HLA class II-restricted T cell receptors for RAS with G12R mutations

Abstract

To provide a further method for treating cancer.SOLUTION: Disclosed is an isolated or purified T cell receptor (TCR), wherein the TCR has antigenic specificity for a mutated human RAS amino acid sequence with a substitution of glycine at position 12 with arginine. Related polypeptides and proteins, as well as related nucleic acids, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions are also provided. Also disclosed are methods of detecting the presence of cancer in a mammal and methods of treating or preventing cancer in a mammal.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Cross-reference of related applications This patent application claims the benefit of U.S. Provisional Patent Application No. 62 / 795,203, filed January 22, 2019, which is incorporated herein by reference in its entirety.

[0002] 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 74,093-byte ASCII (text) file named "746666_ST25.txt" dated January 21, 2020. [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 Initiative]

[0005] Embodiments of the present invention provide an isolated or purified T cell receptor (TCR) that has antigen specificity to a mutant human RAS amino acid sequence in which glycine at position 12 is substituted with arginine, wherein the mutant human RAS amino acid sequence is the amino acid sequence of 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 (WT) human KRAS, WT human HRAS, or WT 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 the amino acid sequences of (a) all of SEQ ID NOs: 1-3, (b) all of SEQ ID NOs: 4-6, (c) all of SEQ ID NOs: 7-9, (d) all of SEQ ID NOs: 10-12, (e) all of SEQ ID NOs: 1-6, or (f) all of SEQ ID NOs: 7-12.

[0007] Yet another embodiment of the present invention provides an isolated or purified protein comprising at least one polypeptide of the present invention.

[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 involve aligning a first nucleic acid sequence and a second nucleotide sequence in the 5'→3' direction. The present invention provides isolated or purified nucleic acids, wherein the first and second nucleotide sequences each encode the amino acid sequences of SEQ ID NOs: 13 and 14; 14 and 13; 15 and 16; 16 and 15; 30 and 31; 31 and 30; 32 and 33; 33 and 32; 34 and 35; 35 and 34; 36 and 37; 37 and 36; 41 and 42; 42 and 41; 43 and 44; 44 and 43; 45 and 46; 46 and 45; 47 and 48; 48 and 47; 49 and 50; 50 and 49; 51 and 52; or 52 and 51.

[0010] Embodiments of the present invention further provide a method for detecting the presence of cancer in mammals, a method for treating or preventing cancer in mammals, a method for inducing an immune response to cancer in mammals, a method for generating host cells that express a TCR having antigen specificity to the peptide of SEQ ID NO: 39, and a method for generating the TCR, polypeptide, and protein of the present invention. [Brief explanation of the drawing]

[0011] [Figure 1] This graph shows the number of interferon-gamma (IFNγ) positive spots per 2 × 10⁴ (2E⁴) cells detected when 4270 TCR transduced cells were co-cultured with autologous dendritic cells (DCs). Prior to co-culture, autologous DCs were pulsed with serial dilutions (ng / mL) of either WT RAS peptide (squares) or G12R RAS peptide (circles). [Figure 2] This graph shows the concentration (pg / mL) of IFNγ detected when 4270 TCR transduced cells were co-cultured with COS7 cells. Prior to co-culture, COS7 cells were transfected with one of the specified HLA molecules, and then pulsed with the G12R RAS peptide. HLA-DRA01:01 is the second member of the heterodimer for each of DRB1*11:01, DRB1*15:01, DRB3*02:02, DRB4*01:01, and DRB5*01. [Figure 3]This graph shows the number of IFNγ-positive spots per 2E4 cells detected when 4268 TCR transduced cells were co-cultured with autologous dendritic cells (DCs). Prior to co-culture, autologous DCs were pulsed with serial dilutions (ng / mL) of either WT RAS peptide (squares) or G12R RAS peptide (circles). [Figure 4] This graph shows the concentration (pg / mL) of IFNγ detected when 4268 TCR transduced cells were co-cultured with COS7 cells. Prior to co-culture, COS7 cells were transfected with one of the specified HLA molecules, and then pulsed with the G12R RAS peptide. HLA-DRA01:01 is the second member of the heterodimer for each of DRB1*11:01, DRB1*12:01, DRB3*02, DRB4*01, and DRB5*01. [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. There are two transcript variants of KRAS: KRAS variant A and KRAS variant B. WT KRAS variant A has the amino acid sequence of SEQ ID NO: 17. WT KRAS variant B has the amino acid sequence of SEQ ID NO: 17. It has the amino acid sequence of column number 18. Hereafter, references to "KRAS" (mutant or non-mutant (WT)) refer 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: 19.

[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: 20.

[0016] Embodiments of the present invention provide an isolated or purified TCR that has antigen specificity to a mutant human RAS amino acid sequence in which the glycine at position 12 is replaced with arginine, and the mutant human RAS amino acid sequence is the amino acid sequence of mutant human KRAS, mutant human HRAS, or mutant human NRAS, and the TCR is defined by the fact that the 12th position refers 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 have a length of 188 or 189 amino acid residues, respectively, 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 that of the WT human KRAS protein. Amino acid residues 1-86 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 that of the WT human KRAS protein. Amino acid residues 1-94 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 embodiments 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 replaced with arginine, and position 12 is defined by referring to the corresponding WT RAS protein. The WT RAS protein may be any one of the WT KRAS protein (SEQ ID NO: 17 or 18), WT HRAS protein (SEQ ID NO: 19), or WT NRAS protein (SEQ ID NO: 20), for the reason that, as explained above, amino acid residues 1-86 of the WT human NRAS protein and the WT human KRAS protein are 100% identical, and amino acid residues 1-94 of the WT human HRAS protein and the WT human KRAS protein are also 100% identical. Therefore, the amino acid residue at position 12 of the WT KRAS, WT HRAS, and WT NRAS proteins is the same, i.e., glycine.

[0019] The mutant human RAS amino acid sequence has a substitution of arginine for glycine at position 12. In this regard, embodiments of the present invention provide a TCR having antigen specificity for the amino acid sequence of any human RAS protein, polypeptide, or peptide having the G12R mutation.

[0020] Mutations and substitutions of the RAS are defined herein by reference to the amino acid sequence of the corresponding WT RAS protein. Accordingly, mutations and substitutions of the RAS are described herein by reference to the amino acid residue located at a specific position (i.e., position 12) of the WT RAS protein, followed by the position number, followed by the amino acid residue that replaces the above residue in the particular mutation or substitution under consideration. The RAS amino acid sequence (e.g., RAS peptide) may contain fewer amino acid residues than the total amino acid residues of the full-length WT RAS protein. Accordingly, with the understanding that the actual positions of the corresponding residues may differ in specific examples of RAS amino acid sequences, position 12 is defined herein by reference to the WT full-length RAS protein (i.e., any one of sequence numbers 17-20). If the position is as defined by any one of sequence numbers 17-20, the term "G12" refers to the glycine normally present at position 12 of any one of sequence numbers 17-20, and "G12R" indicates that the glycine normally present at position 12 of any one of sequence numbers 17-20 is replaced with arginine. For example, a specific example of the RAS amino acid sequence is, for instance, TEYKLVVVGA G In the case of GVGKSALTIQLI (SEQ ID NO: 25) (an exemplary WT KRAS peptide corresponding to consecutive amino acid residues 2-24 of SEQ ID NO: 17), "G12R" refers to the substitution of the underlined glycine with valine in SEQ ID NO: 25, even if the actual position of the underlined glycine in SEQ ID NO: 25 is 11. Hereinafter, human RAS amino acid sequences with the G12R mutation will be referred to as "G12R RAS".

[0021] Examples of full-length RAS proteins containing the G12R 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 G12R mutation, and the G12R RAS peptide has any length. In embodiments of the present invention, the G12R RAS peptide has any length suitable for binding to any of the HLA class II molecules described herein. For example, the TCR may have antigen specificity for a RAS peptide having the G12R mutation and having a length of about 11 to about 30 amino acid residues, about 12 to about 24 amino acid residues, or about 18 to about 20 amino acid residues. The G12R RAS peptide may contain any sequence of amino acid residues of a mutant RAS protein containing the G12R mutation. In embodiments of the present invention, the TCR may exhibit antigen specificity to mutant RAS peptides having a G12R mutation and having lengths 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 ranges. An example of a specific peptide having a G12R mutation that can be recognized by the TCR of the present invention is MTEYKLVVVGA R This is GVGKSALTIQLI (SEQ ID NO: 39). In embodiments of the present invention, it has antigen specificity for the mutant human RAS amino acid sequence of SEQ ID NO: 39. In embodiments of the present invention, the TCR does not have antigen specificity for the wild-type human RAS amino acid sequence of SEQ ID NO: 40.

[0024] In embodiments of the present invention, the TCR of the present invention can recognize G12R RAS presented by HLA class II molecules. In this regard, the TCR can elicit an immune response when it binds to G12R RAS within the framework of an HLA class II molecule. The TCR of the present invention can recognize G12R RAS presented by an HLA class II molecule and can bind to HLA class II molecules in addition to G12R 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 is an HLA-DRB5:HLA-DRA heterodimer. In particularly preferred embodiments, the HLA class II molecule is HLA-DRB5 * 01: HLA-DRA * 01:01 Allele (i.e., HLA-DRB5) * 01: HLA-DRA * It is expressed by the 01:01 heterodimer.

