated herein by reference in its entirety: a 58,420-byte ASCII (text) file named "754396_ST25.txt" dated 17 May 2021. [Background technology]

[0004] Some cancers, especially when they become metastatic and unresectable, offer very limited treatment options. For example, despite advances in surgical, chemotherapy, and radiation therapy, many cancers, such as those of the pancreas, colorectal, lung, endometrium, ovarian, and prostate, may have a poor prognosis. Therefore, there is an unmet need for further cancer treatments. [Overview of the project]

[0005] Embodiments of the present invention provide an isolated or purified T cell receptor (TCR) comprising (a) all of SEQ ID NOs: 1-3, (b) all of SEQ ID NOs: 4-6, or (c) all of SEQ ID NOs: 1-6, wherein the TCR has antigen specificity to a mutant human RAS amino acid sequence in which glycine at position 12 is replaced with valine, and the mutant human RAS amino acid sequence is the amino acid sequence of mutant human Kirsten rat sarcoma virus oncogene homolog (KRAS), mutant human Harvey rat sarcoma virus oncogene homolog (HRAS), or mutant human neuroblastoma rat sarcoma virus oncogene homolog (NRAS), and the TCR is defined by the 12th position referencing the protein of wild-type human KRAS, wild-type human HRAS, or wild-type human NRAS, respectively.

[0006] 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 (a) all of SEQ ID NOs: 1-3, (b) all of SEQ ID NOs: 4-6, or (c) all of the amino acid sequences of SEQ ID NOs: 1-6.

[0007] Yet another embodiment of the present invention provides an isolated or purified protein comprising a first polypeptide chain containing the amino acid sequences of SEQ ID NOs: 1-3 and a second polypeptide chain containing the amino acid sequences of SEQ ID NOs: 4-6.

[0008] Embodiments of the present invention further provide nucleic acids, recombinant expression vectors, host cells, cell populations, and pharmaceutical compositions related to the TCR, polypeptide, and protein of the present invention.

[0009] Embodiments of the present invention provide an isolated or purified nucleic acid comprising a first nucleic acid sequence and a second nucleotide sequence from 5' to 3', wherein the first and second nucleotide sequences encode the amino acid sequences of SEQ ID NOs. 7 and 8; 8 and 7; 9 and 10; or 10 and 9, respectively.

[0010] Methods for detecting the presence of cancer in mammals, methods for treating or preventing cancer in mammals, methods for inducing an immune response against cancer in mammals, MTEYKLVVVGA V A method for generating host cells that express a TCR having antigen specificity to the peptide GVGKSALTIQLI (SEQ ID NO: 34), and a method for generating the TCR, polypeptide, and protein of the present invention are further provided by embodiments of the present invention. [Brief explanation of the drawing]

[0011] [Figure 1A-1B] Figures 1A and 1B are graphs showing the percentage of TCR mouse constant region (mTCR)-positive / CD3+ cells that were positive for IFN-γ secretion (pg / mL) (Figure 1A) or 4-1BB expression (Figure 1B) after co-culturing target cells with effector cells. Effector cells were T cells transduced with 4304 TCR1. Target cells were DCs pulsed with a specified concentration (ng / mL) of G12V 24-mer peptide (square) (KRAS mut) or the corresponding WT 24-mer peptide (circular) (KRAS wt). [Figure 2] Figure 2 is a graph showing IFN-γ secretion (spot / 3e4 cells) measured by the ELISPOT assay after co-culturing effector cells with target cells. Effector cells were healthy donor PBLs transduced with 4304 TCR1. Target cells were COS7 cells or HEK293 cells independently transfected with one of the HLA class II heterodimers shown in the figure. Target cells were loaded with G12V 24-mer peptide and cultured in or without antibodies blocking the respective pre-transfected HLA class II molecules. Target cells cultured with DMSO were used as a negative control. (i) Autologous DCs from patient 4304 co-cultured with G12V 24-mer peptide were used as a positive control, and (ii) Autologous DCs from patient 4304 co-cultured with DMSO were used as a negative control. [Modes for carrying out the invention]

[0012] The RAS family of proteins belongs to a large family of small GTPases. Although not bound by any specific theory or mechanism, when mutated, RAS proteins are thought to be involved in signal transduction in the early stages of carcinogenesis in many human cancers. A single amino acid substitution can activate the protein. Mutant RAS protein products can be constitutively activated. Mutant RAS proteins can be expressed in any of the following human cancers, for example, pancreatic cancer (e.g., pancreatic cancer), colorectal cancer, lung cancer (e.g., lung adenocarcinoma), endometrial cancer, ovarian cancer (e.g., epithelial ovarian cancer), and prostate cancer. Examples of human RAS family proteins include KRAS, HRAS, and NRAS.

[0013] KRAS is also known as GTPase KRas, V-Ki-Ras2 Kirsten rat sarcoma virus oncogene, or KRAS2. KRAS has two transcript variants: KRAS variant A and KRAS variant B. Wild-type (WT) KRAS variant A has the amino acid sequence of SEQ ID NO: 11. WT KRAS variant B has the amino acid sequence of SEQ ID NO: 12. Hereafter, "KRAS" (mutant or non-mutant (WT)) refers to both variant A and variant B unless otherwise specified. When activated, mutant KRAS binds to guanosine-5'-triphosphate (GTP) and converts GTP to guanosine-5'-diphosphate (GDP).

[0014] HRAS is another member of the RAS protein family. HRAS is also known as Harvey rat sarcoma virus oncoprotein, V-Ha-Ras Harvey rat sarcoma virus oncogene homolog, or Ras family small GTP-binding protein H-Ras. WT HRAS has the amino acid sequence of SEQ ID NO: 13.

[0015] NRAS is yet another member of the RAS protein family. NRAS is also known as GTPase NRas, V-Ras neuroblastoma RAS virus oncogene homolog, or NRAS1. WT NRAS has the amino acid sequence of SEQ ID NO: 14.

[0016] Embodiments of the present invention provide isolated or purified TCRs that have antigen specificity to the amino acid sequence of a mutant human RAS in which glycine at position 12 is substituted with valine, wherein the amino acid sequence of the mutant human RAS is the amino acid sequence of a mutant human KRAS, a mutant human HRAS, or a mutant human NRAS, and the TCR is defined by the 12th position referring to the protein of WT human KRAS, WT human HRAS, or WT human NRAS, respectively. Hereafter, references to "TCR" also refer to the functional portion and functional variants of the TCR unless otherwise specified.

[0017] The mutant human RAS amino acid sequence may be the mutant human KRAS amino acid sequence, the mutant human HRAS amino acid sequence, or the mutant human NRAS amino acid sequence. The amino acid sequences of the KRAS, NRAS, and HRAS proteins in wild-type (WT) humans each have a length of 188 to 189 amino acid residues and exhibit a high degree of identity with respect to each other. For example, the amino acid sequence of the WT human NRAS protein is 86.8% identical to the amino acid sequence of the WT human KRAS protein. Amino acid residues 1 to 86 of the WT human NRAS protein and the WT human KRAS protein are 100% identical. The amino acid sequence of the WT human HRAS protein is 86.3% identical to the amino acid sequence of the WT human KRAS protein. Amino acid residues 1 to 94 of the WT human HRAS protein and the WT human KRAS protein are 100% identical. Hereafter, unless otherwise specified, "RAS" (mutant or non-mutant (WT)) refers collectively to KRAS, HRAS, and NRAS.

[0018] In an embodiment of the present invention, the mutant human RAS amino acid sequence includes a human RAS amino acid sequence in which glycine at position 12 is substituted with valine, and position 12 is defined with reference to the corresponding WT RAS protein. The WT RAS protein may be any one of the WT KRAS protein (SEQ ID NO: 11 or 12), the WT HRAS protein (SEQ ID NO: 13), or the WT NRAS protein (SEQ ID NO: 14), because, as explained above, amino acid residues 1 to 86 of the WT human NRAS protein and the WT human KRAS protein are 100% identical, and amino acid residues 1 to 94 of the WT human HRAS protein and the WT human KRAS protein are also 100% identical. Therefore, the amino acid residue at position 12 of each of the WT KRAS, WT HRAS, and WT NRAS proteins is the same, that is, glycine.

[0019] The mutant human RAS amino acid sequence has a substitution of glycine at position 12 with valine. In this regard, embodiments of the present invention provide TCRs having antigen specificity for the amino acid sequence of any human RAS protein, polypeptide, or peptide having a G12V mutation.

[0020] Mutations and substitutions of RAS are defined herein by reference to the amino acid sequence of the corresponding WT RAS protein. Thus, mutations and substitutions of RAS are described herein by reference to the amino acid residue present at a specific position in the WT RAS protein (i.e., position 12), followed by the position number, followed by the amino acid residue that replaces that residue in the particular mutation or substitution under consideration. A RAS amino acid sequence (e.g., a RAS peptide) may contain fewer amino acid residues than the total amino acid residues of the full-length WT RAS protein. Thus, herein, position 12 is defined by reference to the WT full-length RAS protein (i.e., any one of SEQ ID NOs: 11-14), understanding that in certain examples of RAS amino acid sequences, the actual position of the corresponding residue may be different. When the position is as defined by any one of SEQ ID NOs: 11-14, the term "G12" refers to the fact that glycine is normally present at position 12 of any one of SEQ ID NOs: 11-14, and "G12V" indicates that the glycine normally present at position 12 of any one of SEQ ID NOs: 11-14 has been replaced by valine. For example, in a particular example of a RAS amino acid sequence such as TEYKLVVVGA G GVGKSALTIQLI (SEQ ID NO: 36) (an exemplary WT KRAS peptide corresponding to contiguous amino acid residues 2-24 of SEQ ID NO: 11), "G12V" refers to the substitution of the underlined glycine in SEQ ID NO: 36 by valine, even if the actual position of the underlined glycine in SEQ ID NO: 36 is 11. Hereinafter, a human RAS amino acid sequence having a G12V mutation is referred to as "G12V RAS" or "G12V".

[0021] Examples of full-length RAS proteins having a G12V mutation are listed in Table 1 below.

[0022]

Table 1

[0023] In embodiments of the present invention, the TCR has antigen specificity for the RAS peptide having the G12V mutation, and the G12V RAS peptide has any length. In embodiments of the present invention, the G12V RAS peptide has any length suitable for binding to any of the HLA class II molecules described herein. For example, the TCR may have antigen specificity for a RAS peptide having the G12V mutation, having a length of about 11 to about 30 amino acid residues, about 12 to about 24 amino acid residues, or about 18 to about 20 amino acid residues. The G12V RAS peptide may contain any sequence of amino acid residues of a mutant RAS protein containing the G12V mutation. In embodiments of the present invention, the TCR is a RAS peptide having a G12V mutation and may exhibit antigen specificity to mutant RAS peptides having lengths in the ranges of approximately 30 amino acid residues, approximately 29 amino acid residues, approximately 28 amino acid residues, approximately 27 amino acid residues, approximately 26 amino acid residues, approximately 25 amino acid residues, approximately 24 amino acid residues, approximately 23 amino acid residues, approximately 22 amino acid residues, approximately 21 amino acid residues, approximately 20 amino acid residues, approximately 19 amino acid residues, approximately 18 amino acid residues, approximately 17 amino acid residues, approximately 16 amino acid residues, approximately 15 amino acid residues, approximately 14 amino acid residues, approximately 13 amino acid residues, approximately 12 amino acid residues, approximately 11 amino acid residues, or any two of the above values. An example of a specific peptide having a G12V mutation that can be recognized by the TCR of the present invention is MTEYKLVVVGA V GVGKSALTIQLI (SEQ ID NO: 34). In embodiments of the present invention, the TCR has antigen specificity for the mutant human RAS amino acid sequence of SEQ ID NO: 34. In embodiments of the present invention, the TCR is MTEYKLVVVGAG G VGKSALTIQLI (SEQ ID NO: 35) does not exhibit antigen specificity for the wild-type human RAS amino acid sequence.

[0024] In embodiments of the present invention, the TCR of the present invention can recognize G12V RAS presented by HLA class II molecules. In this regard, the TCR can elicit an immune response when it binds to G12V RAS within the framework of an HLA class II molecule. The TCR of the present invention can recognize G12V RAS presented by an HLA class II molecule and can bind to HLA class II molecules in addition to G12V RAS.

