HLA class I restrictive T cell receptors for mutated RAS

Abstract

An isolated or purified T cell receptor (TCR) is disclosed, wherein the TCR has antigen specificity for a mutated RAS amino acid sequence presented by a human leukocyte antigen (HLA) class I molecule. Also provided are related polypeptides and proteins, as well as related nucleic acids, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions. Methods of detecting the presence of cancer in a mammal and methods of treating or preventing cancer in a mammal are also disclosed.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims the benefit of U.S. Provisional Patent Application No. 62 / 594,244, filed on December 4, 2017, which is incorporated herein by reference in its entirety.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0004] This invention was made with government support through the National Cancer Institute of the National Institutes of Health under Grant No. BC010985. The government has certain rights in this invention. Electronically Submitted Materials Incorporated by Reference

[0005] Incorporated herein by reference in its entirety is a computer-readable nucleotide / amino acid sequence listing filed concurrently with this document and identified as follows: a 37,098-byte ASCII (text) file named "741039_ST25.txt" and dated December 3, 2018. Background of the Invention

[0007] Some cancers may have very limited treatment options, especially when the cancer becomes metastatic and unresectable. Although treatments such as, for example, surgery, chemotherapy, and radiation therapy have advanced, the prognosis of many cancers, such as, for example, pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer, and prostate cancer, may be poor. Therefore, there is an unmet need for additional treatments for cancer. Brief description of the invention

[0009] Embodiments of the present invention provide an isolated or purified T cell receptor (TCR) comprising an amino acid sequence of (i) SEQ ID NO: 1-3, (ii) SEQ ID NO: 4-6 or (iii) SEQ ID NO: 1-6, wherein the TCR has antigenic specificity for a mutated human RAS amino acid sequence presented by a human leukocyte antigen (HLA) class I molecule, and wherein the mutated human RAS amino acid sequence is a mutated human Kirsten rat sarcoma viral oncogene homolog (KRAS) amino acid sequence, a mutated human Harvey rat sarcoma viral oncogene homolog (HRAS) amino acid sequence or a mutated human neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence.

[0010] Another embodiment of the present invention provides an isolated or purified polypeptide comprising a functional portion of the TCR of the present invention, wherein the functional portion comprises the following amino acid sequence: (a) all of SEQ ID NO: 1-3, (b) all of SEQ ID NO: 4-6, or (c) all of SEQ ID NO: 1-6.

[0011] Another embodiment of the present invention provides an isolated or purified protein comprising at least one polypeptide of the present invention.

[0012] Other embodiments of the present invention provide nucleic acids, recombinant expression vectors, host cells, cell populations and pharmaceutical compositions related to the TCR, polypeptides and proteins of the present invention.

[0013] Embodiments of the invention also provide methods of detecting the presence of cancer in a mammal and methods of treating or preventing cancer in a mammal.

[0014] Brief description of several views in the accompanying drawings

[0015] Figure 1A Depicted are graphs illustrating CD137 expression by peripheral blood CD8+ T cells after co-culture with autologous DC pulsed with dimethyl sulfoxide (DMSO) or with KRAS WT 24-mer peptide or KRAS G12V24-mer peptide. The numbers in the boxes represent the percentage of T cells expressing CD137 under each co-culture condition.

[0016] Figure 1B Figure 2 shows that after T cells were co-cultured with autologous DCs pulsed with DMSO, KRAS WT 24-mer peptide, or KRAS G12V 24-mer peptide, 2x10 4 Figure 3 is a graph of the number of IFN-γ spots per cell. T cells cultured with PMA:ionomycin and T cells cultured with DCs pulsed with DMSO served as controls.

[0017] Figure 2 Graphs depicting experimental data (dot plots) are shown, illustrating the percentage of CD8+ peripheral blood cells that have been subjected to IVS with KRAS G12V 24-mer peptides bound to KRAS G12V tetramer-allophycocyanin (APC) or KRAS G12V tetramer-phycoerythrin (PE) (right graph). The left graph shows T cells that have been modified to express a TCR that recognizes KRAS G12D 10-mer bound to KRAS G12V tetramer-APC or KRAS G12V tetramer-PE in the context of HLA-A*11:01.

[0018] Figure 3Depicted are graphs showing TCR expression on the surface of allogeneic T cells from a first donor and a second donor (left and right panels, respectively) transduced with the TCR of Example 2, as measured by flow cytometry by staining for the constant region of the murine TCR β chain (mTCR β). The numbers in the boxes represent the percentage of cells positive for mTCR β expression.

[0019] Figure 4 Figure 2 shows the difference between KRAS expression and G12 IFN-γ production (as measured by ELISPOT assay (IFN-γ spots / 2E4 cells) and 4-1BB expression (% mTCRβ+CD137+) of T cells from two different donors (Donor 1 and Donor 2) transduced with the TCR of Example 2 after co-culture of the mutant cancer cell lines overnight. Each target cancer cell line was either untransduced or transduced with HLA-A*11:01. ">" indicates greater than 500 spots.

[0020] Figure 5A is a graph showing the concentration (pg / mL) of IFN-γ secreted after co-culturing T cells transduced with the TCR of Example 2 with target cells pulsed with the indicated concentrations (ng / mL) of KRAS G12V 9-mer peptide (solid circles) or KRAS WT 9-mer peptide (open circles).

[0021] Figure 5B is a graph showing the concentration (pg / mL) of IFN-γ secreted after co-culturing T cells transduced with the TCR of Example 2 with target cells pulsed with the indicated concentrations (ng / mL) of KRAS G12V 10-mer peptide (solid circles) or KRAS WT 10-mer peptide (open circles). DETAILED DESCRIPTION OF THE INVENTION

[0023] RAS family proteins belong to a large family of small GTPases. Not bound by a specific theory or mechanism, it is believed that when mutated, RAS proteins can participate in the early signal transduction of tumor formation in many human cancers. Single amino acid substitutions can activate proteins. Mutated RAS protein products can be constitutively activated. Mutated RAS proteins can be expressed in any of a variety of human cancers, such as 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. Human RAS family proteins include Kirsten rat sarcoma viral oncogene homolog (KRAS), Harvey rat sarcoma viral oncogene homolog (HRAS), and neuroblastoma rat sarcoma viral oncogene homolog (NRAS).

[0024] KRAS is also known as GTPase KRas, V-Ki-Ras2 Kirsten rat sarcoma viral oncogene or KRAS2. There are two transcript variants of KRAS: KRAS variant A and KRAS variant B. Wild-type (WT) KRAS variant A has the amino acid sequence of SEQ ID NO: 9. Wild-type (WT) KRAS variant B has the amino acid sequence of SEQ ID NO: 10. In the following, unless otherwise specified, reference to "KRAS" (mutated or unmutated (WT)) refers to variant A and variant B. When activated, mutant KRAS binds guanosine-5'-triphosphate (GTP) and converts GTP to guanosine-5'-diphosphate (GDP).

[0025] HRAS is another member of the RAS protein family. HRAS is also known as Harvey rat sarcoma viral oncoprotein, V-Ha-Ras Harvey rat sarcoma viral oncogene homolog or Ras family small GTP binding protein H-Ras. WT HRAS has the amino acid sequence of SEQ ID NO:11.