[0026] In another embodiment of the present invention, the HLA class II molecule is an HLA-DQ heterodimer. The HLA-DQ heterodimer is a cell surface receptor comprising an α-chain and a β-chain. The α-chain of HLA-DQ is encoded by the HLA-DQA1 gene. Examples of HLA-DQA1 alleles include DQA1 * 01:01, DQA1 * 01:02, DQA1 * 01:03, DQA1 * 01:04, DQA1 * 02:01, DQA1 * 03:01, DQA1 * 03:02, DQA1 * 03:03, DQA1 * 04:01, DQA1 * 05:01, DQA1 * 05:05, and DQA1 * 06:01. The β-chain of HLA-DQ is encoded by the HLA-DQB1 gene. Examples of HLA-DQB1 alleles include HLA-DQB1 * 02:01, HLA-DQB1 * 02:02, HLA-DQB1 * 02:03, HLA-DQB1 * 03:01, HLA-DQB1 * 03:02, HLA-DQB1 * 03:03, HLA-DQB1 * 03:04, HLA-DQB1 * 03:05, HLA-DQB1 * 04:01, HLA-DQB1 * 04:02, HLA-DQB1 * 05:01, HLA-DQB1 * 05:02, HLA-DQB1 * 05:03, HLA-DQB1 * 05:04, HLA-DQB1 * 06:01, HLA-DQB1 * 06:02, HLA-DQB1 * 06:03, HLA-DQB1 * 06:04, HLA-DQB1 * 06:05, and HLA-DQB1 *06:09 can be cited. In embodiments of the present invention, the HLA class II molecule is an HLA-DQA1:HLA-DQB1 heterodimer. In particularly preferred embodiments, the HLA class II molecule is HLA-DQA1 * 05:05:HLA-DQB1 * 03:01 Allele (i.e., HLA-DQA1) * 05:05:HLA-DQB1 * It is expressed by the 03:01 heterodimer.

[0027] 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. G12R RAS is expressed in cancer cells but not in normal non-cancer cells. While not bound by any particular theory or mechanism, the TCR of the present invention advantageously targets the destruction of cancer cells, while minimizing or eliminating the destruction of normal non-cancer cells, for example, by minimizing or eliminating toxicity, thereby... It is thought that this will reduce the risk. Furthermore, since G12R mutations are likely to occur in the early stages of tumorigenesis, G12R RAS mutations may be expressed in substantially all of a patient's cancer cells. The TCR of the present invention can advantageously and successfully treat or prevent G12R RAS-positive cancers that do not respond to other types of treatment, such as chemotherapy, surgery, or radiation therapy. In addition, the TCR of the present invention can treat unmanipulated tumor cells (e.g., G12R RAS and HLA-DRB5 that have not been treated with interferon (IFN)-γ). * 01: HLA-DRA * 01:01 (either one or both), G12R RAS and HLA-DQA1 * 05:05:HLA-DQB1 * It can provide highly avid recognition of tumor cells (not transfected with a vector encoding one or both of 03:01, not pulsed with G12R RAS peptide, or a combination thereof) with high binding activity of G12R RAS. Furthermore, HLA-DRB5 *The 01 allele is expressed at a frequency of approximately 18% in the human population, and is associated with HLA-DQA1. * 05:05:HLA-DQB1 * 03:01 is expressed in approximately 11% of people of Caucasian ethnicity in the United States. Therefore, the TCR of the present invention may not be suitable for immunotherapy using a TCR that recognizes the G12R RAS presented by other MHC molecules. * 01: HLA-DRA * 01:01 Allele and HLA-DQA1 * 05:05:HLA-DQB1 * The number of immunotherapy-eligible cancer patients can be increased to include patients expressing one or both of the 03:01 alleles. HLA-DRB5 in various cancers in the United States * 01: HLA-DRA * 01:01 and HLA-DQA1 * 05:05:HLA-DQB1 * Based on estimations of the frequency of the 03:01 allele and G12R RAS expression, it is estimated that more than 1,000 pancreatic cancer patients each year may be eligible for treatment with one of the TCRs of the present invention. For example, the KRAS G12R mutation is expressed in more than 8% of pancreatic cancer patients.

[0028] The term "antigen specificity," as used herein, means that the TCR specifically binds to and is immunologically recognizable to the G12R 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 G12R 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 G12R RAS has been introduced so that the target cells express the G12R RAS, approximately 1 × 10⁻¹⁶ cells express the TCR. 4 ~Approx. 1×10 5If 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 G12R 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 higher concentrations of G12R RAS peptide. The HLA class II molecule is any of the HLA class II molecules described herein (for example, HLA-DRB5). * 01: HLA-DRA * 01:01 heterodimer or HLA-DQA1 * 05:05:HLA-DQB1 * 03:01 (It may be a heterodimer.)

[0029] Alternatively, or additionally, a TCR is considered to have "antigen specificity" for G12R RAS if, when co-cultured with (a) antigen-negative HLA class II molecule-positive target cells pulsed with a low concentration of G12R RAS peptide or (b) antigen-negative HLA class II molecule-positive target cells into which a nucleotide sequence encoding G12R RAS has been introduced to enable the target cells to express G12R RAS, the T cells expressing the TCR secrete at least twice as much IFN-γ compared to the amount of IFN-γ expressed by the negative control. This may be done. The negative control may be, for example, (i)(a) antigen-negative HLA class II molecule-positive target cells pulsed with the same concentration of an unrelated peptide (e.g., several other peptides having a different sequence from the G12R 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) antigen-negative HLA class II molecule-positive target cells pulsed with the same concentration of G12R RAS peptide or (b) untransduced TCR cells (e.g., derived from PBMCs that do not express the TCR), co-cultured with antigen-negative HLA class II molecule-positive target cells into which a nucleotide sequence encoding G12R RAS has been introduced so that the target cells express G12R RAS. The HLA class II molecule expressed by the 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. HLA class II molecules are any of the HLA class II molecules described herein (e.g., HLA-DRB5) * 01: HLA-DRA * 01:01 heterodimer or HLA-DQA1 * 05:05:HLA-DQB1 * 03:01 (heterodimer) may be used. IFN-γ secretion can be measured by methods known in the art, such as enzyme-linked immunosorbent assay (ELISA).

[0030] Alternatively, or additionally, a TCR may be considered to have "antigen specificity" for G12R RAS if, when co-cultured with (a) antigen-negative HLA class II molecule-positive target cells pulsed with a low concentration of G12R RAS peptide or (b) antigen-negative HLA class II molecule-positive target cells into which a nucleotide sequence encoding G12R RAS has been introduced so that the target cells express G12R RAS, at least twice as many 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.

[0031] Alternatively, or additionally, a TCR may be considered to have "antigen specificity" for G12R RAS if, after stimulation with target cells expressing G12R RAS, the T cells expressing the TCR upregulate the expression of one or more T cell activation markers, for example by flow cytometry. Examples of T cell activation markers include 4-1BB, OX40, CD107a, CD69, and cytokines that are upregulated upon antigen stimulation (e.g., tumor necrosis factor (TNF), interleukin (IL)-2, etc.).

[0032] Embodiments of the present invention provide a TCR comprising two polypeptides (i.e., polypeptide chains), such as an alpha (α) chain of the TCR, a beta (β) chain of the TCR, a gamma (γ) chain of the TCR, a delta (δ) chain of the 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 the G12R RAS. In some embodiments, the TCR does not exist in nature.

[0033] 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 the 4268 TCR) containing the amino acid sequence of SEQ ID NO: 1, CDR2 (CDR2 of the α chain of the 4268 TCR) containing the amino acid sequence of SEQ ID NO: 2, and CDR3 (CDR3 of the α chain of the 4268 TCR) containing the amino acid sequence of SEQ ID NO: 3, and CDR1 (CDR1 of the β chain of the 4268 TCR) containing the amino acid sequence of SEQ ID NO: 4 It comprises a second polypeptide chain containing CDR2 of the β-chain of 268 TCRs, and CDR3 containing the amino acid sequence of SEQ ID NO: 6 (CDR3 of the β-chain of 4268 TCRs).

[0034] In another embodiment of the present invention, the TCR comprises a first polypeptide chain comprising CDR1 (CDR1 of the α-chain of 4270 TCR) containing the amino acid sequence of SEQ ID NO: 7, CDR2 (CDR2 of the α-chain of 4270 TCR) containing the amino acid sequence of SEQ ID NO: 8, and CDR3 (CDR3 of the α-chain of 4270 TCR) containing the amino acid sequence of SEQ ID NO: 9, and a second polypeptide chain comprising CDR1 (CDR1 of the β-chain of 4270 TCR) containing the amino acid sequence of SEQ ID NO: 10, CDR2 (CDR2 of the β-chain of 4270 TCR) containing the amino acid sequence of SEQ ID NO: 11, and CDR3 (CDR3 of the β-chain of 4270 TCR) containing the amino acid sequence of SEQ ID NO: 12.