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

[0026] The TCR of the present invention may offer one or more of a variety of advantages, including when expressed by cells used for adoptive cell transfer. G12V RAS is expressed in cancer cells but not in normal non-cancerous cells. Although not bound by any particular theory or mechanism, the TCR of the present invention is advantageously thought to target the destruction of cancer cells while minimizing or eliminating the destruction of normal non-cancerous cells, for example, by minimizing or eliminating toxicity, thereby reducing the overall impact. Furthermore, since G12V mutations are likely to occur in the early stages of tumorigenesis, G12V RAS mutations may be expressed in substantially all cancer cells of a patient. The TCR of the present invention is advantageously capable of successfully treating or preventing G12V RAS-positive cancers that do not respond to other types of treatment, such as chemotherapy, surgery, or radiation therapy. Furthermore, the TCR of the present invention can provide highly avid recognition with high binding activity of G12V RAS, which may provide the ability to recognize unmanipulated tumor cells (e.g., tumor cells not treated with interferon (IFN)-γ, not transfected with a vector encoding one or both of the G12V RAS and HLA-DRB1*01:HLA-DRA*01 heterodimers, not pulsed with the G12V RAS peptide, or a combination thereof). KRAS mutations are found in approximately 70% of pancreatic cancers, 36% of colorectal cancers, and 20% of lung cancers. Most commonly, mutations occur at codon 12 (coding glycine, G). G12V RAS mutations are found in approximately 27% and 8% of patients with pancreatic and colorectal cancers, respectively. Furthermore, the HLA-DRB1*01 allele is commonly expressed in humans of Caucasian ethnicity. For example, this allele is expressed in approximately 20% of people of Caucasian descent in the United States. Given that this allele is expressed in approximately 20% of people of Caucasian descent in the United States, that variant RAS is expressed in approximately 30% of all cancer patients, and that approximately 25% of RAS mutations are G12V RAS, the TCR has the potential to treat approximately 1.5% (0.2 × 0.3 × 0.25 = 0.015) of all people of Caucasian descent with cancer in the United States.Therefore, the TCRs of the present invention can increase the number of immunotherapy-eligible cancer patients, including patients expressing the HLA-DRB1*01 allele, which may not be eligible for immunotherapy using TCRs that recognize RAS presented by other MHC molecules. Furthermore, the TCRs, polypeptides, and proteins of the present invention include the amino acid sequences of the human CDR and variable region, thereby reducing the risk of rejection by the human immune system compared to TCRs, polypeptides, and proteins that include, for example, the amino acid sequences of the mouse CDR and variable region.

[0027] The term "antigen specificity," as used herein, means that the TCR specifically binds to and is immunologically recognizable to the G12V RAS with high binding activity. For example, when the TCR is co-cultured with (a) antigen-negative HLA class II molecule-positive target cells pulsed with a low concentration of G12V RAS peptide (e.g., approximately 0.05 ng / mL to approximately 10 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 8 ng / mL, 10 ng / mL, or any two of the above values), or (b) antigen-negative HLA class II molecule-positive target cells into which a nucleotide sequence encoding the G12V RAS has been introduced so that the target cells express the G12V RAS, approximately 1 × 10⁻¹⁶ cells express the TCR. 4 ~Approx. 1×10 5 If a T cell secretes at least approximately 200 pg / mL or more of IFN-γ (for example, 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 within a range defined by any two of the above values), it may be considered to have "antigen specificity" for G12V RAS. Cells expressing the TCR of the present invention can also secrete IFN-γ when co-cultured with antigen-negative HLA class II molecule-positive target cells pulsed with a higher concentration of G12V RAS peptide. The HLA class II molecule may be any of the HLA class II molecules described herein.

[0028] Alternatively, a TCR may be considered to have "antigen specificity" for G12V RAS if, when co-cultured with (a) antigen-negative HLA class II molecule-positive target cells pulsed with a low concentration of G12V RAS peptide or (b) antigen-negative HLA class II molecule-positive target cells into which a nucleotide sequence encoding G12V RAS has been introduced so that the target cells express G12V RAS, the T cells expressing the TCR secrete at least twice (e.g., five times) more IFN-γ compared to the amount of IFN-γ expressed by the negative control. The negative control may be, for example, (i)(a) an unrelated peptide at the same concentration (e.g., several other peptides having a different sequence from the G12V RAS peptide) or (b) T cells expressing the TCR, co-cultured with antigen-negative HLA class II molecule-positive target cells into which a nucleotide sequence encoding the unrelated peptide has been introduced so that the target cells express the unrelated peptide; or (ii)(a) an antigen-negative HLA class II molecule-positive target cell pulsed with the same concentration of the G12V RAS peptide or (b) an untransduced TCR cell (e.g., derived from a PBMC that does not express the TCR), co-cultured with antigen-negative HLA class II molecule-positive target cells into which a nucleotide sequence encoding the G12V RAS has been introduced so that the target cells express the G12V RAS. The HLA class II molecule expressed by the target cells of the negative control is the same HLA class II molecule expressed by the target cells co-cultured with the T cells under test. The HLA class II molecule may be any of the HLA class II molecules described herein. IFN-γ secretion can be measured by methods known in the art, such as enzyme-linked immunosorbent assay (ELISA).

[0029] Alternatively, a TCR may be considered to have "antigen specificity" for G12V RAS if, when co-cultured with (a) antigen-negative HLA class II molecule-positive target cells pulsed with a low concentration of G12V RAS peptide or (b) antigen-negative HLA class II molecule-positive target cells into which a nucleotide sequence encoding G12V RAS has been introduced so that the target cells express G12V RAS, at least twice (e.g., five times) more T cells expressing the TCR secrete IFN-γ compared to the number of negative control T cells secreting IFN-γ. The concentrations of HLA class II molecules, peptides, and negative controls may be as described herein in relation to other aspects of the present invention. The number of IFN-γ-secreting cells can be measured by methods known in the art, for example, by ELISPOT.

[0030] Alternatively, if a TCR expressing a target cell expressing a G12V RAS is measured, for example by flow cytometry, after stimulation, the T cells expressing the TCR upregulate the expression of one or more T cell activation markers, then the TCR may be considered to have "antigen specificity" for the G12V 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.).

[0031] Embodiments of the present invention provide a TCR comprising two polypeptides (i.e., polypeptide chains), such as an alpha (α) chain of TCR, a beta (β) chain of TCR, a gamma (γ) chain of TCR, a delta (δ) chain of TCR, or a combination thereof. The TCR polypeptide of the present invention may contain any amino acid sequence, as long as the TCR has antigen specificity for G12V RAS. In some embodiments, the TCR does not exist in nature.

[0032] In embodiments of the present invention, the TCR comprises two polypeptide chains, each containing a variable region comprising complementarity-determining regions (CDRs) 1, 2, and 3 of the TCR. In embodiments of the present invention, the TCR comprises a first polypeptide chain comprising CDR1 (CDR1 of the α-chain of 4304 TCR1) containing the amino acid sequence of SEQ ID NO: 1, CDR2 (CDR2 of the α-chain of 4304 TCR1) containing the amino acid sequence of SEQ ID NO: 2, and CDR3 (CDR3 of the α-chain of 4304 TCR1) containing the amino acid sequence of SEQ ID NO: 3, and a second polypeptide chain comprising CDR1 (CDR1 of the β-chain of 4304 TCR1) containing the amino acid sequence of SEQ ID NO: 4, CDR2 (CDR2 of the β-chain of 4304 TCR1) containing the amino acid sequence of SEQ ID NO: 5, and CDR3 (CDR3 of the β-chain of 4304 TCR1) containing the amino acid sequence of SEQ ID NO: 6. In this regard, the TCR of the present invention may include one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 6. In embodiments of the present invention, the TCR includes (a) all of SEQ ID NOs: 1 to 3, (b) all of SEQ ID NOs: 4 to 6, or (c) all of SEQ ID NOs: 1 to 6. In particularly preferred embodiments, the TCR includes all of SEQ ID NOs: 1 to 6.

[0033] In embodiments of the present invention, the TCR includes the amino acid sequence of the variable region of the TCR, which includes the CDR described above. In this regard, the TCR may include the amino acid sequences of (i) SEQ ID NO: 7 (predicted sequence of the variable region of the α chain of 4304 TCR1 without an N-terminal signal peptide); (ii) SEQ ID NO: 8 (predicted sequence of the variable region of the β chain of 4304 TCR1 without an N-terminal signal peptide); (iii) SEQ ID NO: 9 (variable region of the α chain of 4304 TCR1 with an N-terminal signal peptide); (iv) SEQ ID NO: 10 (variable region of the β chain of 4304 TCR1 with an N-terminal signal peptide); (v) both SEQ ID NOs. 7 and 8; or (vi) both SEQ ID NOs. 9 and 10. Preferably, the TCR includes the amino acid sequences of (i) both SEQ ID NOs. 7 and 8 or (ii) both SEQ ID NOs. 9 and 10.

[0034] The TCR of the present invention may further comprise an α-chain constant region and a β-chain constant region. The constant regions may be derived from any suitable species, such as human or mouse. In embodiments of the present invention, the TCR further comprises mouse α and β chain constant regions or human α and β chain constant regions. As used herein, the terms “mouse” or “human” refer to the TCR or any component of the TCR described herein (e.g., CDR, variable region, constant region, α-chain, and / or β-chain), respectively, meaning a mouse or human-derived TCR (or its components), i.e., a TCR (or its components) originating from or formerly expressed by mouse T cells or human T cells, respectively.

[0035] Embodiments of the present invention provide a chimeric TCR comprising a human variable region and a mouse constant region, which has antigen specificity to a mutant human RAS amino acid sequence in which glycine at position 12 is substituted with valine. The mouse constant region may provide any one or more advantages. For example, the mouse constant region may reduce mispairing between the TCR of the present invention and the endogenous TCR of the host cell into which the TCR of the present invention is introduced. Alternatively, the mouse constant region may further increase the expression of the TCR of the present invention compared to the same TCR having a human constant region. The chimeric TCR may comprise the amino acid sequences of SEQ ID NO: 23 (WT mouse α-chain constant region), SEQ ID NO: 24 (WT mouse β-chain constant region), or both SEQ ID NOs. 23 and 24. Preferably, the TCR of the present invention comprises both SEQ ID NOs. 23 and 24. The chimeric TCR may comprise any of the mouse constant regions described herein in combination with any of the CDR regions described herein with respect to other embodiments of the present invention. In this regard, the TCR may comprise (a) all of SEQ ID NOs: 1-3 and 23, (b) all of SEQ ID NOs: 4-6 and 24, or (c) all of SEQ ID NOs: 1-6 and 23-24. In another embodiment of the present invention, the chimeric TCR may comprise any of the mouse constant regions described herein in combination with any of the variable regions described herein in relation to other aspects of the present invention. In this regard, the TCR may comprise (i) both of SEQ ID NOs: 7 and 23, (ii) both of SEQ ID NOs: 8 and 24, (iii) both of SEQ ID NOs: 9 and 23, (iv) both of SEQ ID NOs: 10 and 24, (v) all of SEQ ID NOs: 7-8 and 23-24, or (vi) all of SEQ ID NOs: 9-10 and 23-24.

[0036] In embodiments of the present invention, the TCR includes a substituted constant region. In this regard, the TCR may include any amino acid sequence of the TCRs described herein, having one, two, three, or four amino acid substitutions in the constant regions of one or both of the α and β chains. Preferably, the TCR includes a mouse constant region having one, two, three, or four amino acid substitutions in the mouse constant region of one or both of the α and β chains. In a particularly preferred embodiment, the TCR includes a mouse constant region having one, two, three, or four amino acid substitutions in the mouse constant region of the α chain and one amino acid substitution in the mouse constant region of the β chain. In some embodiments, the TCR including the substituted constant region is advantageous compared to the parent TCR including the unsubstituted (wild-type) constant region, resulting in G12V RAS + The present invention provides one or more of the following: increased target recognition, increased expression by host cells, reduced mispairing with endogenous TCRs, and increased antitumor activity. Generally, the substituted amino acid sequences of the mouse constant regions of the α and β chains of the TCR, SEQ ID NOs. 19 and 20, respectively, correspond to all or part of the unsubstituted mouse constant region amino acid sequences, SEQ ID NOs. 23 and 24, respectively, with SEQ ID NOs. 19 having one, two, three, or four amino acid substitutions (multiple) compared to SEQ ID NOs. 23, and SEQ ID NOs. 20 having one amino acid substitution compared to SEQ ID NOs. 24. In this regard, embodiments of the present invention provide a TCR comprising (a)(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 (sequence number 19, constant region of the α chain); (b) Sequence number 20 (sequence number β chain, constant region of the β chain), where X at position 57 is Ser or Cys; or (c) a TCR comprising the amino acid sequences of both Sequence numbers 19 and 20. In embodiments of the present invention, a TCR comprising Sequence number 19 does not include Sequence number 23 (unsubstituted mouse constant region of the α chain). In embodiments of the present invention, the TCR containing SEQ ID NO: 20 does not contain SEQ ID NO: 24 (the non-substituted mouse constant region of the β chain).