[0026] NRAS is another member of the RAS protein family. NRAS is also known as GTPase NRas, V-Ras neuroblastoma RAS viral oncogene homolog or NRAS1. WT NRAS has the amino acid sequence of SEQ ID NO:12.

[0027] Embodiments of the present invention provide an isolated or purified TCR having antigenic specificity for a mutant human RAS amino acid sequence (hereinafter referred to as "mutated RAS") presented by a human leukocyte antigen (HLA) class I molecule, wherein the mutant human RAS amino acid sequence is a mutant human KRAS, a mutant human HRAS or a mutant human NRAS amino acid sequence. In the following, unless otherwise specified, the "TCR" referred to also refers to the functional part and functional variant of the TCR.

[0028] The TCR of the present invention may have antigenic specificity for any mutated human RAS protein, polypeptide or peptide amino acid sequence. In an embodiment of the present invention, the mutated human RAS amino acid sequence is a mutated human KRAS amino acid sequence, a mutated human HRAS amino acid sequence or a mutated human NRAS amino acid sequence. The amino acid sequences of WT human KRAS, NRAS and HRAS proteins each have a length of 188-189 amino acid residues and are highly identical to each other. For example, the amino acid sequence of WT human NRAS protein is 86.8% identical to the amino acid sequence of WT human KRAS protein. The amino acid residues 1-86 of WT human NRAS protein and WT human KRAS protein are 100% identical. The amino acid sequence of WT human HRAS protein is 86.3% identical to the amino acid sequence of WT human KRAS protein. The amino acid residues 1-94 of WT human HRAS protein and WT human KRAS protein are 100% identical. In the following, unless otherwise specified, the "RAS" (mutated or unmutated (WT)) mentioned are collectively referred to as KRAS, HRAS and NRAS.

[0029] In an embodiment of the present invention, the mutant human RAS amino acid sequence includes a WT RAS amino acid sequence in which glycine at position 12 is substituted, wherein position 12 is defined by reference to a WT RAS protein, respectively. The WT RAS protein may be any of a WT KRAS protein (SEQ ID NO: 9 or 10), a WT HRAS protein (SEQ ID NO: 11), or a WT NRAS protein (SEQ ID NO: 12), because as explained above, amino acid residues 1-86 of the WT human NRAS protein and the WT human KRAS protein are 100% identical, and amino acid residues 1-94 of the WT human HRAS protein and the WT human KRAS protein are 100% identical. Therefore, the amino acid residue at position 12 of each of the WT KRAS, WT HRAS, and WT NRAS proteins is the same, i.e., glycine.

[0030] The glycine at position 12 of the WT RAS amino acid sequence can be substituted with any amino acid residue other than glycine. In an embodiment of the present invention, the substitution is to replace the glycine at position 12 of the WT RAS amino acid sequence with valine. In this regard, embodiments of the present invention provide TCRs with antigenic specificity for any WT RAS protein, polypeptide or peptide amino acid sequence having a G12V mutation.

[0031] In this article, mutations and substitutions of RAS are defined by reference to the amino acid sequence of WT RAS protein. Therefore, in this article, mutations and substitutions of RAS are described by reference to the amino acid residue present in a specific position in the WT RAS protein, followed by the position number, followed by the amino acid residue replacing the residue in the specific mutation or substitution. A RAS amino acid sequence (e.g., a RAS peptide) may contain less than all the amino acid residues of the full-length WT RAS protein. Therefore, in the case where it is understood that the actual position of the corresponding residue in a specific instance of the RAS amino acid sequence may be different, in this article, the 12th position is defined by reference to the WT full-length RAS protein (i.e., any one of SEQ ID NO:9-12). When the position is as defined by any one of SEQ ID NO:9-12, the term "G12" refers to the glycine at the 12th position usually present in any one of SEQ ID NO:9-12, and "G12V" indicates that the glycine at the 12th position usually present in any one of SEQ ID NO:9-12 is replaced by valine. For example, in a specific example of a RAS amino acid sequence, for example (an exemplary WT KRAS peptide corresponding to consecutive amino acid residues 2 to 24 of SEQ ID NO:9), "G12V" means that the underlined glycine in SEQ ID NO:28 is replaced by valine, even though the actual position of the underlined glycine in SEQ ID NO:28 is 11.

[0032] Examples of full-length RAS proteins with the G12V mutation are shown in Table 1 below.

[0033] Table 1

[0034] Mutated full-length RAS protein SEQ ID NO: G12V KRAS variant A 13 G12V KRAS variant B 14 G12V HRAS 15 G12V NRAS 16

[0035] In an embodiment of the present invention, TCR has antigenic specificity to the RAS peptide with the above-mentioned G12V mutation, wherein the mutated RAS peptide has any length. In an embodiment of the present invention, the mutated RAS peptide has any length suitable for binding to any HLA class I molecule described herein. For example, TCR can have antigenic specificity to the RAS peptide with G12V mutation, and the RAS peptide has a length of about 9 to about 10 amino acid residues. The mutated RAS peptide can include any continuous amino acid residues of the mutated RAS protein including the G12V mutation. In an embodiment of the present invention, TCR can have antigenic specificity to the RAS peptide with G12V mutation, and the mutated RAS peptide has a length of about 9 amino acid residues or about 10 amino acid residues. Examples of specific peptides each having a G12V mutation that can be recognized by the G12V TCR of the present invention are 9-merVVGAVGVGK (SEQ ID NO: 29) and 10-mer VVVGAVGVGK.

[0036] In an embodiment of the present invention, the TCR of the present invention is capable of recognizing a mutated RAS presented by an HLA class I molecule. In this regard, the TCR can elicit an immune response when binding to a mutated RAS in the context of an HLA class I molecule. In addition to a mutated RAS, the TCR of the present invention can also bind to an HLA class I molecule.

[0037] In an embodiment of the present invention, the HLA class I molecule is an HLA-A molecule. The HLA-A molecule is a heterodimer of an α chain and a β2 microglobulin. The HLA-A α chain can be encoded by an HLA-A gene. The β2 microglobulin non-covalently binds to the α1, α2, and α3 domains of the α chain to construct an HLA-A complex. The HLA-A molecule can be any HLA-A molecule. In an embodiment of the present invention, the HLA class I molecule is an HLA-A11 molecule. The HLA-A11 molecule can be any HLA-A11 molecule. Examples of HLA-A11 molecules may include, but are not limited to, HLA-A*11:01, HLA-A*11:02, HLA-A*11:03, or HLA-A*11:04. Preferably, the HLA class I molecule is an HLA-A*11:01 molecule.