[0035] 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 12. 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, (c) all of SEQ ID NOs: 7 to 9, (d) all of SEQ ID NOs: 10 to 12, (e) all of SEQ ID NOs: 1 to 6, or (f) all of SEQ ID NOs: 7 to 12. In particularly preferred embodiments, the TCR includes (i) all of SEQ ID NOs: 1 to 6 or (ii) all of SEQ ID NOs: 7 to 12.

[0036] In embodiments of the present invention, the TCR includes the amino acid sequence of the variable region of the TCR, which includes the above-mentioned CDR. In this regard, the TCR may include the amino acid sequences of SEQ ID NO: 13 (variable region of the α chain of the 4268 TCR having an N-terminal signal peptide); SEQ ID NO: 14 (variable region of the β chain of the 4268 TCR having an N-terminal signal peptide); SEQ ID NO: 15 (variable region of the α chain of the 4270 TCR having an N-terminal signal peptide); SEQ ID NO: 16 (variable region of the β chain of the 4270 TCR having an N-terminal signal peptide); SEQ ID NO: 43 (variable region of the α chain of the 4268 TCR without an N-terminal signal peptide); SEQ ID NO: 44 (variable region of the β chain of the 4268 TCR without an N-terminal signal peptide); SEQ ID NO: 41 (variable region of the α chain of the 4270 TCR without an N-terminal signal peptide); SEQ ID NO: 42 (variable region of the β chain of the 4270 TCR without an N-terminal signal peptide); both SEQ ID NOs: 13 and 14; both SEQ ID NOs: 15 and 16; both SEQ ID NOs: 41 and 42; or both SEQ ID NOs: 43 and 44. Preferably, the TCR includes the amino acid sequences of (i) both SEQ ID NOs: 13 and 14; (ii) both SEQ ID NOs: 15 and 16; (iii) both SEQ ID NOs: 41 and 42; or (iv) both SEQ ID NOs: 43 and 44.

[0037] The 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 TCR (or component thereof) derived from mouse or human, i.e., a TCR (or component thereof) originating from or formerly expressed by mouse T cells or human T cells, respectively.

[0038] 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 arginine. The mouse constant region may provide any one or more advantages. For example, the mouse constant region can reduce mismatching between the TCR of the present invention and the endogenous TCR of the host cell into which the TCR of the present invention is introduced. Alternatively or additionally, the mouse constant region can increase the expression of the TCR of the present invention compared to the same TCR having the human constant region. The chimeric TCR is represented by SEQ ID NO: 28 (WT mouse α-chain constant region). The TCR may include the amino acid sequence of SEQ ID NO: 29 (WT mouse β-chain constant region), or both SEQ ID NOs: 28 and 29. Preferably, the TCR of the present invention includes the amino acid sequences of both SEQ ID NOs: 28 and 29. The chimeric TCR may include any of the mouse constant regions described herein in combination with any of the CDR regions described herein in relation to other aspects of the present invention. In this regard, the TCR may include (a) all of SEQ ID NOs: 1-3 and 28; (b) all of SEQ ID NOs: 4-6 and 29; (c) all of SEQ ID NOs: 7-9 and 28; (d) all of SEQ ID NOs: 10-12 and 29; (e) all of SEQ ID NOs: 1-6 and 28-29; or (f) all of the amino acid sequences of SEQ ID NOs: 7-12 and 28-29. In another embodiment of the present invention, the chimeric TCR may include 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 include (i) both SEQ ID NOs: 13 and 28; (ii) both SEQ ID NOs: 14 and 29; (iii) both SEQ ID NOs: 15 and 28; (iv) both SEQ ID NOs: 16 and 29; (v) all of SEQ ID NOs: 13-14 and 28-29; or (vi) all of the amino acid sequences of SEQ ID NOs: 15-16 and 28-29.

[0039] In another embodiment of the present invention, the TCR is: SEQ ID NO: 34 (4268 TCRα chain having a constant region and an N-terminal signal peptide in a WT mouse), SEQ ID NO: 35 (4268 TCRβ chain having a constant region and an N-terminal signal peptide in a WT mouse), SEQ ID NO: 36 (4270 TCRα chain having a constant region and an N-terminal signal peptide in a WT mouse), SEQ ID NO: 37 (β chain of 4270 TCR having a constant region and an N-terminal signal peptide in a WT mouse), SEQ ID NO: 49 (α chain of 4268 TCR having a constant region and not having an N-terminal signal peptide in a WT mouse), SEQ ID NO: 50 (β chain of 4268 TCR having a constant region and not having an N-terminal signal peptide in a WT mouse), SEQ ID NO: 51 (α chain of 4270 TCR having a constant region and not having an N-terminal signal peptide in a WT mouse), SEQ ID NO: 52 (4270 TCR having a constant region and not having an N-terminal signal peptide in a WT mouse) The amino acid sequence(s) may include the β-chain of the TCR, both of SEQ ID NOs. 34-35, both of SEQ ID NOs. 36-37, both of SEQ ID NOs. 49-50, or both of SEQ ID NOs. 51-52.

[0040] 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, TCRs are (a)(i) X at position 180 of SEQ ID NO: 30 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 30 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 246 of SEQ ID NO: 30 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 247 of SEQ ID NO: 30 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp, containing an α chain (α chain of 4268 TCR having an N-terminal signal peptide); (b) SEQ ID NO: 31 is a β chain (4268 TCR having an N-terminal signal peptide) containing the amino acid sequence of SEQ ID NO: 31, where X at position 198 is Ser or Cys. (c) (i) X at position 188 of SEQ ID NO: 32 is Thr or Cys; (ii) X at position 252 of SEQ ID NO: 32 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 254 of SEQ ID NO: 32 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 255 of SEQ ID NO: 32 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (d) β-chain containing the amino acid sequence of SEQ ID NO: 33 (4270 TCR α-chain having an N-terminal signal peptide) (e) Both (a) and (b); (f) Both (c) and (d); (g) (i) X at position 161 of SEQ ID NO: 45 is Thr or Cys; (ii) X at position 225 of SEQ ID NO: 45 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or (iii) X at position 227 of SEQ ID NO: 45 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 228 of SEQ ID NO: 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp, and the α chain containing the amino acid sequence of SEQ ID NO: 45 (α chain of 4268 TCR without an N-terminal signal peptide); (h) X at position 178 of SEQ ID NO: 46 is Ser or Cys, and the β chain containing the amino acid sequence of SEQ ID NO: 46 (4268 TCR without an N-terminal signal peptide) α-chain of TCR (α-chain of 4270 TCR without an N-terminal signal peptide), containing the amino acid sequence of SEQ ID NO: 47, where (i) X at position 168 of SEQ ID NO: 47 is Thr or Cys; (ii) X at position 232 of SEQ ID NO: 47 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 234 of SEQ ID NO: 47 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 235 of SEQ ID NO: 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (j) β-chain of SEQ ID NO: 48 (α-chain of 4270 TCR without an N-terminal signal peptide), containing the amino acid sequence of SEQ ID NO: 47, where X at position 171 of SEQ ID NO: 48 is Ser or Cys. The β-chain of the TCR may include both (k)(g) and (h); or both (l)(i) and (j).

[0041] 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, with respect to the G12R 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. 26 and 27, respectively, correspond to all or part of the unsubstituted mouse constant region amino acid sequences, SEQ ID NOs. 28 and 29, respectively, with SEQ ID NOs. 26 having 1, 2, 3, or 4(or more) amino acid substitutions compared to SEQ ID NOs. 28, and SEQ ID NOs. 27 having 1 amino acid substitution compared to SEQ ID NOs. 29. 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; (b) SEQ ID NO: 27 (β-chain constant region) where X at position 57 is Ser or Cys; or (c) a TCR comprising the amino acid sequences of both SEQ ID NOs: 26 and 27. In embodiments of the present invention, a TCR comprising SEQ ID NO: 26 does not include SEQ ID NO: 28 (α-chain unsubstituted mouse constant region). In embodiments of the present invention, the TCR containing SEQ ID NO: 27 does not contain SEQ ID NO: 29 (non-substituted mouse constant region of the β chain).

[0042] 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 in order 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, providing a disulfide bond that is not present in TCRs containing an unsubstituted mouse constant region. In this regard, the TCR has one or both of the native Thr (Thr48) at position 48 of SEQ ID NO: 28 and the native Ser (Ser57) at position 57 of SEQ ID NO: 29 substituted with Cys. It may be a cysteine-substituted TCR. Preferably, both the native Thr48 of SEQ ID NO: 28 and the native Ser57 of SEQ ID NO: 29 are substituted with Cys. Examples of constant region sequences of cysteine-substituted TCRs are shown in Table 2. In embodiments of the present invention, a cysteine-substituted TCR includes (i) SEQ ID NO: 26, (ii) SEQ ID NO: 27, or (iii) both SEQ ID NOs: 26 and 27, both of which are defined in Table 2. The cysteine-substituted TCR of the present invention may include a substituted constant region in addition to either the CDR or variable region described herein.