[0037] In embodiments of the present invention, the TCR comprises an α chain including a variable region and a constant region, and a β chain including a variable region and a constant region.In this regard, TCR is an α chain containing the amino acid sequence of SEQ ID NO: 25 (α chain of 4304 TCR1 having an N-terminal signal peptide), where (a) (i) X at position 175 of SEQ ID NO: 25 is Thr or Cys; (ii) X at position 239 of SEQ ID NO: 25 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 241 of SEQ ID NO: 25 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 242 of SEQ ID NO: 25 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) a β chain containing the amino acid sequence of SEQ ID NO: 26 (4304 having an N-terminal signal peptide), where X at position 186 of SEQ ID NO: 26 is Ser or Cys. (c) the β-chain of TCR1; (d) both SEQ ID NOs: 25 and 56; (i) X at position 156 of SEQ ID NO: 27 is Thr or Cys; (ii) X at position 220 of SEQ ID NO: 27 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 222 of SEQ ID NO: 27 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 223 of SEQ ID NO: 27 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (e) the β-chain of SEQ ID NO: 28 containing the amino acid sequence of SEQ ID NO: 28 (without an N-terminal signal peptide); (e) the β-chain containing the amino acid sequence of SEQ ID NO: 28 containing the amino acid sequence of SEQ ID NO: 28 (without an N-terminal signal peptide) (f) Predicted sequence of the β-chain of TCR1; (g) Both SEQ ID NOs: 27 and 28; (h) SEQ ID NOs: 29 (α-chain of cysteine-substituted LVL-modified 4304 TCR1 with an N-terminal signal sequence); (i) SEQ ID NOs: 31 (Predicted sequence of the α-chain of cysteine-substituted LVL-modified 4304 TCR1 without an N-terminal signal sequence); or (j) SEQ ID NOs: 32 (Predicted sequence of the β-chain of cysteine-substituted LVL-modified 4304 TCR1 without an N-terminal signal sequence).

[0038] In embodiments of the present invention, the substituted constant region includes a cysteine ​​substitution in the constant region of one or both of the α-chain and β-chain to provide a cysteine-substituted TCR. Opposing cysteines in the α-chain and β-chain link the constant regions of the α-chain and β-chain of the substituted TCR together and provide a disulfide bond that is not present in TCRs containing the unsubstituted mouse constant region. In this regard, the TCR may be a cysteine-substituted TCR in which one or both of the native Thr at position 48 of SEQ ID NO: 23 (Thr48) and the native Ser at position 57 of SEQ ID NO: 24 (Ser57) are substituted with Cys. Preferably, both the native Thr48 of SEQ ID NO: 23 and the native Ser57 of SEQ ID NO: 24 are substituted with Cys. Examples of constant region sequences of cysteine-substituted TCRs are shown in Table 2. In embodiments of the present invention, the cysteine-substituted TCR comprises (i) SEQ ID NO: 19, (ii) SEQ ID NO: 20, or (iii) both SEQ ID NOs: 19 and 20, both of which are defined in Table 2. The cysteine-substituted TCR of the present invention may include a substituted steady-state region in addition to either the CDR or variable region described herein.

[0039] In embodiments of the present invention, the cysteine-substituted chimeric TCR includes a full-length α chain and a full-length β chain. Examples of the sequences of the α and β chains of the cysteine-substituted chimeric TCR are shown in Table 2. In embodiments of the present invention, the TCR includes (i) SEQ ID NO: 25, (ii) SEQ ID NO: 26, (iii) SEQ ID NO: 27, (iv) SEQ ID NO: 28, (v) both SEQ ID NOs. 25 and 26, or (vi) both SEQ ID NOs. 27 and 28, all of which SEQ ID NOs. 25-28 are as defined in Table 2.

[0040] [Table 2]

[0041] In embodiments of the present invention, the substituted amino acid sequence includes the substitution of one, two, or three amino acids with hydrophobic amino acids in the transmembrane (TM) domain of the constant region of the α chain to provide a TCR substituted with hydrophobic amino acids (also referred herein as an "LVL-modified TCR"). Hydrophobic amino acid substitutions(s) in the TM domain of the TCR can increase the hydrophobicity of the TM domain of the TCR compared to a TCR that does not have hydrophobic amino acid substitutions(s) in the TM domain. In this regard, the TCR is an LVL-modified TCR in which one, two, or three of the native Ser112, Met114, and Gly115 of SEQ ID NO: 23 may be independently substituted with Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; preferably Leu, Ile, or Val. Preferably, all three native Ser112, Met114, and Gly115 of SEQ ID NO: 23 may be independently substituted with Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; preferably Leu, Ile, or Val. In embodiments of the present invention, the LVL-modified TCR comprises (i) SEQ ID NO: 19, (ii) SEQ ID NO: 20, or (iii) both SEQ ID NOs: 19 and 20, both of which are defined in Table 3. The LVL-modified TCR of the present invention may include a substitution steady-state region in addition to either the CDR or variable region described herein.

[0042] In embodiments of the present invention, the LVL-modified TCR includes a full-length α chain and a full-length β chain. Examples of the sequences of the α and β chains of the LVL-modified TCR are shown in Table 3. In embodiments of the present invention, the TCR includes (i) SEQ ID NO: 25, (ii) SEQ ID NO: 26, (iii) SEQ ID NO: 27, (iv) SEQ ID NO: 28, (v) both SEQ ID NOs. 25 and 26, or (vi) both SEQ ID NOs. 27 and 28, all of which SEQ ID NOs. 25-28 are as defined in Table 3.

[0043] [Table 3]

[0044] In embodiments of the present invention, the substituted amino acid sequence includes a cysteine ​​substitution in the constant regions of one or both the α and β chains, in combination with the substitution of one, two, or three amino acids by hydrophobic amino acids in the transmembrane (TM) domain of the constant region of the α chain (also referred to herein as a "cysteine-substituted LVL-modified TCR"). In this regard, the TCR is a cysteine-substituted LVL-modified chimeric TCR in which the native Thr48 of SEQ ID NO: 23 is substituted with Cys; one, two, or three of the native Ser112, Met114, and Gly115 of SEQ ID NO: 23 are independently substituted with Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; preferably Leu, Ile, or Val; and the native Ser57 of SEQ ID NO: 24 is substituted with Cys. Preferably, all three native Ser112, Met114, and Gly115 of SEQ ID NO: 23 may be independently substituted with Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; preferably Leu, Ile, or Val. In embodiments of the present invention, the cysteine-substituted LVL-modified TCR comprises (i) SEQ ID NO: 19, (ii) SEQ ID NO: 20, or (iii) both SEQ ID NOs: 19 and 20, both of which are defined in Table 4. The cysteine-substituted LVL-modified TCR of the present invention may include a substituted constant region in addition to either the CDR or variable region described herein.

[0045] In embodiments, the cysteine-substituted LVL-modified TCR includes a full-length α-chain and a full-length β-chain. Examples of the sequences of the α-chain and β-chain of the cysteine-substituted LVL-modified TCR are shown in Tables 4 and 8. In embodiments of the present invention, the TCR includes (i) SEQ ID NO: 25, (ii) SEQ ID NO: 26, (iii) SEQ ID NO: 27, (iv) SEQ ID NO: 28, (v) SEQ ID NO: 29, (vi) SEQ ID NO: 30, (vii) SEQ ID NO: 31, (viii) SEQ ID NO: 32, (ix) both SEQ ID NOs: 25 and 26, or (x) both SEQ ID NOs: 27 and 28, (xi) both SEQ ID NOs: 29 and 30, or (xii) both SEQ ID NOs: 31 and 32, where SEQ ID NOs: 25-28 are all as defined in Table 4.

[0046] [Table 4-1]

[0047] [Table 4-2]

[0048] In embodiments of the present invention, the cysteine-substituted LVL-modified TCR includes (a) SEQ ID NO: 21 (α-chain constant region of the cysteine-substituted LVL-modified TCR); (b) SEQ ID NO: 22 (β-chain constant region of the cysteine-substituted LVL-modified TCR); or (c) both (a) and (b).

[0049] Furthermore, the present invention provides polypeptides comprising any of the functional portions of the TCRs described herein. The term "polypeptide," as used herein, refers to a single chain of amino acids comprising an oligopeptide and linked by one or more peptide bonds.

[0050] With respect to the polypeptide of the present invention, the functional portion may be any portion of the TCR comprising a sequence of amino acids, insofar as it specifically binds to the G12V RAS. When the term “functional portion” is used in relation to the TCR, it refers to any portion or fragment of the TCR of the present invention that retains the biological activity of the TCR (parent TCR) to which the portion or fragment is a part. The functional portion includes, for example, a portion of the TCR that specifically binds to the G12V RAS or retains the ability to detect, treat, or prevent cancer to the same degree, the same degree, or a higher degree as the parent TCR (for example, within the framework of any of the HLA class II molecules described herein). With respect to the parent TCR, the functional portion may constitute, for example, about 10%, about 25%, about 30%, about 50%, about 70%, about 80%, about 90%, about 95%, or more of the parent TCR.

[0051] The functional portion may contain additional amino acids at its amino or carboxyl terminus, or both, that are not present in the amino acid sequence of the parent TCR. Preferably, the additional amino acids do not interfere with the biological function of the functional portion, such as having the ability to specifically bind to G12V RAS and / or detect cancer, or to treat or prevent cancer. More preferably, the additional amino acids enhance the biological activity compared to the biological activity of the parent TCR.

[0052] The polypeptide may include a functional portion of either or both of the α-chain and β-chain of the TCR of the present invention, for example, a functional portion including one or more of the variable regions CDR1, CDR2, and CDR3 of the α-chain and / or β-chain of the TCR of the present invention. In embodiments of the present invention, the polypeptide may include the amino acid sequence of SEQ ID NO: 1 (CDR1 of the α-chain of 4304 TCR1), the amino acid sequence of SEQ ID NO: 2 (CDR2 of the α-chain of 4304 TCR1), the amino acid sequence of SEQ ID NO: 3 (CDR3 of the α-chain of 4304 TCR1), the amino acid sequence of SEQ ID NO: 4 (CDR1 of the β-chain of 4304 TCR1), the amino acid sequence of SEQ ID NO: 5 (CDR2 of the β-chain of 4304 TCR1), the amino acid sequence of SEQ ID NO: 6 (CDR3 of the β-chain of 4304 TCR1), or a combination thereof. In this regard, the polypeptide of the present invention may include one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 6. In embodiments of the present invention, the polypeptide comprises (a) all of SEQ ID NOs: 1-3, (b) all of SEQ ID NOs: 4-6, or (c) all of SEQ ID NOs: 1-6. In preferred embodiments, the polypeptide comprises all of SEQ ID NOs: 1-6.

[0053] In embodiments of the present invention, the polypeptide of the present invention may include, for example, the variable region of the TCR of the present invention, which includes a combination of the above-mentioned CDR regions. In this regard, the polypeptide may include (i) SEQ ID NO: 7 (predicted sequence of the variable region of the α chain of 4304 TCR1 without an N-terminal signal peptide); (ii) SEQ ID NO: 8 (predicted sequence of the variable region of the β chain of 4304 TCR1 without an N-terminal signal peptide); (iii) SEQ ID NO: 9 (variable region of the α chain of 4304 TCR1 with an N-terminal signal peptide); (iv) SEQ ID NO: 10 (variable region of the β chain of 4304 TCR1 with an N-terminal signal peptide); (v) both SEQ ID NOs. 7 and 8; or (vi) the amino acid sequences of both SEQ ID NOs. 9 and 10. Preferably, the polypeptide includes the amino acid sequences of (i) both SEQ ID NOs. 7 and 8 or (ii) both SEQ ID NOs. 9 and 10.