[0038] The TCRs of the present invention may provide any one or more of a variety of advantages, including use in adoptive cell transfer when expressed by cells. Mutated RAS is expressed by cancer cells and not by normal non-cancerous cells. Without being bound by a particular theory or mechanism, it is believed that the TCRs of the present invention advantageously target the destruction of cancer cells while minimizing or eliminating the destruction of normal non-cancerous cells, thereby reducing (e.g., minimizing or eliminating) toxicity. In addition, the TCRs of the present invention may advantageously and successfully treat or prevent mutated RAS-positive cancers that are unresponsive to other types of treatments (such as, for example, chemotherapy, surgery, or radiation). RAS G12 Mutation is one of the most common hotspot mutations found in many cancer types. For example, KRAS G12V mutations are expressed in about 27% and about 9% of pancreatic cancer and colorectal cancer patients, respectively. Moreover, RAS family members share G12 hotspot mutations in different cancer types (e.g., NRAS in melanoma). In addition, the TCR of the present invention can provide high affinity recognition of mutated RAS, which can provide the ability to recognize untreated tumor cells (e.g., tumor cells not treated with interferon (IFN)-γ, not transfected with one or two vectors encoding mutated RAS and HLA-A*11:01, not pulsed with RAS peptides with G12V mutations, or a combination thereof). In addition, the HLA-A*11:01 allele is expressed in about 14% and about 9% of Caucasians and Hispanics, respectively. In the United States, the HLA-A*11:01 allele is expressed by up to about 45% of Asian races. Thus, the TCRs of the invention can increase the number of cancer patients eligible for immunotherapy to include those expressing the HLA-A*11:01 allele, which may not be suitable for immunotherapy using TCRs that recognize RAS presented by other MHC molecules. In addition, the TCRs, polypeptides and proteins of the invention comprise human amino acid sequences, which can reduce the risk of rejection by the human immune system compared to, for example, TCRs, polypeptides and proteins comprising mouse amino acid sequences.

[0039] As used herein, the phrase "antigen specificity" means that the TCR is able to specifically bind to and immunologically recognize a mutant RAS with high affinity. For example, if about 1×10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 4 About 1×10 5If a T cell expressing a TCR secretes at least about 200 pg / mL or more (e.g., 200 pg / mL or more, 300 pg / mL or more, 400 pg / mL or more, 500 pg / mL or more, 600 pg / mL or more, 700 pg / mL or more, 1000 pg / mL or more, 5,000 pg / mL or more, 7,000 pg / mL or more, 10,000 pg / mL or more, 20,000 pg / mL or more, or a range defined by any two of the foregoing values) of IFN-γ, the TCR can be considered to be "antigenic specific" for the mutant RAS. Cells expressing the TCR of the invention can also secrete IFN-γ when co-cultured with antigen-negative, HLA class I molecule-positive target cells pulsed with a higher concentration of a mutant RAS peptide. The HLA class I molecule can be any HLA class I molecule described herein (e.g., an HLA-A*11:01 molecule).

[0040] Alternatively or additionally, a TCR can be considered to be "antigen-specific" for mutant RAS if T cells expressing the TCR secrete at least twice the amount of IFN-γ expressed by a negative control when co-cultured with (a) antigen-negative, HLA class I-positive target cells pulsed with a low concentration of mutant RAS peptide or (b) antigen-negative, HLA class I-positive target cells into which a nucleotide sequence encoding mutant RAS has been introduced so that the target cells express mutant RAS. Negative controls can be, for example, (i) T cells expressing a TCR co-cultured with (a) antigen-negative, HLA class I-positive target cells pulsed with the same concentration of an irrelevant peptide (e.g., some other peptide having a different sequence from the mutated RAS peptide), or (b) antigen-negative, HLA class I-positive target cells into which a nucleotide sequence encoding an irrelevant peptide has been introduced so that the target cells express the irrelevant peptide; or (ii) untransduced T cells (e.g., derived from PBMCs that do not express a TCR) co-cultured with (a) antigen-negative, HLA class I-positive target cells pulsed with the same concentration of a mutated RAS peptide, or (b) antigen-negative, HLA class I-positive target cells into which a nucleotide sequence encoding a mutated RAS has been introduced so that the target cells express the mutated RAS. The HLA class I molecules expressed by the target cells of the negative control are the same as the HLA class I molecules expressed by the target cells co-cultured with the T cells being tested. The HLA class I molecule can be any HLA class I molecule described herein (eg, an HLA-A*11:01 molecule). IFN-γ secretion can be measured by methods known in the art, such as, for example, enzyme-linked immunosorbent assay (ELISA).

[0041] Alternatively or additionally, a TCR may be considered to be "antigen specific" for a mutant RAS if at least twice the number of T cells expressing the TCR secrete IFN-γ when co-cultured with (a) antigen-negative, HLA class I-positive target cells pulsed with a low concentration of a mutant RAS peptide or (b) an antigen-negative, HLA class I-positive target cell into which a nucleotide sequence encoding a mutant RAS has been introduced so that the target cell expresses the mutant RAS, compared to the number of negative control T cells that secrete IFN-γ. The HLA class I molecule, peptide concentration, and negative control may be as described herein for other aspects of the invention. The number of cells secreting IFN-γ may be measured by methods known in the art, such as, for example, ELISPOT.

[0042] Alternatively or additionally, if a T cell expressing the TCR upregulates the expression of one or more T cell activation markers (as measured by, for example, flow cytometry after stimulation with a target cell expressing the mutant RAS), the TCR can be considered to be "antigen-specific" for the mutant RAS. Examples of T cell activation markers include 4-1BB, OX40, CD107a, CD69, and cytokines that are upregulated upon antigen stimulation (e.g., tumor necrosis factor (TNF), interleukin (IL) -2, etc.).

[0043] Embodiments of the present invention provide TCR, which comprises two polypeptides (i.e., polypeptide chains), such as the α chain of TCR, the β chain of TCR, the γ chain of TCR, the δ chain of TCR or a combination thereof. The polypeptide of the TCR of the present invention can comprise any amino acid sequence, provided that the TCR has antigen specificity for the mutated RAS.

[0044] In an embodiment of the present invention, TCR comprises two polypeptide chains, each of which comprises a variable region containing a complementary determining region (CDR) 1, CDR2 and CDR3 of TCR. In an embodiment of the present invention, TCR comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprises a CDR1 (CDR1 of an α chain) containing a SEQ ID NO:1 amino acid sequence, a CDR2 (CDR2 of an α chain) containing a SEQ ID NO:2 amino acid sequence and a CDR3 (CDR3 of an α chain) containing a SEQ ID NO:3 amino acid sequence, and the second polypeptide chain comprises a CDR1 (CDR1 of a β chain) containing a SEQ ID NO:4 amino acid sequence, a CDR2 (CDR2 of a β chain) containing a SEQ ID NO:5 amino acid sequence and a CDR3 (CDR3 of a β chain) containing a SEQ ID NO:6 amino acid sequence. In this regard, the TCR of the present invention may include any one or more of the amino acid sequences selected from SEQ ID NO:1-6. In an embodiment of the invention, the TCR comprises the following amino acid sequence: (a) all of SEQ ID NOs: 1-3, (b) all of SEQ ID NOs: 4-6, or (c) all of SEQ ID NOs: 1-6. In a particularly preferred embodiment, the TCR comprises the amino acid sequence of all of SEQ ID NOs: 1-6.