[0043] In embodiments of the present invention, the cysteine-substituted chimeric TCR includes a full-length alpha chain and a full-length beta chain. Examples of the sequences of the alpha and beta 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: 30, (ii) SEQ ID NO: 31, (iii) SEQ ID NO: 32, (iv) SEQ ID NO: 33, (v) both SEQ ID NOs: 30 and 31, (vi) both SEQ ID NOs: 32 and 33, (vii) SEQ ID NO: 45, (viii) SEQ ID NO: 46, (ix) SEQ ID NO: 47, (x) SEQ ID NO: 48, (xi) both SEQ ID NOs: 45 and 46, or (xii) both SEQ ID NOs: 47 and 48, where SEQ ID NOs: 30-33 and 45-48 are all as defined in Table 2.

[0044] [Table 2]

[0045] In embodiments of the 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 one or both constant regions of the α and β chains, in order to provide a TCR substituted with hydrophobic amino acids (also referred herein as an "LVL-modified TCR"). Hydrophobic amino acid substitutions 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 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: 28 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: 28 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 includes (i) SEQ ID NO: 26, (ii) SEQ ID NO: 27, or (iii) both SEQ ID NOs: 26 and 27. These are all defined in Table 3. The LVL-modified TCR of the present invention may include a substitutional steady-state region in addition to either the CDR or the variable region described herein.

[0046] In embodiments of the present invention, the LVL-modified TCR includes a full-length alpha chain and a full-length beta chain. Examples of the sequences of the alpha and beta chains of the LVL-modified TCR are shown in Table 3. In embodiments of the present invention, the LVL-modified TCR includes (i) SEQ ID NO: 30, (ii) SEQ ID NO: 31, (iii) SEQ ID NO: 32, (iv) SEQ ID NO: 33, (v) both SEQ ID NOs: 30 and 31, (vi) both SEQ ID NOs: 32 and 33, (vii) SEQ ID NO: 45, (viii) SEQ ID NO: 46, (ix) SEQ ID NO: 47, (x) SEQ ID NO: 48, (xi) both SEQ ID NOs: 45 and 46, or (xii) both SEQ ID NOs: 47 and 48, where SEQ ID NOs: 30-33 and 45-48 are all as defined in Table 3.

[0047] [Table 3-1]

[0048] [Table 3-2]

[0049] In embodiments of the present invention, the substituted amino acid sequence includes a cysteine ​​substitution in the constant region of one or both the α and β chains (also referred to herein as a "cysteine-substituted LVL-modified TCR"), in combination with the substitution of one, two, or three amino acids with hydrophobic amino acids in the transmembrane (TM) domain of one or both constant regions of the α and β chains. In this regard, the TCR has the native Thr48 of SEQ ID NO: 28 substituted with Cys; and one, two, or three of the native Ser112, Met114, and Gly115 of SEQ ID NO: 28 are independently Ala, Val, Leu, Ile, Pro, Phe, Me t, or Trp; preferably substituted with Leu, Ile, or Val; the native Ser57 of SEQ ID NO: 29 is substituted with Cys, and is a cysteine-substituted LVL-modified chimeric TCR. Preferably, all three native Ser112, Met114, and Gly115 of SEQ ID NO: 28 may be independently substituted with Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; preferably Leu, Ile, or Val. In embodiments of the present invention, the cysteine-substituted LVL-modified TCR includes (i) SEQ ID NO: 26, (ii) SEQ ID NO: 27, or (iii) both SEQ ID NOs: 26 and 27, both of which are as defined in Table 4. The cysteine-substituted LVL-modified TCR of the present invention may include a substituted constant region in addition to either the CDR or variable region described herein.

[0050] In the embodiments, the cysteine-substituted LVL-modified TCR includes a full-length alpha chain and a full-length beta chain. In embodiments of the present invention, the cysteine-substituted LVL-modified TCR includes (i) SEQ ID NO: 30, (ii) SEQ ID NO: 31, (iii) SEQ ID NO: 32, (iv) SEQ ID NO: 33, (v) both SEQ ID NOs: 30 and 31, (vi) both SEQ ID NOs: 32 and 33, (vii) SEQ ID NO: 45, (viii) SEQ ID NO: 46, (ix) SEQ ID NO: 47, (x) SEQ ID NO: 48, (xi) both SEQ ID NOs: 45 and 46, or (xii) both SEQ ID NOs: 47 and 48, where SEQ ID NOs: 30-33 and 45-48 are all defined in Table 4.

[0051] [Table 4-1]

[0052] [Table 4-2]

[0053] 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.

[0054] With respect to the polypeptide of the present invention, the functional portion may be any portion of the TCR that is part of it, including a sequence of amino acids, as long as it specifically binds to the G12R 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 has the biological activity of the TCR (parent TCR) that is part of it. This refers to the retained portion or fragment. Functional portions are, for example, similar in degree to, identical in degree to, or higher in degree to, the parent TCR (e.g., HLA-DRB5). * 01: HLA-DRA * 01:01 heterodimer or HLA-DQA1 * 05:05:HLA-DQB1 *03:01 (Within the framework of a heterodimer) It includes a portion of the TCR that specifically binds to the G12R RAS or retains the ability to detect, treat, or prevent cancer. With respect to the parent TCR, the functional portion may constitute, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more of the parent TCR.

[0055] 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, for example, specifically binding to the G12R RAS and / or having the ability to detect cancer, treat or prevent cancer. More preferably, the additional amino acids enhance the biological activity compared to the biological activity of the parent TCR.

[0056] The polypeptide may comprise a functional portion of either or both of the α-chain and β-chain of the TCR of the present invention, for example, a functional portion comprising one or more of the CDR1, CDR2, and CDR3 of the variable region(s) of the α-chain and / or β-chain of the TCR of the present invention. In embodiments of the present invention, the polypeptide may comprise the amino acid sequence of SEQ ID NO: 1 (CDR1 of the α-chain), SEQ ID NO: 2 (CDR2 of the α-chain), SEQ ID NO: 3 (CDR3 of the α-chain), SEQ ID NO: 4 (CDR1 of the β-chain), SEQ ID NO: 5 (CDR2 of the β-chain), SEQ ID NO: 6 (CDR3 of the β-chain), or a combination thereof. In another embodiment of the present invention, the polypeptide may comprise the amino acid sequence of SEQ ID NO: 7 (CDR1 of the α-chain), SEQ ID NO: 8 (CDR2 of the α-chain), SEQ ID NO: 9 (CDR3 of the α-chain), SEQ ID NO: 10 (CDR1 of the β-chain), SEQ ID NO: 11 (CDR2 of the β-chain), SEQ ID NO: 12 (CDR3 of the β-chain), or a combination thereof.

[0057] In this regard, the polypeptide of the present invention may contain one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 12. In embodiments of the present invention, the TCR is (a) all of SEQ ID NOs: 1-3, (b) all of SEQ ID NOs: 4-6, (c) all of SEQ ID NOs: 7-9, (d) all of SEQ ID NOs: 10-12, (e) all of SEQ ID NOs: 1-6, or (f) all of SEQ ID NOs: 7-12. In a preferred embodiment, the polypeptide comprises (i) all of SEQ ID NOs: 1-6 or (ii) all of SEQ ID NOs: 7-12.

[0058] In embodiments of the present invention, the polypeptide of the present invention may include, for example, a variable region of the TCR of the present invention that includes a combination of the above-mentioned CDR regions. In this regard, the polypeptide may include the amino acid sequences of (i) SEQ ID NO: 13 (α-chain variable region), (ii) SEQ ID NO: 14 (β-chain variable region), (iii) both SEQ ID NOs: 13 and 14, (iv) SEQ ID NO: 15 (α-chain variable region), (v) SEQ ID NO: 16 (β-chain variable region), (vi) both SEQ ID NOs: 15 and 16, (vii) SEQ ID NO: 41 (α-chain variable region), (viii) SEQ ID NO: 42 (β-chain variable region), (ix) both SEQ ID NOs: 41 and 42, (x) SEQ ID NO: 43 (α-chain variable region), (xi) SEQ ID NO: 44 (β-chain variable region), or (xii) both SEQ ID NOs: 43 and 44. Preferably, the polypeptide contains the amino acid sequences of (i) both SEQ ID NOs: 13 and 14, (ii) both SEQ ID NOs: 15 and 16, (iii) both SEQ ID NOs: 41 and 42, or (iv) both SEQ ID NOs: 43 and 44.

[0059] In 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: 28 (WT mouse constant region of the α chain), SEQ ID NO: 29 (WT mouse constant region of the β chain), SEQ ID NO: 26 (substituted mouse constant region of the α chain), SEQ ID NO: 27 (substituted mouse constant region of the β chain), both SEQ ID NOs. 28 and 29, or both SEQ ID NOs. 26 and 27. Preferably, the polypeptide comprises either the CDR region or the variable region described herein in relation to other embodiments of the present invention. In combination with the above, the polypeptide further comprises the amino acid sequences of both SEQ ID NOs: 26 and 27 or both SEQ ID NOs: 28 and 29. In embodiments of the present invention, one or both of SEQ ID NOs: 26 and 27 of the polypeptide are as defined in any one of Tables 2 to 4.