[0054] In embodiments of the present invention, the polypeptide of the present invention may further comprise the constant region of the TCR of the present invention described above. In this regard, the polypeptide may further comprise the amino acid sequences of SEQ ID NO: 23 (WT mouse constant region of the α chain), SEQ ID NO: 24 (WT mouse constant region of the β chain), SEQ ID NO: 19 (substituted mouse constant region of the α chain), SEQ ID NO: 20 (substituted mouse constant region of the β chain), SEQ ID NO: 21 (α chain constant region of cysteine-substituted LVL-modified TCR), SEQ ID NO: 22 (β chain constant region of cysteine-substituted LVL-modified TCR), both SEQ ID NOs. 19 and 20, both SEQ ID NOs. 21 and 22, or both SEQ ID NOs. 23 and 24. Preferably, the polypeptide further comprises the amino acid sequences of both SEQ ID NOs. 19 and 20, both SEQ ID NOs. 21 and 22, or both SEQ ID NOs. 23 and 24 in combination with any of the CDR regions or variable regions described herein in relation to other embodiments of the present invention. In embodiments of the present invention, one or both SEQ ID NOs. 19 and 20 of the polypeptide are as defined in any one of Tables 2 to 4.

[0055] In embodiments of the present invention, the polypeptide of the present invention may comprise the full length of the α-chain or β-chain of the TCR described herein. In this regard, the polypeptide of the present invention may comprise the amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, or SEQ ID NO: 32. Alternatively, the polypeptide of the present invention may comprise both chains of the TCR described herein. For example, the polypeptide may comprise both SEQ ID NOs. 25-26, both SEQ ID NOs. 27-28, SEQ ID NOs. 29-30, or both SEQ ID NOs. 31-32.

[0056] For example, the polypeptide of the present invention is an α chain containing the amino acid sequence of SEQ ID NO: 25, wherein (a) (i) X at position 175 of SEQ ID NO: 25 is Thr or Cys; (ii) X at position 239 of SEQ ID NO: 25 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 241 of SEQ ID NO: 25 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 242 of SEQ ID NO: 25 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) a β chain containing the amino acid sequence of SEQ ID NO: 26, wherein X at position 186 of SEQ ID NO: 26 is Ser or Cys; (c) both SEQ ID NOs: 25 and 56; (d) (i) X at position 156 of SEQ ID NO: 27 is Thr or Cys; ii) X at position 220 of SEQ ID NO: 27 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 222 of SEQ ID NO: 27 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 223 of SEQ ID NO: 27 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp, and the α chain contains the amino acid sequence of SEQ ID NO: 27; (e) X at position 171 of SEQ ID NO: 28 is Ser or Cys, and the β chain contains the amino acid sequence of SEQ ID NO: 28; (f) both SEQ ID NOs: 27 and 28; (g) SEQ ID NO: 29; (h) SEQ ID NO: 30; (i) SEQ ID NO: 31; (j) SEQ ID NO: 32; (k) both SEQ ID NOs: 29 and 30; or (l) both SEQ ID NOs: 31 and 32. In embodiments of the present invention, one or more of the polypeptide sequence numbers 25 to 28 are as defined in any one of Tables 2 to 4.

[0057] The present invention further provides a protein comprising at least one polypeptide described herein. “Protein” means a molecule comprising one or more polypeptide chains.

[0058] In embodiments, the protein of the present invention may comprise a first polypeptide chain containing the amino acid sequences of SEQ ID NOs: 1-3 and a second polypeptide chain containing the amino acid sequences of SEQ ID NOs: 4-6.

[0059] In another embodiment of the present invention, (i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 7 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 8; or (ii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 9 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 10.

[0060] The protein of the present invention may further comprise any of the constant regions described herein in relation to other aspects of the present invention. In this regard, in embodiments of the present invention, (i) the first polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 19 and the second polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 20; (ii) the first polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 21 and the second polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 22; or (ii) the first polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 23 and the second polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 24. In embodiments of the present invention, one or both of SEQ ID NOs. 19 and 20 of the protein are as defined in any one of Tables 2 to 4.

[0061] Or, further, (a) the first polypeptide chain is (i) X at position 175 of SEQ ID NO: 25 is Thr or Cys; (ii) X at position 239 of SEQ ID NO: 25 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 241 of SEQ ID NO: 25 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 242 of SEQ ID NO: 25 is Gly, Ala, V (b) The first polypeptide chain is (i) the amino acid sequence of SEQ ID NO: 25, which is al, Leu, Ile, Pro, Phe, Met, or Trp; (c) both (a) and (b); (d) the first polypeptide chain is (i) the amino acid sequence of SEQ ID NO: 27, which is Thr or Cys; (ii) the amino acid sequence of SEQ ID NO: 27, which is Ser, Ala, or Val (iii) The amino acid sequence of SEQ ID NO: 27 is such that the X at position 222 of SEQ ID NO: 27 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp, and (iv) The X at position 223 of SEQ ID NO: 27 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (e) The second polypeptide chain is such that the X at position 171 of SEQ ID NO: 28 is Ser or k is Cys, and includes the amino acid sequence of SEQ ID NO: 28; (f) is both (d) and (e); (g) the first polypeptide chain includes the amino acid sequence of SEQ ID NO: 29; (h) the second polypeptide chain includes the amino acid sequence of SEQ ID NO: 30; (i) the first polypeptide chain includes the amino acid sequence of SEQ ID NO: 31; (j) the second polypeptide chain includes the amino acid sequence of SEQ ID NO: 32; (k) is both (g) and (h); or (l) is both (i) and (j). In embodiments of the present invention, one or more of SEQ ID NOs 25 to 28 are as defined in any one of Tables 2 to 4.

[0062] The protein of the present invention may be a TCR. Alternatively, the protein of the present invention may be a fusion protein if, for example, the protein comprises a single polypeptide chain containing the amino acid sequences of both the α and β chains of a TCR, or if the first and / or second polypeptide chain(s) of the protein further comprises an amino acid sequence encoding another amino acid sequence, such as an immunoglobulin or a portion thereof. In this regard, the present invention also provides a fusion protein comprising at least one of the polypeptides of the present invention described herein, together with at least one other polypeptide. The other polypeptide may exist as a separate protein of the fusion protein, or as a polypeptide expressed in frame (tandem) with 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.

[0063] The fusion protein may comprise one or more copies of the polypeptide of the present invention and / or one or more copies of other polypeptides. For example, the fusion protein may comprise one, two, three, four, five, or more copies of the polypeptide of the present invention and / or other polypeptides. Preferred methods for producing the fusion protein are known in the art and include, for example, recombinant methods.

[0064] In some embodiments of the present invention, the TCR, polypeptide, and protein of the present invention may be expressed as a single protein comprising a linker peptide linking the α and β chains. In this regard, the TCR, polypeptide, and protein of the present invention may further comprise a linker peptide. The linker peptide may advantageously promote the expression of recombinant TCR, polypeptide, and / or protein in host cells. The linker peptide may comprise any suitable amino acid sequence. The linker peptide may be a cleavable linker peptide. For example, the linker peptide may be a furin-SGSG-P2A linker peptide comprising the amino acid sequence RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 33). Once a construct comprising the linker peptide is expressed by a host cell, the linker peptide may be cleaved to obtain separated α and β chains. In embodiments of the present invention, the TCR, polypeptide, or protein may comprise an amino acid sequence comprising a full-length α chain, a full-length β chain, and a linker peptide located between the α and β chains.

[0065] The protein of the present invention may be a recombinant antibody or its antigen-binding portion, comprising at least one of the polypeptides of the present invention described herein. As used herein, “recombinant antibody” means a recombinant (e.g., genetically engineered) protein comprising at least one of the polypeptides of the present invention and the polypeptide chain of the antibody or its antigen-binding portion. The polypeptide of the antibody or its antigen-binding portion may be the heavy chain, light chain, variable or constant region of the heavy or light chain, single-chain variable region (scFv), or fragments of Fc, Fab, or F(ab)2' of the antibody. The polypeptide chain of the antibody or its antigen-binding portion may exist as separate polypeptides of the recombinant antibody. Alternatively, the polypeptide chain of the antibody or its antigen-binding portion may exist as a polypeptide expressed in frame (tandem) with 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, comprising any of the antibodies and antibody fragments described herein.

[0066] Functional variants of the TCR, polypeptide, or protein described herein are included within the scope of the present invention. The term “functional variant,” as used herein, means a TCR, polypeptide, or protein having substantial or significant sequence identity or similarity to the parent TCR, polypeptide, or protein, wherein the functional variant retains the biological activity of the TCR, polypeptide, or protein to which the functional variant is. Functional variants include, for example, variants of the TCR, polypeptide, or protein (parent TCR, polypeptide, or protein) described herein, which retain, to the same degree, or to a greater degree, the ability of the parent TCR to have antigen specificity or to specifically bind to the G12V RAS to which the parent polypeptide or protein specifically binds. With respect to the parental TCR, polypeptide, or protein, the functional variant may be identical in amino acid sequence to, for example, the parental TCR, polypeptide, or protein by at least about 30%, about 50%, about 75%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or more.

[0067] A functional variant may include, for example, the amino acid sequence of a parent TCR, polypeptide, or protein having at least one conserved amino acid substitution. Conserved 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 replaced by another amino acid having the same chemical or physical properties. For example, a conserved amino acid substitution may be the substitution of an acidic amino acid with another acidic amino acid (e.g., Asp or Glu), the substitution of an amino acid having a nonpolar side chain with another amino acid having a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), the substitution of a basic amino acid with another basic amino acid (Lys, Arg, etc.), the substitution of an amino acid having a polar side chain with another amino acid having a polar side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.).

[0068] Alternatively, the functional variant may further include an amino acid sequence of a parent TCR, polypeptide, or protein having at least one non-conservative amino acid substitution. In this case, it is preferable that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. Preferably, the non-conservative amino acid substitution enhances the biological activity of the functional variant, resulting in increased biological activity compared to the parent TCR, polypeptide, or protein.

[0069] A TCR, polypeptide, or protein may essentially consist of one of the specified amino acid sequences described herein, so that other components of the TCR, polypeptide, or protein, such as other amino acids, do not substantially alter the biological activity of the TCR, polypeptide, or protein. In this regard, the TCR, polypeptide, or protein of the present invention may essentially consist of, for example, the amino acid sequences of SEQ ID NOs. 25, 26, 27, 28, 29, 30, 31, 32, both SEQ ID NOs. 25 and 26, both SEQ ID NOs. 27 and 28, both SEQ ID NOs. 29 and 30, or both SEQ ID NOs. 31 and 32. Alternatively, for example, the TCR, polypeptide, or protein of the present invention may essentially consist of one of the amino acid sequences of (i) SEQ ID NOs. 7, (ii) SEQ ID NOs. 8, (iii) SEQ ID NOs. 9, (iv) SEQ ID NOs. 10, (v) both SEQ ID NOs. 7 and 8, or (vi) both SEQ ID NOs. 9 and 10. Furthermore, the TCR, polypeptide, or protein of the invention may essentially consist of (a) all of SEQ ID NOs: 1-3, (b) all of SEQ ID NOs: 4-6, or (c) all of SEQ ID NOs: 1-6.

[0070] The TCRs, polypeptides, and proteins of the present invention may be of any length, that is, they may contain any number of amino acids, as long as they retain their biological activity, such as the ability to specifically bind to G12V RAS; to detect cancer in mammals; or to treat or prevent cancer in mammals. For example, the polypeptide may have an amino acid length in the range of about 50 to about 5000, for example, 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. In this regard, the polypeptides of the present invention also include oligopeptides.

[0071] The TCRs, polypeptides, and proteins of the present invention may contain synthetic amino acids instead of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino-n-decanoic acid, homoserine, S-acetylaminomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, and indoline. Examples include -2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyllysine, 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.

[0072] The TCR, polypeptide, and protein of the present invention may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via disulfide crosslinks, etc., or converted to an acid addition salt, and / or optionally dimerized, polymerized, or conjugated.