[0045] In an embodiment of the present invention, the TCR comprises an amino acid sequence of a variable region of a TCR containing the CDRs shown above. The TCR may comprise a human variable region, such as a human α chain variable region and a human β chain variable region. In this regard, the TCR may comprise the following amino acid sequence: SEQ ID NO: 7 (variable region of the α chain); SEQ ID NO: 8 (variable region of the β chain); or SEQ ID NO: 7 and 8. Preferably, the TCR comprises the amino acid sequence of both SEQ ID NO: 7 and 8.

[0046] The TCR of the present invention may also include an α chain constant region and a β chain constant region. The constant region may be derived from any suitable species (such as, for example, humans or mice). In an embodiment of the present invention, TCR also includes mouse α chain and β chain constant regions or human α chain and β chain constant regions. As used herein, when referring to any component of TCR or TCR described herein (such as complementary determining region (CDR), variable region, constant region, α chain and / or β chain), the term "mouse" or "human" means that TCR (or its components) are derived from mice or humans, that is, TCR (or its components) are derived from mouse T cells or human T cells, respectively, or are expressed once.

[0047] Embodiments of the present invention provide chimeric TCRs comprising human variable regions and mouse constant regions, wherein the TCR has antigenic specificity for mutated human RAS amino acid sequences presented by HLA class I molecules. Mouse constant regions may provide any one or more advantages. For example, mouse constant regions may reduce the mismatch of the TCR of the present invention with the endogenous TCR of the host cell into which the TCR of the present invention is introduced. Alternatively or additionally, the mouse constant region may increase the expression of the TCR of the present invention compared to the same TCR with a human constant region. The chimeric TCR may comprise the amino acid sequences of SEQ ID NO: 19 (wild-type (WT) mouse α chain constant region), SEQ ID NO: 20 (WT mouse β chain constant region) or SEQ ID NO: 19 and 20. Preferably, the TCR of the present invention comprises the amino acid sequences of SEQ ID NO: 19 and 20. The chimeric TCR may comprise any mouse constant region described herein in combination with any CDR region described herein for other aspects of the present invention. In this regard, the TCR may comprise the following amino acid sequence: (a) all of SEQ ID NOs: 1-3 and 19; (b) all of SEQ ID NOs: 4-6 and 20; or (c) all of SEQ ID NOs: 1-6 and 19-20. In another embodiment of the invention, the chimeric TCR may comprise any of the murine constant regions described herein in combination with any of the variable regions described herein for other aspects of the invention. In this regard, the TCR may comprise the following amino acid sequence: (i) both of SEQ ID NOs: 7 and 19; (ii) both of SEQ ID NOs: 8 and 20; or (vi) all of SEQ ID NOs: 7-8 and 19-20.

[0048] In another embodiment of the invention, the TCR comprises the following amino acid sequence: SEQ ID NO:23 (alpha chain with WT mouse constant region), SEQ ID NO:24 (beta chain with WT mouse constant region), or both SEQ ID NOs:23-24.

[0049] In an embodiment of the present invention, TCR comprises a substituted constant region. In this regard, TCR may comprise any TCR described herein in the constant region of one or both of the α chain and the β chain with one, two, three or four amino acid substitutions in the amino acid sequence. Preferably, TCR comprises a mouse constant region, which has one, two, three or four amino acid substitutions in the mouse constant region of one or both of the α chain and the β chain. In a particularly preferred embodiment, TCR comprises a mouse constant region, which has 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, compared to the parent TCR comprising an unsubstituted (wild-type) constant region, the TCR comprising a substituted constant region advantageously provides one or more of the following: Increase the mutated RAS+ Target recognition, increased host cell expression, reduced mismatching with endogenous TCRs, and increased anti-tumor activity. In general, the substituted amino acid sequences of the mouse constant regions of the TCR α chain and β chain, SEQ ID NOs: 17 and 18, correspond to all or part of the unsubstituted mouse constant region amino acid sequences, SEQ ID NOs: 19 and 20, respectively, wherein SEQ ID NO: 17 has one, two, three or four amino acid substitutions compared to SEQ ID NO: 19, and SEQ ID NO: 18 has one amino acid substitution compared to SEQ ID NO: 20. In this regard, embodiments of the invention provide a TCR comprising the following amino acid sequence: (a) SEQ ID NO: 17 (constant region of the α chain), wherein (i) X at position 48 is Thr or Cys, (ii) X at position 112 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp, (iii) X at position 114 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp and (iv) X at position 115 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) SEQ ID NO: 18 (constant region of the β chain), wherein X at position 57 is Ser or Cys; or (c) both SEQ ID NOs: 17 and 18. In embodiments of the invention, the TCR comprising SEQ ID NO: 17 does not comprise SEQ ID NO: 19 (unsubstituted murine constant region of the α chain). In an embodiment of the invention, the TCR comprising SEQ ID NO: 18 does not comprise SEQ ID NO: 20 (unsubstituted murine constant region of the beta chain).

[0050] In an embodiment of the invention, the TCR comprises an alpha chain comprising a variable region and a constant region and a beta chain comprising a variable region and a constant region. In this regard, the TCR may comprise (a) an alpha chain comprising the amino acid sequence of SEQ ID NO: 21, wherein: (i) X at position 185 of SEQ ID NO: 21 is Thr or Cys, (ii) X at position 249 of SEQ ID NO: 21 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp, (iii) X at position 251 of SEQ ID NO: 21 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp, and (iv) X at position 252 of SEQ ID NO: 21 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) a beta chain comprising the amino acid sequence of SEQ ID NO: 22, wherein X at position 190 of SEQ ID NO: 22 is Ser or Cys; or (c) both (a) and (b). In an embodiment of the invention, a TCR comprising SEQ ID NO: 21 does not comprise SEQ ID NO: 23 (unsubstituted alpha chain). In an embodiment of the invention, a TCR comprising SEQ ID NO: 22 does not comprise SEQ ID NO: 24 (unsubstituted beta chain).

[0051] In an embodiment of the present invention, the constant region of the replacement comprises cysteine ​​substitution in the constant region of one or both of the α chain and the β chain to provide a TCR substituted with cysteine. The relative cysteine ​​in the α chain and the β chain provides a disulfide bond, which connects the constant regions of the α chain and the β chain of the substituted TCR to each other and is not present in the TCR comprising the unsubstituted mouse constant region. In this regard, TCR can be a TCR substituted with cysteine, wherein one or both of the natural Thr (Thr48) at position 48 of SEQ ID NO:19 and the natural Ser (Ser57) at position 57 of SEQ ID NO:20 can be replaced by Cys. Preferably, the natural Thr48 of SEQ ID NO:19 and the natural Ser57 of SEQ ID NO:20 are both replaced by Cys. Examples of TCR constant region sequences substituted with cysteine ​​are shown in Table 2. In embodiments of the invention, the cysteine ​​substituted TCR comprises (i) SEQ ID NO: 17, (ii) SEQ ID NO: 18, or (iii) both SEQ ID NO: 17 and 18, wherein SEQ ID NO: 17 and 18 are as defined in Table 2. In addition to any CDR or variable region described herein, the cysteine ​​substituted TCR of the invention may comprise a substituted constant region.