[0060] In embodiments of the present invention, the polypeptide of the present invention may include 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 include the amino acid sequences of SEQ ID NOs. 30, 31, 32, 33, 34, 35, 36, 37, both SEQ ID NOs. 30-31, both SEQ ID NOs. 32-33, both SEQ ID NOs. 34-35, both SEQ ID NOs. 36-37, 45, 46, 47, 48, 49, 50, 51, 52, both SEQ ID NOs. 45 and 46, both SEQ ID NOs. 47 and 48, both SEQ ID NOs. 49 and 50, or both SEQ ID NOs. 51 and 52. Alternatively, the polypeptide of the present invention may include both chains of the TCR described herein.

[0061] For example, the polypeptide of the present invention is the amide of SEQ ID NO: 30, wherein (a) (i) X at position 180 of SEQ ID NO: 30 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 30 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 246 of SEQ ID NO: 30 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 247 of SEQ ID NO: 30 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp. (b) The amino acid sequence of SEQ ID NO 31, where X at position 198 of SEQ ID NO 31 is Ser or Cys; (c) (i) X at position 188 of SEQ ID NO 32 is Thr or Cys; (ii) X at position 252 of SEQ ID NO 32 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 254 of SEQ ID NO 32 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 255 of SEQ ID NO 32 is Gly, Ala, Val, Leu, Il (d) The amino acid sequence of SEQ ID NO: 32 is e, Pro, Phe, Met, or Trp; (e) The amino acid sequence of SEQ ID NO: 33 is Ser or Cys at position 191 of SEQ ID NO: 33; (f) Both (a) and (b); (g) Both (c) and (d); (i) The X at position 161 of SEQ ID NO: 45 is Thr or Cys; (ii) The X at position 225 of SEQ ID NO: 45 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) The X at position 227 of SEQ ID NO: 45 is Met, Ala, Val, Le (iv) The amino acid sequence of SEQ ID NO: 45, where X at position 228 of SEQ ID NO: 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (h) The amino acid sequence of SEQ ID NO: 46, where X at position 178 of SEQ ID NO: 46 is Ser or Cys; (i) (i) The X at position 168 of SEQ ID NO: 47 is Thr or Cys; (ii) The X at position 232 of SEQ ID NO: 47 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp;(iii) The amino acid sequence of SEQ ID NO: 47, wherein X at position 234 of SEQ ID NO: 47 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) The amino acid sequence of SEQ ID NO: 47, wherein X at position 235 of SEQ ID NO: 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (j) The amino acid sequence of SEQ ID NO: 48, wherein X at position 171 of SEQ ID NO: 48 is Ser or Cys; (k) may include both (g) and (h); or (l) may include both (i) and (j). In embodiments of the present invention, one or more of the polypeptide SEQ ID NOs: 30-33 and 45-48 are as defined in any one of Tables 2-4.

[0062] The present invention further provides a protein comprising at least one polypeptide described herein. “Protein” means a molecule comprising one or more polypeptide chains.

[0063] In embodiments, the protein of the present invention may comprise (a) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 1-3 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 4-6; or (b) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 7-9 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 10-12.

[0064] In another embodiment of the present invention, the protein may comprise (i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 13 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 14; (ii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 15 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 16; (iii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 41 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 42; or (iv) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 43 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 44.

[0065] The protein of the present invention may further comprise any of the constant regions described herein in relation to other embodiments 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: 26 and the second polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 27; or (ii) the first polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 28 and the second polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 29. In embodiments of the present invention, one or both of SEQ ID NOs. 26 and 27 of the protein are as defined in any one of Tables 2 to 4.

[0066] Alternatively or additionally, the proteins of the embodiments of the present invention are: (a) (i) X at position 180 of SEQ ID NO: 30 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 30 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 246 of SEQ ID NO: 30 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 247 of SEQ ID NO: 30 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp (b) A first polypeptide chain containing the amino acid sequence of SEQ ID NO: 30, wherein the X at position 198 of SEQ ID NO: 31 is Ser or Cys; (c) (i) The X at position 188 of SEQ ID NO: 32 is Thr or Cys; (ii) The X at position 252 of SEQ ID NO: 32 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) The X at position 254 of SEQ ID NO: 32 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp (i) a first polypeptide chain containing the amino acid sequence of SEQ ID NO: 32, wherein X at position 255 of SEQ ID NO: 32 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (d) a second polypeptide chain containing the amino acid sequence of SEQ ID NO: 33, wherein X at position 191 of SEQ ID NO: 33 is Ser or Cys; (e) both (a) and (b); (f) both (c) and (d); (g) (i) X at position 161 of SEQ ID NO: 45 is Thr or Cys; (ii) X at position 225 of SEQ ID NO: 45 is Ser or Ala (iii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 45, wherein X at position 227 of SEQ ID NO: 45 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 45, wherein X at position 228 of SEQ ID NO: 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (h) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 46, wherein X at position 178 of SEQ ID NO: 46 is Ser or Cys;(i)(i) The X at position 168 of sequence number 47 is either Thr or Cys; (ii) The X at position 232 of sequence number 47 is either Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) The X at position 234 of sequence number 47 is either Met, Ala, Val, Leu, Ile, Pro, or P; (i) and (h); or (l) may include both (i) and (j). In embodiments of the present invention, one or more of the sequence numbers 30-33 and 45-48 are defined in any one of Tables 2-4.

[0067] The protein of the present invention may be a TCR. Alternatively, for example, the protein may be a fusion protein if it comprises a single polypeptide chain containing the amino acid sequences of both SEQ ID NOs: 30 and 31 and both SEQ ID NOs: 32 and 33, 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.

[0068] 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.

[0069] 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 facilitate 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 comprising the amino acid sequence of SEQ ID NO: 38. 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.

[0070] 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', etc. 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 be in frame (tandem) with the polypeptide of the present invention. It may exist as a polypeptide expressed in (m). The polypeptide of the antibody or its antigen-binding portion may be any antibody or polypeptide of any antibody fragment, which includes any of the antibodies and antibody fragments described herein.

[0071] The 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 the variant. 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 G12R 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.

[0072] 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.).

[0073] Alternatively, or additionally, the functional variant may include an amino acid sequence of a parent TCR, polypeptide, or protein having at least one non-conserved amino acid substitution. In this case, it is preferable that the non-conserved amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. Preferably, the non-conserved amino acid substitution can enhance the biological activity of the functional variant, resulting in increased biological activity compared to the parent TCR, polypeptide, or protein.

[0074] A TCR, polypeptide, or protein may essentially consist of one of the specified amino acid sequences described herein, and as a result, other components of the TCR, polypeptide, or protein, such as other amino acids, will 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. 30, 31, 32, 33, 34, 35, 36, 37, both SEQ ID NOs. 30-31, both SEQ ID NOs. 32-33, both SEQ ID NOs. 34-35, or both SEQ ID NOs. 36-37. 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. 13, (ii) SEQ ID NOs. 14, (iii) SEQ ID NOs. 15, (iv) SEQ ID NOs. 16, (v) both SEQ ID NOs. 13 and 14, or (vi) both SEQ ID NOs. 15 and 16. Furthermore, the TCR, polypeptide, or protein of the present invention may include (a) one or more of SEQ ID NOs: 1 to 12; (b) all of SEQ ID NOs: 1 to 3; (c) all of SEQ ID NOs: 4 to 6; (d) all of sequence numbers 7-9; (e) all of sequence numbers 10-12; (f) all of sequence numbers 1-6; or (g) essentially consisting of all amino acid sequences of sequence numbers 7-12.

[0075] 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 G12R RAS; to detect cancer in mammals; or to treat or prevent cancer in mammals. For example, the polypeptides may have amino acid lengths 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.

[0076] 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.

[0077] The TCR, polypeptide, or protein of the present invention may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via disulfide crosslinking, etc., or converted to an acid addition salt, and / or optionally dimerized, polymerized, or conjugated.

[0078] 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, companies such as Synpep (Dublin, CA), Peptide Technologies Corp. (Gaithersburg, MD), and Multiple Peptide Systems (San Diego, CA) may commercially synthesize the TCRs, polypeptides, and / or proteins described herein. 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 of the nucleic acids or vectors described herein in relation to other aspects of the present invention. Another embodiment of the present invention provides a TCR, polypeptide, or protein encoded by any of the nucleic acids or vectors described herein in relation to other aspects of the present invention expressed in a cell. The present invention provides isolated or purified TCRs, polypeptides, or proteins obtained as a result. Yet another embodiment of the present invention provides a method for producing any of the TCRs, polypeptides, or proteins described herein, comprising culturing any of the host cells or populations of host cells described herein so as to produce the TCRs, polypeptides, or proteins.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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. 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, beta-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, beta-D-mannosylqueosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6 -Isopentenyl adenine, uracil-5-oxyacetic acid (v), weybutoxosin, pseudouracil, queosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5 Examples include, but are not limited to, methyl oxyacetate, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the present invention can be purchased from companies such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0083] 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.

[0084] 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.