[0073] The TCRs, polypeptides, and / or proteins of the present invention can be obtained by methods known in the art, for example, by de novo synthesis. Alternatively, polypeptides and proteins can be recombinantly produced using standard recombinant methods and nucleic acids described herein. For example, Green and Sambrook, Molecular Cloning: A Laboratory Manual ,4 th See ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Alternatively, the TCRs, polypeptides, and / or proteins described herein may be commercially synthesized by companies such as Peptide Technologies Corp. (Gaithersburg, MD), GenScript (Piscataway, NJ), and RayBiotech Life (Peachtree Corners, GA). In this regard, the TCRs, polypeptides, and proteins of the present invention may be synthetic, recombinant, isolated, and / or purified. Embodiments of the present invention provide isolated or purified TCRs, polypeptides, or proteins encoded by any nucleic acid or vector described herein in relation to other aspects of the present invention. Another embodiment of the present invention provides isolated or purified TCRs, polypeptides, or proteins obtained as a result of intracellular expression of any nucleic acid or vector described herein in relation to other aspects of the present invention. Yet another embodiment of the present invention provides a method for producing any TCR, polypeptide, or protein described herein, comprising culturing any host cell or population of host cells described herein so as to produce the TCR, polypeptide, or protein.

[0074] The scope of the present invention also includes conjugates, such as bioconjugates, that contain any of the TCRs, polypeptides, or proteins (including any functional portion or variant thereof), nucleic acids, recombinant expression vectors, host cells, populations of host cells, or antibodies or their antigen-binding portions. Conjugates and methods for synthesizing conjugates in general are known in the art.

[0075] Embodiments of the present invention provide nucleic acids comprising nucleotide sequences encoding any of the TCRs, polypeptides, or proteins described herein. “Nucleic acid,” as used herein, includes “polynucleotide,” “oligonucleotide,” and “nucleic acid molecule,” and generally means polymers of DNA or RNA, which may be single-stranded or double-stranded, may contain natural, unnatural, or modified nucleotides, and may contain natural, unnatural, or modified internucleotide bonds, such as phosphoramidate bonds or phosphorothioate bonds, instead of phosphodiesters found between nucleotides in unmodified oligonucleotides. In embodiments, the nucleic acid comprises complementary DNA (cDNA). Generally, it is preferable that the nucleic acid does not contain any insertions, deletions, inversions, and / or substitutions. However, in some examples, as discussed herein, it may be preferable for the nucleic acid to contain one or more insertions, deletions, inversions, and / or substitutions.

[0076] Preferably, the nucleic acids of the present invention are recombinants. As used herein, the term “recombinant” means (i) a molecule constructed outside of a living cell by linking a natural or synthetic nucleic acid segment to a nucleic acid molecule that can be replicated in a living cell, or (ii) a molecule obtained by replication of the one described in (i) above. For the purposes of this specification, replication may be in vitro or in vivo.

[0077] In embodiments of the present invention, the nucleic acid comprises the nucleotide sequences of SEQ ID NO: 38 (encoding both the α and β chains of 4304 TCR1 separated by a cleavable linker peptide), SEQ ID NO: 39 (encoding the variable region of the α chain of 4304 TCR1), SEQ ID NO: 40 (encoding the variable region of the β chain of 4304 TCR1), or both SEQ ID NOs: 39 and 40.

[0078] 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., cited above. For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to increase the biological stability of the molecule or the physical stability of the double strands formed during hybridization. Examples of modified nucleotides that can be used to make nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosin, 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-mannosylqueosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6Examples include, but are not limited to, isopentenyl adenine, uracil-5-oxyacetic acid(v), weybutoxosin, pseudouracil, queosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the present invention may be purchased from any of the various commercial entities.

[0079] Nucleic acids may comprise any nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein. In embodiments of the present invention, the nucleic acid comprises a codon-optimized nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein. While not bound by any particular theory or mechanism, codon optimization of nucleotide sequences is thought to increase the translation efficiency of mRNA transcripts. Codon optimization of nucleotide sequences may involve replacing native codons with other codons that encode the same amino acids but can be translated by tRNAs that are more readily available in the cell, thereby increasing translation efficiency. Alternatively, nucleotide sequence optimization may also increase translation efficiency by reducing the secondary structure of mRNA that interferes with translation.

[0080] Furthermore, the present invention provides nucleic acids comprising a nucleotide sequence complementary to any of the nucleotide sequences of the nucleic acids described herein, or a nucleotide sequence that hybridizes to any of the nucleotide sequences of the nucleic acids described herein under stringent conditions.

[0081] Nucleotide sequences that hybridize under stringent conditions preferably hybridize under high-stringent conditions. “High-stringent conditions” means that the nucleotide sequence specifically hybridizes to the target sequence (any nucleotide sequence of the nucleic acids described herein) in a detectably greater amount than in non-specific hybridization. High-stringent conditions include conditions that distinguish polynucleotides having precisely complementary sequences or containing only a few scattered mismatches from random sequences that coincidentally have several small regions (e.g., 3-10 bases) that match the nucleotide sequence. Such complementary small regions melt more readily than full-length complements of 14-17 bases or more, and are therefore readily distinguishable by high-stringent hybridization. Relatively high-stringent conditions include low-salt and / or high-temperature conditions, such as those provided by about 0.02-0.1 M NaCl or equivalent, at a temperature of about 50-70°C. Such highly stringent conditions tolerate only slight mismatches (if any) between the nucleotide sequence and the template or target chain, making them particularly suitable for detecting the expression of any of the TCRs of the present invention. Generally, it is understood that conditions can be made more stringent by adding gradually increasing amounts of formamide.

[0082] Embodiments of the present invention also provide nucleic acids comprising nucleotide sequences that are at least about 70% identical, for example, 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 essentially consist of any of the nucleotide sequences described herein.

[0083] Embodiments of the present invention provide an isolated or purified nucleic acid comprising a first nucleic acid sequence and a second nucleotide sequence from 5' to 3', wherein the first and second nucleotide sequences each encode the amino acid sequences of SEQ ID NOs: 7 and 8; 8 and 7; 9 and 10; 10 and 9; 25 and 26; 26 and 25; 27 and 28; 28 and 27; 29 and 30; 30 and 29; 31 and 32; or 32 and 31.

[0084] In embodiments of the present invention, the isolated or purified nucleic acid further comprises a third nucleotide sequence interposed between a first nucleotide sequence and a second nucleotide sequence, the third nucleotide sequence encoding a cleavable linker peptide. In embodiments of the present invention, the cleavable linker peptide comprises the amino acid sequence RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 33).

[0085] The nucleic acids of the present invention can be incorporated into recombinant expression vectors. In this regard, the present invention provides recombinant expression vectors comprising any of the nucleic acids of the present invention. In embodiments of the present invention, the recombinant expression vector comprises nucleotide sequences encoding an α chain, a β chain, and a linker peptide.

[0086] For the purposes of this specification, the term “recombinant expression vector” means a genetically modified oligonucleotide or polynucleotide construct comprising a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide, such that when the vector is brought into contact with a host cell under conditions sufficient to express the mRNA, protein, polypeptide, or peptide in the host cell, the cell will be able to express the mRNA, protein, polypeptide, or peptide. The vectors of the present invention do not exist in nature as a whole. However, parts of the vectors may exist in nature. The recombinant expression vectors of the present invention may contain any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, may be synthesized or partially obtained from natural sources, and may contain natural, non-natural, or modified nucleotides. The recombinant expression vectors may contain naturally occurring nucleotide-nucleotide bonds, non-natural nucleotide-nucleotide bonds, or both types of bonds. Preferably, non-natural or modified nucleotides or nucleotide-nucleotide bonds do not interfere with the transcription or replication of the vector.

[0087] The recombinant expression vector of the present invention may be any suitable recombinant expression vector and can be used to transform or transfect any suitable host cell. Suitable vectors include plasmids and viruses, etc., designed for propagation and proliferation, or for expression, or both. Vectors may be selected from the group consisting of the pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 may also be used. Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo(Clontech). Preferably, the recombinant expression vector is a viral vector, such as a retroviral vector. In a particularly preferred embodiment, the recombinant expression vector is an MSGV1 vector. In embodiments of the present invention, the recombinant expression vector is a transposon or a lentiviral vector.

[0088] The recombinant expression vectors of the present invention can be prepared, for example, using the standard recombinant DNA techniques described above by Green and Sambrook et al. The circular or linear expression vector constructs can be prepared to contain a replication system that functions in prokaryotic or eukaryotic host cells. The replication system may be derived from, for example, ColEl, 2μ plasmid, λ, SV40, bovine papillomavirus, etc.

[0089] Preferably, the recombinant expression vector includes regulatory sequences, for example, start and stop codons for transcription and translation specific to the type of host cell into which the vector is introduced (e.g., bacteria, fungi, plants, or animals), as needed, and taking into consideration whether the vector is DNA-based or RNA-based.

[0090] Recombinant expression vectors may contain one or more marker genes that enable the selection of transformed or transfected host cells. Marker genes include those for biocide resistance, such as resistance to antibiotics and heavy metals, and nutritional complementation in the host to provide protrophotrophy. Suitable marker genes for the expression vectors of the present invention include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0091] Recombinant expression vectors may include native or non-native promoters operably ligated to nucleotide sequences encoding TCRs, polypeptides, or proteins, or to nucleotide sequences complementary to or hybridizing with TCRs, polypeptides, or protein-encoding nucleotide sequences. For example, the selection of strong, weak, inducible, tissue-specific, and developmental stage-specific promoters is within the scope of the skills of those skilled in the art. Similarly, the combination of nucleotide sequences and promoters is also within the scope of the skills of those skilled in the art. Promoters may be non-viral promoters, or they may be viral promoters, such as cytomegalovirus (CMV) promoters, SV40 promoters, RSV promoters, and promoters found in the terminal repeat sequences of mouse stem cell viruses.

[0092] The recombinant expression vectors of the present invention can be designed for transient expression, stable expression, or both. Furthermore, recombinant expression vectors can be prepared for constitutive or inducible expression.

[0093] Furthermore, recombinant expression vectors may be constructed to include suicide genes. As used herein, the term “suicide gene” means a gene that causes cells expressing a suicide gene to die. A suicide gene may be a gene that confers sensitivity to an agent, such as a drug, to cells expressing the gene, and causes the cells to die when they come into contact with or are exposed to the agent. Suicide genes are known in the art and include, for example, the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, nitroreductase, and the inducible caspase 9 gene system.

[0094] Another embodiment of the present invention further provides a host cell containing either the nucleic acid or recombinant expression vector described herein. As used herein, the term “host cell” means any type of cell that may contain the recombinant expression vector of the present invention. The host cell may be a eukaryotic cell, e.g., a plant, animal, fungus, or algae, or a prokaryotic cell, e.g., a bacterium or protist. The host cell may be a cultured cell, or a primary cell, i.e., a cell directly isolated from an organism, e.g., a human. The host cell may be an adherent cell, or a suspension cell, i.e., a cell growing in a suspension. Suitable host cells are known in the art and include, for example, DH5α Escherichia coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. For the purpose of amplifying or replicating the recombinant expression vector, the host cell is preferably a prokaryotic cell, e.g., a DH5α cell. For the purpose of producing recombinant TCRs, polypeptides, or proteins, the host cell is preferably a mammalian cell. Most preferably, the host cell is a human cell. The host cell may be any cell type, may originate from any type of tissue, and may be at any developmental stage, but the host cell is preferably a peripheral blood lymphocyte (PBL) or peripheral blood mononuclear cell (PBMC). More preferably, the host cell is a T cell. In embodiments of the present invention, the host cell is a human lymphocyte. In another embodiment of the present invention, the host cell is selected from the group consisting of T cells, natural killer T (NKT) cells, invariant natural killer T (iNKT) cells, and natural killer (NK) cells. Yet another embodiment of the present invention is MTEYKLVVVGA V A method is provided for generating host cells that express a TCR having antigen specificity to the peptide GVGKSALTIQLI (SEQ ID NO: 34), comprising contacting the cells with the vectors described herein under conditions that enable the introduction of any of the vectors described herein into the cells.