[0052] In an embodiment of the invention, the chimeric TCR substituted with cysteine ​​comprises a full-length α chain and a full-length β chain. Examples of cysteine-substituted chimeric TCR α chain and β chain sequences are shown in Table 2. In an embodiment of the invention, the TCR comprises (i) SEQ ID NO: 21, (ii) SEQ ID NO: 22, or (iii) both SEQ ID NO: 21 and 22, wherein SEQ ID NO: 21-22 are as defined in Table 2.

[0053] Table 2

[0054]

[0055] In an embodiment of the present invention, the substituted amino acid sequence comprises one, two or three amino acid substitutions in the transmembrane (TM) domain of the constant region of one or both of the α chain and the β chain with hydrophobic amino acids, to provide a TCR substituted with hydrophobic amino acids (also referred to herein as "LVL modified TCR"). Compared with TCRs lacking hydrophobic amino acid substitutions in the TM domain, hydrophobic amino acid substitutions in the TM domain of the TCR can increase the hydrophobicity of the TM domain of the TCR. In this regard, the TCR is a LVL modified TCR, wherein one, two or three of the natural Ser112, Met114 and Gly115 of SEQ ID NO: 19 can be independently replaced by Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; preferably replaced by Leu, Ile or Val. Preferably, all three of the native Ser112, Met114 and Gly115 of SEQ ID NO: 19 can be independently substituted by Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; preferably by Leu, Ile or Val. In an embodiment of the invention, the LVL modified TCR comprises (i) SEQ ID NO: 17, (ii) SEQ ID NO: 18 or (iii) both SEQ ID NO: 17 and 18, wherein SEQ ID NO: 17 and 18 are both as defined in Table 3. In addition to any CDR or variable region described herein, the LVL modified TCR of the invention may comprise a substituted constant region.

[0056] In an embodiment of the present invention, the LVL modified TCR comprises a full-length α chain and a full-length β chain. Examples of LVL modified TCR α chain and β chain sequences are shown in Table 3. In an embodiment of the present invention, the LVL modified TCR comprises (i) SEQ ID NO: 21, (ii) SEQ ID NO: 22 or (iii) both SEQ ID NO: 21 and 22, wherein SEQ ID NO: 21-22 are as defined in Table 3.

[0057] Table 3

[0058]

[0059]

[0060] In an embodiment of the present invention, the substituted amino acid sequence includes a cysteine ​​substitution in the constant region of one or both of the α chain and the β chain in combination with a substitution of one, two or three amino acids in the transmembrane (TM) domain of the constant region of one or both of the α chain and the β chain with a hydrophobic amino acid (also referred to herein as a "cysteine-substituted LVL-modified TCR"). In this regard, the TCR is a cysteine-substituted LVL-modified chimeric TCR, wherein the native Thr48 of SEQ ID NO: 19 is substituted with Cys; one, two or three of the native Ser112, Met114 and Gly115 of SEQ ID NO: 19 are independently substituted with Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; preferably substituted with Leu, Ile or Val; and the native Ser57 of SEQ ID NO: 20 is substituted with Cys. Preferably, all three of the native Ser112, Met114 and Gly115 of SEQ ID NO: 19 can be independently substituted by Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; preferably by Leu, Ile or Val. In an embodiment of the present invention, the cysteine-substituted LVL-modified TCR comprises (i) SEQ ID NO: 17, (ii) SEQ ID NO: 18 or (iii) both SEQ ID NO: 17 and 18, wherein SEQ ID NO: 17 and 18 are both as defined in Table 4. In addition to any CDR or variable region described herein, the cysteine-substituted LVL-modified TCR of the present invention may also comprise a substituted constant region.

[0061] In an embodiment, the cysteine-substituted LVL-modified TCR comprises a full-length α chain and a full-length β chain. In an embodiment of the invention, the cysteine-substituted LVL-modified TCR comprises (i) SEQ ID NO: 21, (ii) SEQ ID NO: 22, or (iii) both SEQ ID NO: 21 and 22, wherein SEQ ID NO: 21 and 22 are as defined in Table 4.

[0062] Table 4

[0063]

[0064]

[0065] Embodiments of the invention also provide polypeptides comprising a functional portion of any TCR described herein.The term "polypeptide" as used herein includes oligopeptides and refers to a single chain of amino acids linked by one or more peptide bonds.

[0066] For the polypeptide of the present invention, the functional part can be any part of the continuous amino acids of the TCR that is a part of TCR, provided that the functional part can specifically bind to the RAS of mutation.When referring to the use of TCR, the term "functional part" refers to any part or fragment of the TCR of the present invention, and the part or fragment retains the biological activity of the TCR (parental TCR) that produces the part. The functional part covers, for example, RAS (for example, in the context of HLA-A*11:01 molecules) that retains specific binding mutations in TCR or those parts of the ability to detect, treat or prevent cancer to a degree similar to that of parental TCR, the same degree or a higher degree. When referring to parental TCR, the functional part can include, for example, about 10%, about 25%, about 30%, about 50%, about 70%, about 80%, about 90%, about 95% or more of parental TCR.

[0067] The functional portion may include additional amino acids at the amino or carboxyl terminus of the portion or at both termini that are not found in the amino acid sequence of the parent TCR. Ideally, the additional amino acids do not interfere with the biological function of the functional portion, such as specific binding to mutated RAS and / or the ability to detect, treat or prevent cancer, etc. More ideally, the additional amino acids enhance the biological activity compared to the biological activity of the parent TCR.

[0068] 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, such as a functional portion of one or more of the CDR1, CDR2 and CDR3 of the variable region in the α chain and / or β chain of the TCR of the present invention. In an embodiment of the present invention, the polypeptide may comprise an 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.

[0069] In this regard, the polypeptide of the present invention may comprise any one or more of the amino acid sequences selected from SEQ ID NO: 1-6. In an embodiment of the present invention, the TCR comprises the following amino acid sequences: (a) all of SEQ ID NO: 1-3, (b) all of SEQ ID NO: 4-6, or (c) all of SEQ ID NO: 1-6. In a preferred embodiment, the polypeptide comprises the amino acid sequence of all of SEQ ID NO: 1-6.

[0070] In an embodiment of the present invention, the polypeptide of the present invention may comprise, for example, a variable region of a TCR of the present invention, comprising a combination of the CDR regions shown above. In this regard, the polypeptide may comprise the following amino acid sequence: (i) SEQ ID NO: 7 (variable region of the α chain), (ii) SEQ ID NO: 8 (variable region of the β chain), or (iii) SEQ ID NO: 7 and 8. Preferably, the polypeptide comprises the amino acid sequences of both SEQ ID NO: 7 and 8.

[0071] In an embodiment of the present invention, the polypeptide of the present invention may also include the constant region of the TCR of the present invention shown above. In this regard, the polypeptide may also include the following amino acid sequence: SEQ ID NO: 19 (WT mouse constant region of α chain), SEQ ID NO: 20 (WT mouse constant region of β chain), SEQ ID NO: 17 (substituted mouse constant region of α chain), SEQ ID NO: 18 (substituted mouse constant region of β chain), SEQ ID NOs: 19 and 20, or SEQ ID NOs: 17 and 18. Preferably, the polypeptide also includes SEQ ID NOs: 19 and 20 or SEQ ID NOs: 17 and 18 amino acid sequences in combination with any CDR region or variable region described herein for other aspects of the present invention.