[0085] Nucleotide sequences that hybridize under stringent conditions preferably hybridize under highly stringent conditions. “Highly stringent conditions” means that the nucleotide sequence hybridizes specifically to a target sequence (a nucleotide sequence of any of the nucleic acids described herein) in a detectably greater amount than in nonspecific hybridization. Highly 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) where the nucleotide sequences match. Such complementary small regions melt more readily than full-length complements of 14-17 bases or more, and are therefore readily distinguishable by highly stringent hybridization. Relatively highly stringent conditions include, for example, low-salt and / or high-temperature conditions, such as those provided by about 0.02-0.1 M NaCl or an 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.

[0086] Furthermore, embodiments of the present invention 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.

[0087] Embodiments of the present invention provide an isolated or purified nucleic acid comprising a first nucleic acid sequence and a second nucleotide sequence in the 5'→3' direction, wherein the first and second nucleotide sequences are, respectively, sequence numbers 13 and 14; 14 and 13; 15 and 16; 16 and 15; 30 and 31; 31 and 30; 32 and 33; 33 and 32; 34 and 35; 35 and 34; 36 and 37; 37 and 36; 41 and 42; 42 and 41; 43 and 44; 44 and 43; 45 and 46; 46 and 45; 47 and 48; 48 and 47; 49 and 50; 50 and 49; The present invention provides nucleic acids encoding amino acid sequences 51 and 52; or 52 and 51.

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

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

[0090] 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 cause the cell to express the mRNA, protein, polypeptide, or peptide, the cell can 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.

[0091] 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.

[0092] The recombinant expression vectors of the present invention can be prepared, for example, using the standard recombinant DNA techniques described above, such as those by Green and Sambrook et al. The expression vector constructs, which may be circular or linear, can be prepared to include 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.

[0093] 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.

[0094] 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, e.g., resistance to antibiotics, heavy metals, etc., 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] Another embodiment of the present invention further provides a host cell containing any of the recombinant expression vectors 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 isolated directly from an organism, e.g., a human. The host cell may be an adherent cell or a suspension cell, i.e., a cell that grows 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. The host cell may be a tertiary cell, but is preferably a peripheral blood lymphocyte (PBL) or a 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 provides a method for generating host cells expressing a TCR having antigen specificity for the peptide of SEQ ID NO: 39, comprising contacting the cells with a vector under conditions that allow introduction of any of the vectors described herein into the cells.

[0099] For the purposes of this specification, T cells may be any T cells, such as cultured T cells, such as primary T cells, or T cells derived from cultured T cell lines, such as Jurkat, SupT1, etc., or T cells obtained from mammals. When obtained from mammals, 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. The T cells may also be concentrated or purified. Preferably, the T cells are human T cells. The T cells may be any type of T cell and may be at any developmental stage, for example, CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells, e.g., Th1 and Th2 cells, CD4 + T cells, CD8 + Examples include, but are not limited to, T cells (e.g., cytotoxic T cells), tumor-infiltrating lymphocytes (TILs), memory T cells (e.g., central memory T cells and effector memory T cells), and naive T cells.

[0100] Furthermore, the present invention provides a population of cells comprising at least one host cell as described herein. The population of cells may be a heterogeneous population comprising at least one other cell, 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 comprising one of the described recombinant expression vectors. Alternatively, the population of cells may be a substantially homogeneous population comprising mainly host cells (e.g., essentially consisting of) a recombinant expression vector. The population may also be a clonal population of cells in which all cells are clones of one host cell comprising a recombinant expression vector, and as a result all cells in the population comprise a recombinant expression vector. In one embodiment of the present invention, the population of cells is a clonal population comprising a host cell comprising a recombinant expression vector as described herein.

[0101] 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, such as those described in, for example, 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 achieved by culturing the T cells with an OKT3 antibody, IL-2, and feeder PBMCs (e.g., irradiated allogeneic PBMCs).

[0102] The TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, and host cells (including populations thereof) of the present invention may be isolated and / or purified. The term “isolated” as used herein means removed from its natural environment. The term “purified” as used herein means increased purity; “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%.

[0103] The TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, and host cells (including populations thereof) of the present invention (hereinafter, all of these are 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, e.g., polypeptides and nucleic acids, or two or more different TCRs. Alternatively, a pharmaceutical composition may contain the TCR materials of the present invention in combination with another pharmaceutically active agent or drug (may include multiple), e.g., chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.

[0104] 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 ndThis 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.

[0105] 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 for parenteral, subcutaneous, intravenous, intramuscular, intra-arterial, subarachnoid, intratumoral, or intraperitoneal administration. 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.

[0106] Preferably, the TCR material of the present invention is administered, for example, by intravenous injection. When the TCR material of the present invention is host cells (or a population thereof) expressing the TCR of the present invention, a pharmaceutically acceptable carrier for the cells for injection may contain any isotonic carrier, for example, ordinary 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.

[0107] 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, for example, 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., G12R 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).

[0108] Many assays for determining the dose to be administered are known in the art. For the purposes of the present invention, for example, a set of mammals given different doses of T cells In this context, an initial dose to be administered to a mammal can be determined 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 when such T cells are administered to a mammal. The extent to which target cells are lysed or IFN-γ is secreted when a specific dose is administered can be assayed by methods known in the art.

[0109] 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 at the level of a single cell or more. In a particular embodiment, 1 × 10 6It is acceptable to administer fewer than one cell.

[0110] 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 crosslinking and / or chemotherapeutic agents, provided that the crosslinking and / or chemotherapeutic agent does not interfere with the function of the TCR material of the present invention, i.e., its ability to bind to the G12R RAS or to detect, treat, or prevent cancer, once conjugated to the TCR material of the present invention.

[0111] 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 the G12R 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 the G12R 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 cells or populations of cells containing a recombinant vector encoding any of the TCRs, polypeptides, or proteins described herein.

[0112] Embodiments of the present invention provide a method for inducing an immune response to cancer in a mammal, comprising administering to the mammal an amount effective for inducing an immune response to cancer in the mammal 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.

[0113] Embodiments of the present invention relate to pharmaceutical compositions, TCRs, polypeptides, or proteins described herein for use in the treatment or prevention of cancer in mammals. The present invention provides any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding one of the TCRs, polypeptides, or proteins described herein, or any host cell or population of cells comprising a recombinant vector encoding one of the TCRs, polypeptides, or proteins described herein.

[0114] 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.

[0115] The terms “treat” and “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 treatment or prevention of any amount or level of cancer in mammals. Furthermore, the treatment or prevention provided by the methods of the present invention may include treatment or prevention of one or more pathological 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 pathological conditions. Alternatively, or additionally, “prevention” may include preventing or delaying the recurrence of cancer or its symptoms or pathological conditions.

[0116] 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 detecting the complex, wherein the detection of the complex indicates the presence of cancer in the mammal.

[0117] 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.

[0118] 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.

[0119] 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).

[0120] 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 autologous to mammals. Preferably, the cells are autologous to mammals.

[0121] 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, and oral cancer. Any cancer may be any of the following: cancer of the 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 tumor, glioma, Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, oropharyngeal cancer, ovarian cancer, penile cancer, pancreatic cancer, cancer of the peritoneum, retinoplasm, and mesentery, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, ureteral cancer, and bladder cancer. Preferred cancers are pancreatic, colorectal, lung, endometrial, ovarian, or prostate cancer. Preferred cancers are those of the pancreas, colorectal, lung, endometrium, ovaries, or prostate. Preferably, lung cancer is lung adenocarcinoma, ovarian cancer is epithelial ovarian cancer, and pancreatic cancer is pancreatic adenocarcinoma. In embodiments of the present invention, the cancer expresses a mutant human RAS amino acid sequence in which glycine at position 12 is substituted with arginine, and the mutant human RAS amino acid sequence is defined by the amino acid sequence of mutant human KRAS, mutant human HRAS, or mutant human NRAS, where position 12 refers to the protein of WT human KRAS, WT human HRAS, or WT human NRAS, respectively. The mutant human KRAS, mutant human HRAS, and mutant human NRAS expressed by the cancer may be as described herein in relation to other embodiments of the present invention.

[0122] 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.

[0123] The following embodiments further illustrate the present invention, but should not be construed as limiting its scope in any way. [Examples]

[0124] Example 1 This example uses HLA-DRB5 * 01: HLA-DRA * This shows the isolation of an antigen-specific TCR for human KRAS with a G12R mutation presented by the 01:01 heterodimer.

[0125] HLA-DRB5 * 01: HLA-DRA * A TCR exhibiting antigen specificity for human KRAS with a G12R mutation presented by a 01:01 heterodimer was isolated from a TIL of a metastatic pancreatic cancer patient (patient 4270). The TCR isolated from the TIL showed specific recognition for KRAS G12R but not for WT KRAS.

[0126] To determine the sequence of the reactive 4270 TCR, reactive TILs were selected by fluorescence-activated cell sorting (FACS) based on the upregulation of 4-1BB, a T cell activation marker. The cells were then lysed, and the TCR transcripts were sequenced using the Sanger sequencing method. Table 5 shows the amino acid sequences of the variable regions of the alpha and beta chains of the 4270 TCR. The CDR is underlined.

[0127] [Table 5]

[0128] Example 2 In this example, cells transduced from the 4270 TCR isolated in Example 1 were G12R This study demonstrates the specific recognition of RAS peptides.