[0095] For the purposes of this specification, the T cells may be any T cells, such as cultured T cells, e.g., primary T cells, or T cells derived from cultured T cell lines, e.g., Jurkat, SupT1, etc., or T cells obtained from mammals, etc. When obtained from a mammal, the T cells can be obtained from a number of sources including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. Also, the T cells may be concentrated or purified. Preferably, the T cells are human T cells. The T cells may be any type of T cells and may be at any stage of development, CD4 + / CD8 + double positive T cells, CD4 + helper T cells, such as Th1 and Th2 cells, CD4 + T cells, CD8 + T cells (e.g., cytotoxic T cells), tumor infiltrating lymphocytes (TIL), memory T cells (e.g., central memory T cells and effector memory T cells), naive T cells, etc., but are not limited thereto.

[0096] Also provided by the present invention is a population of cells comprising at least one host cell described herein. The population of cells may be a heterogeneous population comprising at least one other cell that does not contain any of the recombinant expression vectors, e.g., a host cell (e.g., a T cell), or a cell other than a T cell, e.g., a B cell, macrophage, neutrophil, erythrocyte, hepatocyte, endothelial cell, epithelial cell, muscle cell, brain cell, etc., in addition to a host cell containing any of the described recombinant expression vectors. Alternatively, the population of cells may be a substantially homogeneous population mainly comprising host cells containing (e.g., consisting essentially of) a recombinant expression vector. Also, the population may be a clonal population of cells where all the cells of the population are clones of one host cell containing a recombinant expression vector, such that all the cells of the population contain the recombinant expression vector. In one embodiment of the present invention, the population of cells is a clonal population comprising a host cell containing the recombinant expression vector described herein.

[0097] In embodiments of the present invention, the number of cells in a population can be rapidly increased. The increase in T cell numbers can be achieved by any of numerous methods known in the art, as described, for example, in U.S. Patent Nos. 8,034,334 and 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 increase in T cell numbers is carried out by culturing the T cells with an OKT3 antibody, IL-2, and feeder PBMCs (e.g., irradiated allogeneic PBMCs).

[0098] The TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, and host cells (including populations thereof) of the present invention may be isolated and / or purified. The term “isolated” as used herein means removed from its natural environment. The term “purified” as used herein means increased purity, and “purity” is a relative term and is not necessarily to be interpreted as absolute purity. For example, purity may be at least about 50%, and may be greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 95%, and may be about 100%.

[0099] The TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, and host cells (including populations thereof) of the present invention (hereinafter collectively referred to as "the TCR materials of the present invention") may be formulated into compositions such as pharmaceutical compositions. In this regard, the present invention provides a pharmaceutical composition comprising any of the TCRs, polypeptides, proteins, nucleic acids, expression vectors, and host cells (including populations thereof) described herein, and a pharmaceutically acceptable carrier. A pharmaceutical composition of the present invention containing any of the TCR materials of the present invention may contain one or more TCR materials of the present invention, for example, polypeptides and nucleic acids, or two or more different TCRs. Alternatively, the pharmaceutical composition may contain the TCR materials of the present invention in combination with another pharmaceutically active agent(s) or drug(s), for example, chemotherapeutic agents, for example, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.

[0100] Preferably, the carrier is a pharmaceutically acceptable carrier. With respect to the pharmaceutical composition, the carrier may be any of those conventionally used with the specific TCR material of the present invention under consideration. Methods for preparing the administerable composition are known or obvious to those skilled in the art, e.g., Remington: The Science and Practice of Pharmacy, 22 nd This is described in detail in Ed., Pharmaceutical Press (2012). A pharmaceutically acceptable carrier is preferably one that does not have harmful side effects or toxicity under the conditions of use.

[0101] The choice of carrier is determined in part by the specific TCR material of the present invention and the specific method used to administer the TCR material of the present invention. Accordingly, a variety of suitable formulations of the pharmaceutical composition of the present invention exist. Suitable formulations may include those administered parenterally, subcutaneously, intravenously, intramuscularly, intra-arterially, subarachnoidally, intratumorally, or intraperitoneally. The TCR material of the present invention may be administered using more than one route, and in certain cases, a particular route may provide a more immediate and effective response than another route.

[0102] Preferably, the TCR material of the present invention is administered, for example, by intravenous injection. When the TCR material of the present invention is host cells (or a population thereof) expressing the TCR of the present invention, the pharmaceutically acceptable carrier for the cells for injection may contain any isotonic carrier, for example, normal saline (about 0.90% w / v NaCl in water, about 300 mOsm / L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water, or Ringer's lactate solution. In embodiments, the pharmaceutically acceptable carrier is supplemented with human serum albumen.

[0103] For the purposes of the present invention, the amount or dose of the TCR material of the present invention administered (for example, the number of cells if the TCR material of the present invention is one or more cells) must be sufficient to produce an effect, such as a therapeutic or prophylactic response, in the subject or animal over an appropriate time frame. For example, the dose of the TCR material of the present invention must be sufficient to bind to a cancer antigen (e.g., G12V RAS) or to detect, treat, or prevent cancer for about two hours or more from the time of administration, for example, 12 to 24 hours or longer. In certain embodiments, the period may be longer. The dose is determined by the efficacy of the specific TCR material of the present invention and the condition of the animal (e.g., human), as well as the body weight of the animal being treated (e.g., human).

[0104] Many assays for determining the dose to be administered are known in the art. For the purposes of the present invention, the starting dose to be administered to mammals can be determined by using an assay that includes comparing the extent to which target cells are lysed or IFN-γ is secreted by T cells expressing a given dose of the TCR, polypeptide, or protein of the present invention in a set of mammals given different doses of T cells. The extent to which target cells are lysed or IFN-γ is secreted upon administration of a particular dose can be assayed by methods known in the art.

[0105] The dosage of the TCR material of the present invention is also determined by the presence, nature, and extent of any adverse side effects that may accompany the administration of the specific TCR material of the present invention. Typically, the attending physician determines the dosage of the TCR material of the present invention for treating each individual patient by considering various factors, such as age, weight, overall health, diet, sex, the TCR material of the present invention administered, the route of administration, and the severity of the cancer being treated. In embodiments in which the TCR material of the present invention is a population of cells, the number of cells administered per injection is, for example, about 1 × 10⁶ 6 ~Approx. 1×10 12 It can vary by one or more cells. In a particular embodiment, 1 × 10 6 It is acceptable to administer fewer than one cell.

[0106] Those skilled in the art will readily understand that the TCR material of the present invention may be modified in any number of ways, and that the therapeutic or prophylactic efficacy of the TCR material of the present invention may be increased through such modification. For example, the TCR material of the present invention may be conjugated with a chemotherapeutic agent directly or indirectly via crosslinking. The practice of conjugating compounds with chemotherapeutic agents is known in the art. Those skilled in the art will recognize that sites of the TCR material of the present invention that are not essential to the function of the TCR material of the present invention are suitable sites for conjugating crosslinks and / or chemotherapeutic agents, as long as the binding of the crosslinks and / or chemotherapeutic agents to the TCR material of the present invention does not interfere with the function of the TCR material of the present invention, i.e., its ability to bind to G12V RAS or its ability to detect, treat, or prevent cancer.

[0107] The pharmaceutical compositions, TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, and populations of cells of the present invention are intended to be used in methods for treating or preventing cancer. Although not bound by any particular theory, it is believed that the TCRs of the present invention specifically bind to G12V RAS, and as a result, when expressed by cells, the TCRs (or related polypeptides or proteins of the present invention) can mediate an immune response against target cells expressing G12V RAS. In this regard, embodiments of the present invention provide a method for treating or preventing cancer in a mammal, comprising administering to a mammal an effective amount for treating or preventing cancer in the mammal any of the pharmaceutical compositions, TCRs, polypeptides, or proteins described herein, any nucleic acid or recombinant expression vector containing a nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein, or any host cell or population of cells containing a recombinant vector encoding any of the TCRs, polypeptides, or proteins described herein.

[0108] Embodiments of the present invention provide a method for inducing an immune response against cancer in a mammal, comprising administering to the mammal in an amount effective for inducing an immune response against cancer, any of the following: a pharmaceutical composition described herein, a TCR, polypeptide, or protein, any nucleic acid comprising a nucleotide sequence encoding any of the TCR, polypeptide, or protein described herein, or a recombinant expression vector, or any host cell or population of cells comprising a recombinant vector encoding any of the TCR, polypeptide, or protein described herein.

[0109] Embodiments of the present invention provide any of the following for use in the treatment or prevention of cancer in mammals: a pharmaceutical composition described herein, a TCR, polypeptide, or protein, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCR, polypeptide, or protein described herein, or any host cell or population of cells comprising a recombinant vector encoding any of the TCR, polypeptide, or protein described herein.

[0110] Embodiments of the present invention provide any of the following for use in inducing an immune response against cancer in mammals: a pharmaceutical composition described herein, a TCR, polypeptide, or protein, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCR, polypeptide, or protein described herein, or any host cell or population of cells comprising a recombinant vector encoding any of the TCR, polypeptide, or protein described herein.

[0111] The terms “to treat” and “to prevent,” and any terms derived therefrom, as used herein, do not necessarily imply 100%, i.e., complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that a person skilled in the art would recognize as having potential benefits or therapeutic effects. In this regard, the methods of the present invention can provide any level of treatment or prevention of any amount of cancer in mammals. Furthermore, the treatment or prevention provided by the methods of the present invention may include the 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. Also, for the purposes of this specification, “prevention” may include delaying the onset of cancer or its symptoms or conditions. Or, further, “prevention” may include preventing or delaying the recurrence of cancer or its symptoms or conditions.

[0112] Furthermore, a method for detecting the presence of cancer in mammals is also provided. The method comprises (i) contacting one of the TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, cell populations, or pharmaceutical compositions of the present invention as described herein with a sample containing one or more cells of mammalian origin to form a complex, and (ii) detecting the complex, wherein the detection of the complex indicates the presence of cancer in the mammal.

[0113] With respect to the method of the present invention for detecting cancer in mammals, the cell sample may be a sample containing whole cells, their lysates, or fractions of whole cell lysates, such as a nuclear or cytoplasmic fraction, a total protein fraction, or a nucleic acid fraction.

[0114] For the purposes of the present invention's method for detecting cancer, contact may be performed in vitro or in vivo on a mammal. Preferably, contact is performed in vitro.

[0115] Furthermore, the complex can be detected by any number of methods known in the art. For example, the TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, or population of cells described herein may be labeled with a detectable label, such as a radioisotope, fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

[0116] For the purposes of the method of the present invention, in which host cells or a population of cells are administered, the cells may be homogeneous or self-cells to the mammal. Preferably, the cells are self-cells to the mammal.

[0117] Regarding the method of the present invention, cancers include acute lymphoblastic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vagina, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumors, The cancer may be any cancer, including any of the following: glioma, Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, oropharyngeal cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal, retinal, and mesentery cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, ureteral cancer, and bladder cancer. Preferred cancers are pancreatic, colorectal, lung, endometrial, ovarian, or prostate cancer. Preferably, lung cancer is lung adenocarcinoma, ovarian cancer is epithelial ovarian cancer, and pancreatic cancer is pancreatic adenocarcinoma. In embodiments of the present invention, cancer expresses a mutant human RAS amino acid sequence in which glycine at position 12 is replaced with valine, and the mutant human RAS amino acid sequence is the amino acid sequence of mutant human KRAS, mutant human HRAS, or mutant human NRAS, where position 12 is defined by referring to the WT human KRAS, WT human HRAS, or WT human NRAS protein, respectively. The mutant human KRAS, mutant human HRAS, and mutant human NRAS expressed by cancer may be as described herein in relation to other embodiments of the present invention.

[0118] The mammals referred to in the methods of the present invention may be any mammal. As used herein, the term “mammal” means any mammal, including but not limited to rodents such as mice and hamsters, and mammals of the order Lagomorpha such as rabbits. Preferably, the mammals are carnivores, including cats and dogs. More preferably, the mammals are even-toed ungulates, including cats and pigs, or odd-toed ungulates, including horses. Most preferably, the mammals are mammals of the order Primates, Ceboids or Simoids (monkeys), or Haplorhini (humans and apes). A particularly preferred mammal is humans. [Examples]

[0119] The following embodiments further illustrate the present invention, but of course, they should not be interpreted as limiting its scope in any way.

[0120] Example 1 This example demonstrates the isolation of anti-G12V RAS TCRs from TILs in 4304 colorectal cancer patients.