[0072] In an embodiment of the invention, the polypeptide comprises: (a) an amino acid sequence of SEQ ID NO: 17, wherein (i) X at position 48 of SEQ ID NO: 17 is Thr or Cys, (ii) X at position 112 of SEQ ID NO: 17 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp, (iii) X at position 114 of SEQ ID NO: 17 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp, and (iv) X at position 115 of SEQ ID NO: 17 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) an amino acid sequence of SEQ ID NO: 18, wherein X at position 57 of SEQ ID NO: 18 is Ser or Cys; or (c) both (a) and (b). In an embodiment of the invention, one or both of SEQ ID NOs: 17 and 18 of the polypeptide are as defined in any one of Tables 2-4.

[0073] In an embodiment of the present invention, the polypeptide of the present invention may comprise the entire length of the α or β chain of the TCR described herein. In this regard, the polypeptide of the present invention may comprise the amino acid sequence of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NOs:21 and 22, or SEQ ID NOs:23 and 24. Preferably, the polypeptide comprises the amino acid sequence of SEQ ID NOs:21 and 22 or the amino acid sequence of SEQ ID NOs:23 and 24.

[0074] In an embodiment of the invention, the polypeptide comprises: (a) an amino acid sequence of SEQ ID NO: 21, wherein: (i) X at position 185 of SEQ ID NO: 21 is Thr or Cys, (ii) X at position 249 of SEQ ID NO: 21 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp, (iii) X at position 251 of SEQ ID NO: 21 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp, and (iv) X at position 252 of SEQ ID NO: 21 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) an amino acid sequence of SEQ ID NO: 22, wherein X at position 190 of SEQ ID NO: 22 is Ser or Cys; or (c) both (a) and (b). In an embodiment of the invention, any one or more of SEQ ID NOs: 21-22 of the polypeptide are as defined in any one of Tables 2-4.

[0075] Embodiments of the invention also provide proteins comprising at least one polypeptide described herein. "Protein" means a molecule comprising one or more polypeptide chains.

[0076] In an embodiment, the protein of the present invention may comprise a first polypeptide chain comprising the amino acid sequence of SEQ ID NOs: 1-3 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NOs: 4-6.

[0077] In another embodiment of the present invention, the protein may comprise a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:7 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:8.

[0078] The protein of the present invention may also comprise any constant region described herein for other aspects of the present invention. In this regard, in embodiments of the present invention, the first polypeptide chain may also comprise the amino acid sequence of SEQ ID NO: 17 and the second polypeptide chain may also comprise the amino acid sequence of SEQ ID NO: 18. In embodiments of the present invention, the first polypeptide chain may also comprise the amino acid sequence of SEQ ID NO: 19 and the second polypeptide chain may also comprise the amino acid sequence of SEQ ID NO: 20.

[0079] In an embodiment of the invention, the protein comprises: (a) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 17, wherein: (i) X at position 48 of SEQ ID NO: 17 is Thr or Cys, (ii) X at position 112 of SEQ ID NO: 17 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp, (iii) X at position 114 of SEQ ID NO: 17 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp, and (iv) X at position 115 of SEQ ID NO: 17 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 18, wherein X at position 57 of SEQ ID NO: 18 is Ser or Cys; or (c) both (a) and (b). In an embodiment of the present invention, one or both of SEQ ID NOs: 17 and 18 of the protein are as defined in any one of Tables 2-4.

[0080] Alternatively or additionally, a protein according to an embodiment of the present invention may comprise (a) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 21, wherein: (i) X at position 185 of SEQ ID NO: 21 is Thr or Cys, (ii) X at position 249 of SEQ ID NO: 21 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp, (iii) X at position 251 of SEQ ID NO: 21 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp, and (iv) X at position 252 of SEQ ID NO: 21 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 22, wherein X at position 190 of SEQ ID NO: 22 is Ser or Cys; or (c) both (a) and (b). In an embodiment of the present invention, the protein may comprise a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 23 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 24. In an embodiment of the present invention, one or two of SEQ ID NOs: 21-22 are as defined in any one of Tables 2-4.

[0081] The protein of the present invention can be a TCR. Alternatively, for example, if the protein comprises a single polypeptide chain containing the amino acid sequences of both SEQ ID NO:21 and 22, SEQ ID NO:23 and 24, or if the first and / or second polypeptide chain of the protein also comprises other amino acid sequences (e.g., amino acid sequences encoding immunoglobulins or parts thereof), the protein of the present invention can be a fusion protein. In this regard, embodiments of the present invention also provide fusion proteins comprising at least one polypeptide of the present invention as described herein and at least one other polypeptide. Other polypeptides may exist as separate polypeptides of the fusion protein, or may exist as polypeptides expressed in the same frame (in series) as a polypeptide of the present invention as described herein. Other polypeptides 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.

[0082] Fusion protein can comprise one or more copies of polypeptide of the present invention and / or one or more copies of other polypeptide.For example, fusion protein can comprise 1,2,3,4,5 or more copies of polypeptide of the present invention and / or other polypeptide.Suitable methods for preparing fusion protein are known in the art and include, for example, recombinant methods.

[0083] In some embodiments of the invention, the TCR, polypeptide and protein of the present invention can be expressed as a single protein comprising a joint peptide connecting an alpha chain and a beta chain. In this regard, the TCR, polypeptide and protein of the present invention can also include a joint peptide. The joint peptide can advantageously promote the expression of recombinant TCR, polypeptide and / or protein in a host cell. The joint peptide can include any suitable amino acid sequence. For example, the joint peptide can be a furin-SGSG-P2A joint comprising the amino acid sequence of SEQ ID NO:25. When the construct comprising the joint peptide is expressed by a host cell, the joint peptide can be cut to produce an alpha chain and a beta chain separated. In embodiments of the present invention, TCR, polypeptide or protein can include an amino acid sequence comprising a full-length alpha chain, a full-length beta chain and a joint peptide positioned between the alpha chain and the beta chain.

[0084] The protein of the present invention may be a recombinant antibody or antigen binding portion thereof comprising at least one polypeptide of the present invention described herein. As used herein, "recombinant antibody" refers to a recombinant (e.g., genetically modified) protein comprising at least one of the polypeptides of the present invention and the polypeptide chains of the antibody or its antigen binding portion. The polypeptide of the antibody or its antigen binding portion may be a heavy chain, light chain, variable region or constant region of a heavy chain or light chain, a single chain variable fragment (scFv), or an Fc, Fab, or F(ab)2' fragment, etc. of an antibody. The polypeptide chain of the antibody or its antigen binding portion may exist as a separate polypeptide of a recombinant antibody. Alternatively, the polypeptide chain of the antibody or its antigen binding portion may exist as a polypeptide expressed in the same frame (in series) as the polypeptide of the present invention. The polypeptide of the antibody or its antigen binding portion may be a polypeptide of any antibody or any antibody fragment (including any antibody and antibody fragment described herein).