[0129] The nucleic acid sequence encoding the G12R RAS-reactive 4270 TCR of Example 1, including the cysteine-substituted LVL-modified mouse constant region, was cloned into a retroviral expression vector. The α-chain mouse constant region contained the amino acid sequence of SEQ ID NO: 26, where X at position 48 is Cys, X at position 112 is Leu, X at position 114 is Ile, and X at position 115 is Val. The β-chain constant region contained the amino acid sequence of SEQ ID NO: 27, where X at position 57 is Cys. The linker containing the amino acid sequence RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 38) was located between the α-chain and β-chain constant regions. The retroviral expression vector was transduced into allogeneic PBMCs.

[0130] Transduced cells, WT RAS peptide MTEYKLVVVGA G GVGKSALTIQLI (SEQ ID NO: 40) or G12R RAS peptide MTEYKLVVVGA R Serial dilutions of GVGKSALTIQLI (SEQ ID NO: 39) were co-cultured overnight with pulsed autologous DCs. IFNγ secretion was evaluated using enzyme-linked immunosorbent spot (ELISpot). The results are shown in Figure 1.

[0131] As shown in Figure 1, cells transduced with 4270 TCR specifically recognized the G12R RAS peptide.

[0132] Example 3 In this example, cells transduced from the 4270 TCR isolated in Example 1 were found to be HLA-DRB5 * 01: HLA-DRA * 01:01 Demonstrates recognition of the G12R RAS peptide presented in relation to heterodimers.

[0133] The retroviral expression vector from Example 2 was transduced into allogeneic PBMCs using a virus. One of the HLA molecules expressed by patient 4270 was transferred to the target COS7 cells. Transfected and shown in Figure 2. Next, the transfected COS7 cells were pulsed with the G12R RAS peptide MTEYKLVVVGA R GVGKSALTIQLI (SEQ ID NO: 39). Cells transduced with 4270 TCR were pulsed and co-cultured overnight with the pulsed and transfected COS7 cells. IFNγ secretion was evaluated by ELISA. The results are shown in Figure 2.

[0134] As shown in Figure 2, cells transduced with the 4270 TCR isolated in Example 1 specifically recognized the G12R RAS peptide presented in association with the HLA-DRB5 * 01:HLA-DRA * 01:01 heterodimer.

[0135] Example 4 This example shows the isolation of a TCR with antigen specificity for human KRAS having a G12R mutation presented by the HLA-DQA1 * 05:05:HLA-DQB1 * 03:01 heterodimer.

[0136] HLA-DQA1 * 05:05:HLA-DQB1 * A TCR with antigen specificity for human KRAS having a G12R mutation presented by the HLA-DQA1

[0137] 05:05:HLA-DQB1

[0138]

Table 6

[0139] Example 5 This example demonstrates that cells transduced with the 4268 TCR isolated in Example 4 specifically recognize the G12R RAS peptide.

[0140] A nucleic acid sequence encoding the G12R RAS-reactive 4268 TCR of Example 4 and containing a cysteine-substituted LVL-modified mouse constant region was cloned into a retroviral expression vector. The α-chain mouse constant region contained the amino acid sequence of SEQ ID NO: 26 where X at position 48 was Cys, X at position 112 was Leu, X at position 114 was Ile, and X at position 115 was Val. The β-chain constant region contained the amino acid sequence of SEQ ID NO: 27 where X at position 57 was Cys. A linker containing the amino acid sequence of RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 38) was located between the α-chain constant region and the β-chain constant region. The retroviral expression vector was transduced into allogeneic PBMCs.

[0141] The transduced cells were co-cultured overnight with autologous DCs pulsed with serial dilutions of the WT RAS peptide MTEYKLVVVGA G GVGKSALTIQLI (SEQ ID NO: 40) or the G12R RAS peptide MTEYKLVVVGA R GVGKSALTIQLI (SEQ ID NO: 39). IFNγ secretion was evaluated by ELIspot. The results are shown in Figure 3.

[0142] As shown in Figure 3, cells transduced with the 4268 TCR specifically recognized the G12R RAS peptide.

[0143] Example 6 This example demonstrates that cells transduced with the 4268 TCR isolated in Example 4 recognize HLA-DQA1 * 05:05:HLA-DQB1 *03:01 Demonstrates recognition of the G12R RAS peptide presented in relation to heterodimers.

[0144] The retroviral expression vector from Example 5 was transfected into allogeneic PBMCs using a virus. Target COS7 cells were transfected with one of the HLA molecules expressed by patient 4268, as shown in Figure 4. Subsequently, the transfected COS7 cells were treated with the G12R RAS peptide MTEYKLVVVGA R GVGKSALTIQLI (SEQ ID NO: 39) was pulsed. Cells transduced with 4268 TCRs were co-cultured overnight with pulsed and transfected COS7 cells. IFNγ secretion was evaluated by ELISA. The results are shown in Figure 4.

[0145] As shown in Figure 4, the cells transduced with 4268 TCR isolated in Example 4 were HLA-DQA1 * 05:05:HLA-DQB1 * 03:01 The G12R RAS peptide presented in relation to the heterodimer was specifically recognized.

[0146] 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.

[0147] 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 encompassing one item (A or B) selected from the enumerated items or any combination of two or more of the enumerated items, unless otherwise specified herein or unless clearly contradicted by the context. It should be interpreted as meaning (A and B). The terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., “including but not limited to”) unless otherwise specified herein. Unless otherwise specified herein, the enumeration of ranges of values ​​is intended to function simply 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 carried out 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 simply to elucidate the invention in more detail and does not raise any limitation of the scope of the invention unless otherwise specifically asserted. No expression in the specification should be interpreted as indicating that any unclaimed element is essential for carrying out the invention.

[0148] 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. A method for generating human cells or populations of human cells that express T cell receptors (TCRs), A method comprising introducing a recombinant expression vector into isolated human cells or a population of isolated human cells, wherein the recombinant expression vector comprises a nucleotide sequence encoding the TCR, and the TCR has antigen specificity to a mutant human RAS amino acid sequence in which the glycine at position 12 is replaced with arginine. 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 protein of wild-type human KRAS, wild-type human HRAS, or wild-type human NRAS, and The aforementioned TCR, (a) CDR1 of the α chain containing the amino acid sequence of SEQ ID NO: 1, CDR2 of the α chain containing the amino acid sequence of SEQ ID NO: 2, CDR3 of the α chain containing the amino acid sequence of SEQ ID NO: 3, CDR1 of the β chain containing the amino acid sequence of SEQ ID NO: 4, CDR2 of the β chain containing the amino acid sequence of SEQ ID NO: 5, and CDR3 of the β chain containing the amino acid sequence of SEQ ID NO: 6, or (b) CDR1 of the α chain containing the amino acid sequence of SEQ ID NO: 7, CDR2 of the α chain containing the amino acid sequence of SEQ ID NO: 8, CDR3 of the α chain containing the amino acid sequence of SEQ ID NO: 9, CDR1 of the β chain containing the amino acid sequence of SEQ ID NO: 10, CDR2 of the β chain containing the amino acid sequence of SEQ ID NO: 11, and CDR3 of the β chain containing the amino acid sequence of SEQ ID NO: 12 Methods that include...

2. The method according to claim 1, wherein the mutant human RAS amino acid sequence is Sequence ID No.

39.

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

40.

4. The method according to any one of claims 1 to 3, wherein the mutant human RAS amino acid sequence is presented by a human leukocyte antigen (HLA) class II molecule.

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

6. The method according to claim 4, wherein the HLA class II molecule is an HLA-DRB5:HLA-DRA heterodimer or an HLA-DQA1:HLA-DQB1 heterodimer.

7. The aforementioned HLA class II molecule is HLA-DRB5 ＊ 01:HLA-DRA ＊ 01:01 heterodimer or HLA-DQA1 ＊ 05:05:HLA-DQB1 ＊ The method according to claim 4, wherein the 03:01 heterodimer is present.

8. The aforementioned TCR, (i) an amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 13, (ii) An amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 14, (iii) An amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 15, (iv) An amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 16, (v) Both an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 13 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

14. (vi) Both an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 15 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

16. (vii) An amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 41, (viiii) An amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 42, (ix) An amino acid sequence that is at least 95% identical to the amino acid sequence of sequence number 43, (x) An amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 44, (xi) Both an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 41 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 42, or (xi) Both an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 43 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 44 The method according to any one of claims 1 to 7, including the method described in any one of claims 1 to 7.