[0121] TILs derived from colorectal cancer patient 4304 were independently screened for responsiveness to multiple different neoantigens expressed by the patient. Reactivity to G12V was observed as described below. TILs derived from tumor fragments (numbered F2, F4, F5, F23, and F24) from patient 4304 were co-cultured with dendritic cells (DCs). The DCs were either (i) independently pulsed with one of eight different pools of 24-mer peptides (peptide pool numbers 1-8 (PP1-PP8)) or (ii) independently transduced with one of five different tandem minigene (TMG) constructs. Each peptide in the pool contained a different neoantigen expressed by the patient. One TMG construct encoded multiple different unrelated peptides in tandem. The other four TMG constructs each encoded multiple different 24-mer peptides containing patient-expressed neoantigens in tandem. As a negative control, TILs were co-cultured with DCs treated with dimethyl sulfoxide (DMSO). As a positive control, TILs were treated with phorbol myristate acetate (PMA).

[0122] Reactivity was tested by IFNγ secretion using an enzyme-linked immunosorbent spot (ELISpot) assay. Results for tumor fragment F4 are shown in Table 5. Reactive cells were observed in TILs co-cultured with PP3-pulsed DCs. To determine which PP3 peptide confered reactivity, TILs were co-cultured independently with DCs pulsed separately with each peptide from PP3. Results are shown in Table 6. Reactive cells were observed by co-culturing TILs with DCs pulsed with the G12V 24mer peptide.

[0123] [Table 5]

[0124] [Table 6]

[0125] Positive cells were restimulated and sorted by 4-1BB upregulation for single-cell T cell receptor (TCR) sequencing, then placed in 96-well plates. 4304 TCR1 cells (TRBV29-1*01 / TRBJ1-2*01 / TRBD2*01 / TRAV16*01) were observed.

[0126] The sequences of the variable regions of the alpha and beta chains of the TCR were identified by single-cell TCR sequencing. The amino acid sequences of the alpha and beta chain variable regions are shown in Table 7. CDRs are underlined. N-terminal signal peptides are in bold.

[0127] [Table 7]

[0128] Example 2 This example demonstrates the construction of a retroviral vector encoding the TCR of Example 1.

[0129] The nucleotide sequences encoding the variable regions of the α and β chains of 4304 TCR1 (Table 7) were obtained, and the codons were optimized. The VDJ region of TCRβ was fused to the mouse TCRβ constant chain. The VJ region of TCRα was fused to the mouse TCRα constant chain. Although not bound by any specific theory or mechanism, it is thought that substituting the constant regions of human TCRα and TCRβ chains with the corresponding mouse constant regions improves TCR expression and functionality (Cohen et al., Cancer Res., 66(17):8878-86(2006)).

[0130] Furthermore, the constant chains of mouse TCRα and TCRβ were modified with cysteine. Transmembrane hydrophobic mutations were introduced into the constant chain of mouse TCRα. Although not bound by any specific theory or mechanism, these modifications are thought to preferentially pair the introduced TCR chain, thereby enhancing TCR surface expression and functionality (Cohen et al., Cancer Res., 67(8):3898-903 (2007); Haga-Friedman et al., J.Immu., 188:5538-5546 (2012)). Table 8 shows the full-length α and β chains of each of the four TCRs, including these modifications to the constant region. In Table 8, CDRs are underlined, and modified amino acid residues in the constant region are underlined and shown in bold.

[0131] [Table 8]

[0132] The nucleotide sequences encoding the variable regions of the α and β chains of TCR1 (Table 8) were cloned into an MSGV1-based retroviral vector having the following expression cassette configuration: 5'NcoI-VDJβ-mCβ-Fulin / SerGly / P2A-VJα-mCα-EcoRI3'. To facilitate the cloning of the TCR expression cassette to the 5'NcoI site of the MSGV1 vector, the second amino acid in the N-terminal signal peptide of the TCRβ chain was changed from leucine (L) to alanine (A). The nucleotide sequence encoding the TCRα chain included SEQ ID NO: 39. The nucleotide sequence encoding the TCRβ chain included SEQ ID NO: 40.

[0133] The TCRβ and TCRα chains were separated by the furin Ser / Gly P2A linker peptide (SEQ ID NO: 33). Although not bound by any specific theory or mechanism, the linker peptide is thought to equalize the expression efficiency of the two chains (Szymczak et al., Nat. Biotechnol., 22(5):589-94 (2004)).

[0134] The retroviral vector's TCR expression cassette encoded the TCRβ and TCRα chains separated by the linker peptide from 5' to 3'. The TCR expression cassette contained the nucleotide sequence of SEQ ID NO: 38. The amino acid sequence encoded by the TCR expression cassette is shown in Table 9. In Table 9, the CDR is underlined, the constant region is italicized, and the linker peptide is shown in bold.

[0135] [Table 9]

[0136] Example 3 This example demonstrates the binding activity of the TCR expressed by the retroviral vector of Example 2.

[0137] Healthy donor PBLs were transduced with the retroviral vector from Example 2. Autologous DCs were then transduced with various concentrations of the G12V 24-mer peptide MTEYKLVVVGA shown in Figures 1A-1B. V GVGKSALTIQLI (SEQ ID NO: 34) or the corresponding WT 24-mer peptide MTEYKLVVVGAG G VGKSALTIQLI (SEQ ID NO: 35) was pulsed for 2 hours. These cells were washed twice and co-cultured overnight with transduced T cells in a 1:1 ratio. IFN-γ secretion was measured by ELISA (Figure 1A). 4-1BB upregulation was evaluated by fluorescence-activated cell sorting (FACS) (Figure 1B). As shown in Figures 1A-1B, TCR transduced cells showed specific and highly binding activity to DCs pulsed with the G12V 24mer peptide.

[0138] Example 4 This example demonstrates that the TCR expressed by the retroviral vector of Example 2 recognizes the G12V RAS presented by the HLA-DRB1*01 / HLA-DRA*01 heterodimer.

[0139] Exome and mRNA sequencing were used to determine the MHC class II molecules expressed in patient 4304. The expressed MHC class II molecules are shown in Figure 2.

[0140] Effector cells were healthy donor PBLs transfected with the retroviral vector of Example 2 encoding 4304 TCR1. Target cells were COS7 or HEK293 cells independently transfected with one of the HLA class II heterodimers shown in Figure 2. Target cells were loaded with the G12V 24-mer peptide and cultured in or without antibodies blocking the respective HLA class II molecules that had been pre-transfected. Target cells cultured with DMSO were used as a negative control. (i) Autologous DCs from patient 4304 treated with the G12V 24-mer peptide in the absence of HLA blocking antibodies were used as a positive control, and (ii) Autologous DCs from patient 4304 treated with DMSO were used as a negative control.

[0141] The reactivity was tested using the ELISpot assay by measuring IFNγ secretion. The results are shown in Figure 2. As shown in Figure 2, reactivity was observed when target cells loaded with G12V 24mer transduced with a nucleotide sequence encoding the DRA*01:01 / DRB1*01:01 heterodimer were co-cultured with 4304 TCR1 transduced cells. This reactivity was blocked by an antibody that recognizes the DRA*01:01 / DRB1*01:01 heterodimer.

[0142] All references cited herein, including publications, patent applications, and patents, are incorporated herein by reference as if they were included herein, each reference being individually and specifically indicated to be incorporated herein by reference.

[0143] In connection with the description of the present invention (particularly in connection with the following claims), the terms “a,” “an,” “the,” and “at least one,” as well as similar references, should be interpreted as encompassing both singular and plural forms, unless otherwise specified herein or unless clearly contradicted by the context. The use of the term “at least one” after 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) selected from the enumerated items or any combination of two or more enumerated items (A and B), unless otherwise specified herein or unless clearly contradicted by the context. The terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., “including but not limited to these”), unless otherwise specified herein. The enumeration of value ranges in this specification is intended, unless otherwise specified herein, simply to function as an abbreviation for each distinct value within the range, and each distinct value is incorporated into the specification as if it were individually enumerated herein. All methods described herein may be performed in any preferred order unless otherwise specified herein or unless it is clearly inconsistent with the context. The use of any and all examples or exemplary expressions provided herein (e.g., "etc.") is intended, unless otherwise specifically asserted, merely to further elucidate the invention and not to propose any limitation of the scope of the invention. No expression in the specification should be construed as indicating that any unclaimed element is essential for the practice of the invention.

[0144] Preferred embodiments of the Invention, including the best mode known to the inventors for carrying out the Invention, are described herein. Variations of the preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors anticipate that those skilled in the art will use such variations as appropriate, and they intend that the Invention may be carried out in ways other than those specifically described herein. Accordingly, the Invention includes all variations and equivalents of the subject matter of the Invention enumerated in the claims appended herein, as permitted by applicable law. Furthermore, any combination of the above elements in all possible variations is encompassed by the Invention unless otherwise specified herein or is clearly inconsistent with the context.

Claims

1. An isolated or purified T cell receptor (TCR) comprising an α-chain complementarity-determining region (CDR) 1 containing the amino acid sequence of SEQ ID NO: 1, an α-chain CDR 2 containing the amino acid sequence of SEQ ID NO: 2, an α-chain CDR 3 containing the amino acid sequence of SEQ ID NO: 3, a β-chain CDR 1 containing the amino acid sequence of SEQ ID NO: 4, a β-chain CDR 2 containing the amino acid sequence of SEQ ID NO: 5, and a β-chain CDR 3 containing the amino acid sequence of SEQ ID NO: 6, The aforementioned TCR has antigen specificity for a mutant human RAS amino acid sequence in which the glycine at position 12 is replaced with valine. The aforementioned mutant human RAS amino acid sequence is presented by a human leukocyte antigen (HLA)-DRB1*01:HLA-DRA*01 heterodimer. The aforementioned mutant human RAS amino acid sequence is the amino acid sequence of the mutant human Kirsten rat sarcoma virus oncogene homolog (KRAS), the mutant human Harvey rat sarcoma virus oncogene homolog (HRAS), or the mutant human neuroblastoma rat sarcoma virus oncogene homolog (NRAS). The 12th position is defined by referencing the TCR, which is derived from the proteins of wild-type human KRAS, wild-type human HRAS, or wild-type human NRAS, respectively.

2. The TCR according to claim 1, wherein the mutant human RAS amino acid sequence is MTEYKLVVVGGAVGVGKSALTIQLI (SEQ ID NO: 34).

3. The TCR according to claim 1 or 2, wherein it does not have antigen specificity to the wild-type human RAS amino acid sequence of MTEYKLVVVGAGGVGKSALTIQLI (SEQ ID NO: 35).

4. (i) Sequence ID 7, (ii) Sequence ID 8, (iii) Sequence ID 9, (iv) Sequence ID 10, (v) Both of sequence numbers 7 and 8, or (vi) Both sequence numbers 9 and 10 A TCR according to any one of claims 1 to 3, comprising the amino acid sequence.

5. (a) (i) The X at position 48 of sequence number 19 is either Thr or Cys; (ii) The X at position 112 of sequence number 19 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) The X at position 114 of sequence number 19 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) The α-chain constant region containing the amino acid sequence of SEQ ID NO: 19, wherein X at position 115 of SEQ ID NO: 19 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) A constant region of the β-chain containing the amino acid sequence of SEQ ID NO: 20, wherein X at position 57 of SEQ ID NO: 20 is Ser or Cys; or (c) Both (a) and (b) A TCR according to any one of claims 1 to 4, further comprising:

6. (a) (i) The X at position 175 of sequence number 25 is either Thr or Cys; (ii) The X at position 239 of sequence number 25 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 241 of sequence number 25 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) An α-chain containing the amino acid sequence of SEQ ID NO: 25, wherein X at position 242 of SEQ ID NO: 25 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) A β-chain containing the amino acid sequence of SEQ ID NO: 26, wherein the X at position 186 of SEQ ID NO: 26 is either Ser or Cys; (c) Both (a) and (b); (d) (i) The X at position 156 of sequence number 27 is either Thr or Cys; (ii) The X at position 220 of sequence number 27 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 222 of sequence number 27 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) An α-chain containing the amino acid sequence of SEQ ID NO: 27, wherein X at position 223 of SEQ ID NO: 27 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (e) A β-chain containing the amino acid sequence of SEQ ID NO: 28, wherein the X at position 171 of SEQ ID NO: 28 is either Ser or Cys; (f) Both (d) and (e); (g) Sequence ID 29; (h) Sequence ID 30; (i) Sequence ID 31; (j) Sequence ID 32; (k)(g) and (h) both; or Both (l), (i), and (j); The isolated or purified TCR according to any one of claims 1 to 5, comprising:

7. An isolated or purified polypeptide comprising a functional portion of a TCR according to any one of claims 1 to 6, wherein the functional portion comprises an α-chain CDR1 containing the amino acid sequence of SEQ ID NO: 1, an α-chain CDR2 containing the amino acid sequence of SEQ ID NO: 2, an α-chain CDR3 containing the amino acid sequence of SEQ ID NO: 3, a β-chain CDR1 containing the amino acid sequence of SEQ ID NO: 4, a β-chain CDR2 containing the amino acid sequence of SEQ ID NO: 5, and a β-chain CDR3 containing the amino acid sequence of SEQ ID NO:

6.