[0085] The scope of the present invention includes functional variants of the TCR, polypeptide or protein of the present invention as described herein. The term "functional variant" as used herein refers to a TCR, polypeptide or protein having substantial or significant sequence identity or similarity with a parent TCR, polypeptide or protein, and the functional variant retains the biological activity of the TCR, polypeptide or protein that produces the variant. Functional variants include those variants of, for example, TCR, polypeptide or protein (parent TCR, polypeptide or protein) as described herein, which retain the ability to specifically bind to the mutated RAS (parent TCR has antigen specificity to it, or parent polypeptide or protein specifically binds to it) with a degree similar to that of the parent TCR, polypeptide or protein, the same degree or a higher degree. With regard to parent TCR, polypeptide or protein, functional variants can have, for example, at least about 30%, about 50%, about 75%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or higher amino acid sequence identity with the parent TCR, polypeptide or protein, respectively.

[0086] Functional variants may, for example, include an amino acid sequence of a parent TCR, polypeptide or protein having at least one conservative amino acid substitution. Conservative amino acid substitutions are known in the art, and include an amino acid substitution in which one of the amino acids having certain physical and / or chemical properties is exchanged for another amino acid having the same chemical or physical properties. For example, conservative amino acid substitutions may be acidic amino acid substitutions for another acidic amino acid (e.g., Asp or Glu), amino acids with non-polar side chains for another amino acid with non-polar side chains (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), basic amino acids for another basic amino acid (Lys, Arg, etc.), amino acids with polar side chains for another amino acid with polar side chains (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), etc.

[0087] Alternatively or additionally, the functional variant may comprise an amino acid sequence of a parent TCR, polypeptide or protein having at least one non-conservative amino acid substitution. In this case, preferably, the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. Preferably, the non-conservative amino acid substitution enhances the biological activity of the functional variant, such that the biological activity of the functional variant is increased compared to the parent TCR, polypeptide or protein.

[0088] TCR, polypeptide or protein can be substantially composed of a specified amino acid sequence or a sequence described herein, so that other components (such as other amino acids) of TCR, polypeptide or protein do not substantially change the biological activity of TCR, polypeptide or protein. In this regard, the TCR, polypeptide or protein of the present invention can be, for example, substantially composed of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NOs:21-22 or SEQ ID NOs:23-24 amino acid sequence. In addition, for example, the TCR, polypeptide or protein of the present invention can be substantially composed of (i) SEQ ID NO:7, (ii) SEQ ID NO:8 or (iii) SEQ ID NOs:7 and 8 amino acid sequence. In addition, the TCR, polypeptide or protein of the present invention can be substantially composed of the following amino acid sequence: (a) any one or more of SEQ ID NO:1-6; (b) all of SEQ ID NO:1-3; (c) all of SEQ ID NO:4-6; or (d) all of SEQ ID NO:1-6.

[0089] The TCR, polypeptides and proteins of the present invention can have any length, i.e., can contain any number of amino acids, provided that the TCR, polypeptide or protein retains its biological activity, such as the ability to specifically bind to a mutated RAS; detect cancer in a mammal; or treat or prevent cancer in a mammal, etc. For example, the length of the polypeptide can range from about 50 to about 5000 amino acids, such as about 50, about 70, about 75, about 100, about 125, about 150, about 175, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000 or more amino acids in length. In this regard, the polypeptides of the present invention also include oligopeptides.

[0090] The TCR, polypeptide and protein of the present invention may comprise synthetic amino acids that replace one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine-β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, di ... Indole-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine and α-tert-butylglycine.

[0091] The TCRs, polypeptides and proteins of the invention may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, or converted into acid addition salts and / or optionally dimerized or polymerized or conjugated, e.g., via disulfide bonds.

[0092] The TCRs, polypeptides and / or proteins of the invention can be obtained by methods known in the art, such as, for example, de novo synthesis. In addition, polypeptides and proteins can be recombinantly produced using the nucleic acids described herein using standard recombinant methods. See, for example, Green and Sambrook, MolecularCloning:ALaboratoryManual , 4th edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Alternatively, the TCRs, polypeptides and / or proteins described herein can be commercially synthesized by companies such as Synpep (Dublin, CA), Peptide Technologies Corp. (Gaithersburg, MD) and Multiple Peptide Systems (San Diego, CA). In this regard, the TCRs, polypeptides and proteins of the present invention can be synthetic, recombinant, isolated and / or purified.

[0093] The scope of the invention includes conjugates, such as bioconjugates, comprising any TCR, polypeptide or protein of the invention (including any functional part or variant thereof), nucleic acid, recombinant expression vector, host cell, host cell population or antibody or antigen binding portion thereof. Conjugates and general methods for synthesizing conjugates are known in the art.

[0094] Embodiments of the present invention provide nucleic acids including nucleotide sequences encoding any TCR, polypeptide or protein described herein. "Nucleic acid" used herein includes "polynucleotide", "oligonucleotide" and "nucleic acid molecule" and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, which can contain natural, non-natural or changed nucleotides, and which can contain natural, non-natural or changed internucleotide linkages (such as phosphoramidate linkages or thiophosphate linkages) to replace the phosphodiester between the nucleotides present in unmodified oligonucleotides. In embodiments, nucleic acid includes complementary DNA (cDNA). It is usually preferred that nucleic acid does not include any insertion, deletion, inversion and / or substitution. However, in some cases, as discussed herein, nucleic acid may be suitable for including one or more insertions, deletions, inversions and / or substitutions.

[0095] Preferably, the nucleic acid of the present invention is recombinant. As used herein, the term "recombinant" refers to (i) a molecule constructed outside a living cell by connecting a natural or synthetic nucleic acid segment to a nucleic acid molecule that can replicate in a living cell, or (ii) a molecule replicated from those described in (i) above. For purposes herein, replication can be in vitro replication or in vivo replication.

[0096] Nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, for example, Green and Sambrook et al., supra. For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or nucleotides modified in various ways designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides). Examples of modified nucleotides that can be used to generate nucleic acids include, but are not limited to: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosyl quercetin, inosine, N- 6 -Isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6 Substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosyl quercetin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6-isopentenyl adenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl) uracil and 2,6-diaminopurine. Alternatively, one or more nucleic acids of the present invention can be purchased from companies such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0097] The nucleic acid may include any nucleotide sequence encoding any TCR, polypeptide or protein described herein. In an embodiment of the present invention, the nucleic acid may include a nucleotide sequence of any one of SEQ ID NO:31-32 (Table 5). In an embodiment of the present invention, the nucleic acid includes a nucleotide sequence of both SEQ ID NOs:31-32.

[0098] Table 5

[0099]

[0100] In an embodiment of the present invention, nucleic acid comprises a codon-optimized nucleotide sequence encoding any TCR, polypeptide or protein described herein. Without being bound by any particular theory or mechanism, it is believed that the codon optimization of nucleotide sequence increases the translation efficiency of mRNA transcripts. The codon optimization of nucleotide sequence may involve replacing a natural codon with another codon, which encodes the same amino acid, but can be translated by the tRNA that is more easily available in the cell, thereby increasing translation efficiency. The optimization of nucleotide sequence may also reduce the secondary mRNA structure that interferes with translation, thereby increasing translation efficiency.