9. The aforementioned TCR, (a) (i) The X at position 48 of sequence number 26 is either Thr or Cys; (ii) The X at position 112 of sequence number 26 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) The X at position 114 of sequence number 26 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) An α-chain constant region containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 26, wherein X at position 115 of SEQ ID NO: 26 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) A constant region of the β chain containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 27, wherein X at position 57 of SEQ ID NO: 27 is Ser or Cys; or (c) Both (a) and (b) The method according to any one of claims 1 to 8, further comprising:

10. TCR, (a) (i) The X at position 180 of sequence number 30 is either Thr or Cys; (ii) The X at position 244 of sequence number 30 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 246 of sequence number 30 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) An α-chain containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 30, wherein X at position 247 of SEQ ID NO: 30 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) A β-chain containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 31, wherein the X at position 198 of SEQ ID NO: 31 is Ser or Cys; (c) (i) The X at position 188 of sequence number 32 is either Thr or Cys; (ii) The X at position 252 of sequence number 32 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 254 of sequence number 32 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) An α-chain containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 32, wherein X at position 255 of SEQ ID NO: 32 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (d) A β-chain containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 33, wherein the X at position 191 of SEQ ID NO: 33 is Ser or Cys; (e) Both (a) and (b); (f) Both (c) and (d); (g) (i) The X at position 161 of sequence number 45 is either Thr or Cys; (ii) The X at position 225 of sequence number 45 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 227 of sequence number 45 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) An α-chain containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 45, wherein X at position 228 of SEQ ID NO: 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (h) A β-chain containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 46, wherein the X at position 178 of SEQ ID NO: 46 is Ser or Cys; (i) (i) The X at position 168 of sequence number 47 is either Thr or Cys; (ii) The X at position 232 of sequence number 47 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 234 of sequence number 47 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) An α-chain containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 47, wherein X at position 235 of SEQ ID NO: 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (j) A β-chain containing an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 48, wherein the X at position 171 of SEQ ID NO: 48 is Ser or Cys; (k)(g) and (h) both; or (l)(i) and (j) The method according to any one of claims 1 to 9, including the method described in any one of claims 1 to 9.

11. A method for generating human cells or populations of human cells that express polypeptides, A method comprising introducing a recombinant expression vector into isolated human cells or a population of isolated human cells, wherein the recombinant expression vector comprises a nucleotide sequence encoding a polypeptide containing a functional portion of the TCR, and the functional portion is (a) CDR1 of the α chain containing the amino acid sequence of SEQ ID NO: 1, CDR2 of the α chain containing the amino acid sequence of SEQ ID NO: 2, CDR3 of the α chain containing the amino acid sequence of SEQ ID NO: 3, CDR1 of the β chain containing the amino acid sequence of SEQ ID NO: 4, CDR2 of the β chain containing the amino acid sequence of SEQ ID NO: 5, and CDR3 of the β chain containing the amino acid sequence of SEQ ID NO: 6, or (b) CDR1 of the α chain containing the amino acid sequence of SEQ ID NO: 7, CDR2 of the α chain containing the amino acid sequence of SEQ ID NO: 8, CDR3 of the α chain containing the amino acid sequence of SEQ ID NO: 9, CDR1 of the β chain containing the amino acid sequence of SEQ ID NO: 10, CDR2 of the β chain containing the amino acid sequence of SEQ ID NO: 11, and CDR3 of the β chain containing the amino acid sequence of SEQ ID NO: 12 Includes, The aforementioned functional portion has antigen specificity for a mutant human RAS amino acid sequence in which glycine at position 12 is replaced with arginine. 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), and The 12th position is defined by referencing the proteins of wild-type human KRAS, wild-type human HRAS, or wild-type human NRAS, respectively. method.

12. The aforementioned functional part, (i) an amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 13, (ii) An amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 14, (iii) An amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 15, (iv) An amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 16, (v) Both an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 13 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

14. (vi) Both an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 15 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

16. (vii) An amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 41, (viiii) An amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 42, (ix) An amino acid sequence that is at least 95% identical to the amino acid sequence of sequence number 43, (x) An amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 44, (xi) Both an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 41 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 42, or (xi) Both an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 43 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 44 The method according to claim 11, including the method described in claim 11.

13. The aforementioned functional part, (a) (i) The X at position 48 of sequence number 26 is either Thr or Cys; (ii) The X at position 112 of sequence number 26 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) The X at position 114 of sequence number 26 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) An amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 26, wherein X at position 115 of Sequence ID No. 26 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) an amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 27, wherein X at position 57 of Sequence ID No. 27 is Ser or Cys; or (c) Both (a) and (b) The method according to claim 11 or 12, further comprising:

14. The aforementioned functional part, (a) (i) The X at position 180 of sequence number 30 is either Thr or Cys; (ii) The X at position 244 of sequence number 30 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 246 of sequence number 30 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) An amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 30, wherein X at position 247 of Sequence ID No. 30 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) An amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 31, wherein X at position 198 of Sequence ID No. 31 is Ser or Cys; (c) (i) The X at position 188 of sequence number 32 is either Thr or Cys; (ii) The X at position 252 of sequence number 32 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 254 of sequence number 32 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) An amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 32, wherein X at position 255 of SEQ ID NO: 32 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (d) An amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 33, wherein X at position 191 of Sequence ID No. 33 is Ser or Cys; (e) Both (a) and (b); (f) Both (c) and (d); (g) (i) The X at position 161 of sequence number 45 is either Thr or Cys; (ii) The X at position 225 of sequence number 45 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 227 of sequence number 45 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) An amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 45, wherein X at position 228 of Sequence ID No. 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (h) An amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 46, wherein the X at position 178 of Sequence ID No. 46 is Ser or Cys; (i) (i) The X at position 168 of sequence number 47 is either Thr or Cys; (ii) The X at position 232 of sequence number 47 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 234 of sequence number 47 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) An amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 47, wherein X at position 235 of Sequence ID No. 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (j) An amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 48, wherein X at position 171 of Sequence ID No. 48 is Ser or Cys; (k)(g) and (h) both; or (l)(i) and (j) The method according to any one of claims 11 to 13, including the method described in any one of claims 11 to 13.

15. A method for generating human cells or populations of human cells that express a protein, A method comprising introducing a recombinant expression vector into isolated human cells or a population of isolated human cells, wherein the recombinant expression vector comprises a nucleotide sequence encoding a protein containing a functional portion of a T cell receptor (TCR), and the functional portion is (a) 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, or (b) A first polypeptide chain comprising CDR1 of an α chain containing the amino acid sequence of SEQ ID NO: 7, CDR2 of an α chain containing the amino acid sequence of SEQ ID NO: 8, and CDR3 of an α chain containing the amino acid sequence of SEQ ID NO: 9, and a second polypeptide chain comprising CDR1 of a β chain containing the amino acid sequence of SEQ ID NO: 10, CDR2 of a β chain containing the amino acid sequence of SEQ ID NO: 11, and CDR3 of a β chain containing the amino acid sequence of SEQ ID NO:

12. Includes, The aforementioned functional portion has antigen specificity for a mutant human RAS amino acid sequence in which glycine at position 12 is replaced with arginine. 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), and The 12th position is defined by referencing the proteins of wild-type human KRAS, wild-type human HRAS, or wild-type human NRAS, respectively. method.

16. (i) The first polypeptide chain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 13, and the second polypeptide chain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 14; (ii) The first polypeptide chain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 15, and the second polypeptide chain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

16. (iii) The first polypeptide chain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 41 and the second polypeptide chain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 42; or (iv) The first polypeptide chain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 43 and the second polypeptide chain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

44. The method according to claim 15, including the method described in claim 15.

17. (a) (i) The X at position 48 of sequence number 26 is either Thr or Cys; (ii) The X at position 112 of sequence number 26 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) The X at position 114 of sequence number 26 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) The first polypeptide chain contains an amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 26, wherein X at position 115 of Sequence ID No. 26 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) The second polypeptide chain contains an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 27, wherein the X at position 57 of SEQ ID NO: 27 is Ser or Cys; or (c) Both (a) and (b) The method according to claim 15 or 16.

18. (a) (i) The X at position 180 of sequence number 30 is either Thr or Cys; (ii) The X at position 244 of sequence number 30 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 246 of sequence number 30 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) The first polypeptide chain contains an amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 30, wherein X at position 247 of Sequence ID No. 30 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) The second polypeptide chain contains an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 31, wherein the X at position 198 of SEQ ID NO: 31 is Ser or Cys; (c) (i) The X at position 188 of sequence number 32 is either Thr or Cys; (ii) The X at position 252 of sequence number 32 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 254 of sequence number 32 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) The first polypeptide chain contains an amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 32, wherein X at position 255 of Sequence ID No. 32 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (d) The second polypeptide chain contains an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 33, wherein the X at position 191 of SEQ ID NO: 33 is Ser or Cys; (e) Both (a) and (b); (f) Both (c) and (d); (g) (i) The X at position 161 of sequence number 45 is either Thr or Cys; (ii) The X at position 225 of sequence number 45 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 227 of sequence number 45 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) The first polypeptide chain contains an amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 45, wherein X at position 228 of Sequence ID No. 45 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (h) The first polypeptide chain contains an amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 46, wherein the X at position 178 of Sequence ID No. 46 is Ser or Cys; (i) (i) The X at position 168 of sequence number 47 is either Thr or Cys; (ii) The X at position 232 of sequence number 47 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 234 of sequence number 47 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) A first polypeptide chain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of Sequence ID No. 47, wherein X at position 235 of Sequence ID No. 47 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (j) A second polypeptide chain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 48, wherein X at position 171 of SEQ ID NO: 48 is either Ser or Cys; (k)(g) and (h) both; or (l)(i) and (j) The method according to any one of claims 15 to 17.

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