8. The aforementioned functional part, (i) Sequence ID 7, (ii) Sequence ID 8, (iii) Sequence ID 9, (iv) Sequence ID 10, (v) Both of sequence numbers 7 and 8, or (vi) Both sequence numbers 9 and 10 An isolated or purified polypeptide according to claim 7, comprising the amino acid sequence(s) of the following:

9. (a) (i) The X at position 48 of sequence number 19 is either Thr or Cys; (ii) The X at position 112 of sequence number 19 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) The X at position 114 of sequence number 19 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) The amino acid sequence of Sequence ID No. 19, wherein the X at position 115 of Sequence ID No. 19 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) The amino acid sequence of Sequence ID No. 20 in which X at position 57 of Sequence ID No. 20 is Ser or Cys; or (c) Both (a) and (b) The isolated or purified polypeptide according to claim 7 or 8, further comprising:

10. (a) (i) The X at position 175 of sequence number 25 is either Thr or Cys; (ii) The X at position 239 of sequence number 25 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 241 of sequence number 25 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) An α-chain containing the amino acid sequence of SEQ ID NO: 25, wherein X at position 242 of SEQ ID NO: 25 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) A β-chain containing the amino acid sequence of SEQ ID NO: 26, wherein the X at position 186 of SEQ ID NO: 26 is either Ser or Cys; (c) Both (a) and (b); (d) (i) The X at position 156 of sequence number 27 is either Thr or Cys; (ii) The X at position 220 of sequence number 27 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 222 of sequence number 27 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) An α-chain containing the amino acid sequence of SEQ ID NO: 27, wherein X at position 223 of SEQ ID NO: 27 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (e) A β-chain containing the amino acid sequence of SEQ ID NO: 28, wherein the X at position 171 of SEQ ID NO: 28 is either Ser or Cys; (f) Both (d) and (e); (g) Sequence ID 29; (h) Sequence ID 30; (i) Sequence ID 31; (j) Sequence ID 32; (k)(g) and (h) both; or Both (l), (i), and (j); An isolated or purified polypeptide according to any one of claims 7 to 9, comprising:

11. An isolated or purified protein comprising: a first polypeptide chain comprising CDR1 of an α chain containing the amino acid sequence of SEQ ID NO: 1, CDR2 of an α chain containing the amino acid sequence of SEQ ID NO: 2, and CDR3 of an α chain containing the amino acid sequence of SEQ ID NO: 3; and a second polypeptide chain comprising CDR1 of a β chain containing the amino acid sequence of SEQ ID NO: 4, CDR2 of a β chain containing the amino acid sequence of SEQ ID NO: 5, and CDR3 of a β chain containing the amino acid sequence of SEQ ID NO: 6; The aforementioned protein has antigen specificity for a mutant human RAS amino acid sequence in which the glycine at position 12 is replaced with valine. The aforementioned mutant human RAS amino acid sequence is presented as an HLA-DRB1*01:HLA-DRA*01 heterodimer, The aforementioned mutant human RAS amino acid sequence is the amino acid sequence of the mutant human Kirsten rat sarcoma virus oncogene homolog (KRAS), the mutant human Harvey rat sarcoma virus oncogene homolog (HRAS), or the mutant human neuroblastoma rat sarcoma virus oncogene homolog (NRAS). The 12th position is defined by referring to the proteins of wild-type human KRAS, wild-type human HRAS, or wild-type human NRAS, respectively.

12. (i) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 7, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 8; or (ii) The isolated or purified protein according to claim 11, wherein the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 9 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:

10.

13. (a) The first polypeptide chain, (i) The X at position 48 of sequence number 19 is either Thr or Cys; (ii) The X at position 112 of sequence number 19 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) The X at position 114 of sequence number 19 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) The amino acid sequence of Sequence ID No. 19 further comprises the sequence of Sequence ID No. 19, wherein X at position 115 of Sequence ID No. 19 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) The second polypeptide chain further comprises the amino acid sequence of SEQ ID NO: 20, wherein X at position 57 of SEQ ID NO: 20 is Ser or Cys; or (c) The isolated or purified protein according to claim 11 or 12, which is both (a) and (b).

14. (a) The first polypeptide chain, (i) The X at position 175 of sequence number 25 is either Thr or Cys; (ii) The X at position 239 of sequence number 25 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 241 of sequence number 25 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) The amino acid sequence of Sequence ID No. 25, wherein X at position 242 of Sequence ID No. 25 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) The second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 26, wherein X at position 186 of SEQ ID NO: 26 is either Ser or Cys; (c) Both (a) and (b); (d) The first polypeptide chain, (i) The X at position 156 of sequence number 27 is either Thr or Cys; (ii) The X at position 220 of sequence number 27 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 222 of sequence number 27 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) The amino acid sequence of Sequence ID No. 27, wherein X at position 223 of Sequence ID No. 27 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (e) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 28, wherein the X at position 171 of SEQ ID NO: 28 is either Ser or Cys; (f)(d) and (e) are both; (g) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 29; (h) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 30; (i) The first polypeptide chain contains the amino acid sequence of SEQ ID NO: 31; (j) The second polypeptide chain contains the amino acid sequence of SEQ ID NO: 32; (k)(g) and (h) are both; or The isolated or purified protein according to any one of claims 11 to 13, which is both (l)(i) and (j).

15. Isolated or purified nucleic acid comprising a nucleotide sequence encoding a TCR according to any one of claims 1 to 6, a polypeptide according to any one of claims 7 to 10, or a protein according to any one of claims 11 to 14.

16. An isolated or purified nucleic acid comprising a first nucleotide sequence and a second nucleotide sequence from 5' to 3', wherein the first and second nucleotide sequences each encode the amino sequences of SEQ ID NOs: 7 and 8; 8 and 7; 9 and 10; 10 and 9; 25 and 26; 26 and 25; 27 and 28; 28 and 27; 29 and 30; 30 and 29; 31 and 32; or 32 and 31.

17. The isolated or purified nucleic acid according to claim 16, further comprising a third nucleotide sequence interposed between a first nucleotide sequence and a second nucleotide sequence, wherein the third nucleotide sequence encodes a cleavable linker peptide.

18. The isolated or purified nucleic acid according to claim 17, wherein the cleavable linker peptide comprises the amino acid sequence of RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 33).

19. A recombinant expression vector comprising the nucleic acid described in any one of claims 15 to 18.

20. The recombinant expression vector according to claim 19, which is a transposon or a lentiviral vector.

21. Isolated or purified TCRs, polypeptides, or proteins encoded by the nucleic acid according to any one of claims 15 to 18 or the vector according to claim 19 or 20.

22. Isolated or purified TCRs, polypeptides, or proteins obtained as a result of intracellular expression of the nucleic acid described in any one of claims 15 to 18 or the vector described in claim 19 or 20.

23. A method for generating host cells that express a TCR having antigen specificity to the peptide MTEYKLVVVGAVGVGKSALTIQLI (SEQ ID NO: 34), comprising contacting the cells with the vector under conditions that enable the introduction of the vector described in claim 19 or 20 into isolated or purified host cells.

24. Isolated or purified host cells comprising the nucleic acid according to any one of claims 15 to 18 or the recombinant expression vector according to claim 19 or 20.

25. The host cell according to claim 24, wherein the cell is a human lymphocyte.

26. A host cell according to claim 24 or 25, selected from the group consisting of T cells, natural killer T (NKT) cells, invariant natural killer T (iNKT) cells, and natural killer (NK) cells.

27. A population of isolated or purified cells comprising the host cells described in any one of claims 24 to 26.

28. A method for producing a TCR according to any one of claims 1 to 6, 21, or 22, a polypeptide according to any one of claims 7 to 10, 21, or 22, or a protein according to any one of claims 11 to 14, 21, or 22, comprising culturing a host cell according to any one of claims 24 to 26 or a population of host cells according to claim 27 so as to produce the TCR, polypeptide, or protein.

29. (a) a TCR according to any one of claims 1 to 6, 21, or 22; a polypeptide according to any one of claims 7 to 10, 21, or 22; a protein according to any one of claims 11 to 14, 21, or 22; a nucleic acid according to any one of claims 15 to 18; a recombinant expression vector according to claim 19 or 20; a host cell according to any one of claims 24 to 26; or a population of cells according to claim 27; and (b) a pharmaceutically acceptable carrier.

30. A pharmaceutical composition for inducing an immune response against cancer in mammals, comprising a TCR according to any one of claims 1 to 6, 21, or 22, a polypeptide according to any one of claims 7 to 10, 21, or 22, a protein according to any one of claims 11 to 14, 21, or 22, a nucleic acid according to any one of claims 15 to 18, a recombinant expression vector according to claim 19 or 20, a host cell according to any one of claims 24 to 26, and a population of cells according to claim 27. The aforementioned cancer expresses a mutant human RAS amino acid sequence in which glycine at position 12 is replaced with valine. The aforementioned mutant human RAS amino acid sequence is presented as an HLA-DRB1*01:HLA-DRA*01 heterodimer, The aforementioned mutant human RAS amino acid sequence is the amino acid sequence of the mutant human Kirsten rat sarcoma virus oncogene homolog (KRAS), the mutant human Harvey rat sarcoma virus oncogene homolog (HRAS), or the mutant human neuroblastoma rat sarcoma virus oncogene homolog (NRAS). A pharmaceutical composition defined by the 12th position referring to the protein of wild-type human KRAS, wild-type human HRAS, or wild-type human NRAS, respectively.

31. A pharmaceutical composition for the detection, treatment, or prevention of cancer in mammals, comprising: a TCR according to any one of claims 1 to 6, 21, or 22; a polypeptide according to any one of claims 7 to 10, 21, or 22; a protein according to any one of claims 11 to 14, 21, or 22; a nucleic acid according to any one of claims 15 to 18; a recombinant expression vector according to claim 19 or 20; a host cell according to any one of claims 24 to 26; and a population of cells according to claim 27. The aforementioned cancer expresses a mutant human RAS amino acid sequence in which glycine at position 12 is replaced with valine. The aforementioned mutant human RAS amino acid sequence is presented as an HLA-DRB1*01:HLA-DRA*01 heterodimer, The aforementioned mutant human RAS amino acid sequence is the amino acid sequence of the mutant human Kirsten rat sarcoma virus oncogene homolog (KRAS), the mutant human Harvey rat sarcoma virus oncogene homolog (HRAS), or the mutant human neuroblastoma rat sarcoma virus oncogene homolog (NRAS). A pharmaceutical composition defined by the 12th position referring to the protein of wild-type human KRAS, wild-type human HRAS, or wild-type human NRAS, respectively.

32. The pharmaceutical composition according to claim 30 or 31, wherein the mutant human RAS amino acid sequence is the mutant human Kirsten rat sarcoma virus oncogene homolog (KRAS) amino acid sequence.

33. The pharmaceutical composition according to claim 30 or 31, wherein the mutant human RAS amino acid sequence is a mutant human neuroblastoma rat sarcoma virus oncogene homolog (NRAS) amino acid sequence.

34. The pharmaceutical composition according to claim 30 or 31, wherein the mutant human RAS amino acid sequence is a mutant human Harvey rat sarcoma virus oncogene homolog (HRAS) amino acid sequence.

35. The pharmaceutical composition according to any one of claims 30 to 34, wherein the cancer is cancer of the pancreas, colorectal, lung, endometrium, ovary, or prostate.

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