[0101] The present invention also provides nucleic acids comprising a nucleotide sequence complementary to the nucleotide sequence of any nucleic acid described herein or a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of any nucleic acid described herein.

[0102] The nucleotide sequence that hybridizes under stringent conditions preferably hybridizes under high stringency conditions. "High stringency conditions" means that the nucleotide sequence specifically hybridizes with the target sequence (nucleotide sequence of any nucleic acid described herein) with an amount that is detectably stronger than non-specific hybridization. High stringency conditions include conditions that distinguish the polynucleotides with accurate complementary sequences or polynucleotides containing only some scattered mispairings from the random sequences that happen to have some small regions (such as 3-10 bases) of matching nucleotide sequences. Such small complementary regions are more fusible than the full-length complements of 14-17 or more bases, and high stringency hybridization makes it easy to distinguish. Relatively high stringency conditions will include, for example, low salt and / or high temperature conditions, such as provided by about 0.02-0.1M NaCl or equivalents at a temperature of about 50-70°C. Such high stringency conditions tolerate few mispairings (if any) between the nucleotide sequence and the template or target chain, and are particularly suitable for detecting the expression of any TCR of the present invention. It is generally believed that more stringent conditions can be caused by adding an increased amount of formamide.

[0103] The present invention also provides nucleic acids comprising a nucleotide sequence that is at least about 70% or higher, such as about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to any nucleic acid described herein. In this regard, the nucleic acid can be substantially composed of any nucleotide sequence described herein.

[0104] The nucleic acid of the present invention can be incorporated into a recombinant expression vector. In this regard, the present invention provides a recombinant expression vector comprising any nucleic acid of the present invention. In an embodiment of the present invention, the recombinant expression vector comprises a nucleotide sequence encoding an alpha chain, a beta chain and a linker peptide.

[0105] For the purposes of this article, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that allows a host cell to express mRNA, protein, polypeptide or peptide when the construct contains a nucleotide sequence encoding mRNA, protein, polypeptide or peptide and the vector is contacted with a cell under conditions sufficient to allow the mRNA, protein, polypeptide or peptide to be expressed in the cell. The vector of the present invention is not naturally occurring as a whole. However, a portion of the vector may be naturally occurring. The recombinant expression vector 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, synthetic or partially obtained from a natural source, and may contain natural, non-natural or altered nucleotides. The recombinant expression vector may contain naturally occurring, non-naturally occurring internucleotide linkages, or both types of linkages. Preferably, non-naturally occurring or altered nucleotides or internucleotide linkages do not hinder transcription or replication of the vector.

[0106] The recombinant expression vector of the present invention can be any suitable recombinant expression vector, and can be used for transformation or transfection of any suitable host cell. Suitable vectors include those designed for reproduction and amplification or for expression or for the above two vectors, such as plasmids and viruses. The vector can be selected from: pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX series (Clontech, Palo Alto, CA). Phage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4 and λNM1149 can also be used. Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). Preferably, the recombinant expression vector is a viral vector, such as a retroviral vector. In a particularly preferred embodiment, the recombinant expression vector is a MSGV1 vector.

[0107] The recombinant expression vectors of the present invention can be prepared using standard recombinant DNA techniques described, for example, in Green and Sambrook et al. (see above). Circular or linear expression vector constructs can be prepared to contain replication systems that function in prokaryotic or eukaryotic host cells. The replication system can be derived from, for example, ColE1, 2μ plasmid, λ, SV40, bovine papilloma virus, etc.

[0108] Ideally, recombinant expression vectors contain regulatory sequences, such as transcription and translation start and stop codons, that are specific for the type of host cell (e.g., bacteria, fungi, plants, or animals) into which the vector is to be introduced, as appropriate and taking into account whether the vector is DNA or RNA based.

[0109] The recombinant expression vector can include one or more marker genes that allow selection of transformed or transfected host cells. Marker genes include biocide resistance (e.g., resistance to antibiotics, heavy metals, etc.), complementation in auxotrophic host cells to provide prototrophy, etc. Suitable marker genes for expression vectors of the present invention include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0110] The recombinant expression vector may include a natural or non-natural promoter, which may be operably connected to a nucleotide sequence encoding a TCR, a polypeptide or a protein or may be connected to a nucleotide sequence complementary to or hybridizing with a nucleotide sequence encoding a TCR, a polypeptide or a protein. The selection of promoters, such as strong, weak, inducible, tissue-specific and development-specific, is within the capabilities of those of ordinary skill in the art. Similarly, the combination of nucleotide sequence and promoter is also within the capabilities of the technician. Promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, a SV40 promoter, an RSV promoter and a promoter present in the long terminal repeat of a mouse stem cell virus.

[0111] The recombinant expression vector of the present invention can be designed for transient expression, stable expression or both. In addition, the recombinant expression vector can be prepared for constitutive expression or for inducible expression.

[0112] In addition, the recombinant expression vector can be prepared to include a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the cell expressing the suicide gene to die. A suicide gene can be a gene that imparts sensitivity to a reagent (e.g., a drug) to the cell in which the gene is expressed, and causes cell death when the cell contacts or is exposed to the reagent. Suicide genes are known in the art and include, for example, herpes simplex virus (HSV) thymidine kinase (TK) genes, cytosine deaminase, purine nucleoside phosphorylase, nitroreductase, and inducible caspase 9 gene systems.

[0113] Another embodiment of the present invention also provides a host cell comprising any recombinant expression vector described herein. As used herein, the term "host cell" refers to any type of cell that can contain the recombinant expression vector of the present invention. The host cell can be a eukaryotic cell (e.g., plant, animal, fungus or algae), or can be a prokaryotic cell (e.g., bacteria or protozoa). The host cell can be a cultured cell, or a primary cell, i.e., a cell directly isolated from an organism such as a person. The host cell can be an adherent cell, or a suspension cell, i.e., a cell grown in a suspension. Suitable host cells are known in the art, and include, for example, DH5α Escherichia coli (E.coli) cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. For the purpose of amplifying or replicating the recombinant expression vector, the host cell is preferably a prokaryotic cell, such as a DH5α cell. For the purpose of producing a recombinant TCR, a polypeptide or a protein, the host cell is preferably a mammalian cell. Most preferably, the host cell is a human cell. Although the host cell may be of any cell type, may be derived from any type of tissue, and may be at any developmental stage, the host cell is preferably a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). More preferably, the host cell is a T cell.

[0114] For the purposes of this article, T cells can be any T cells, such as cultured T cells, such as primary T cells, or T cells from cultured T cell lines (such as Jurkat, SupT1, etc.), or T cells obtained from mammals. If obtained from mammals, T cells can be obtained from a variety of sources, including but not limited to: blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells can also be enriched or purified. Preferably, T cells are human T cells. T cells can be any type of T cell and can be at any developmental stage, including but not limited to: CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells (such as Th1 and Th2 cells), CD4 + T cells, CD8 + T cells (such as cytotoxic T cells), tumor infiltrating lymphocytes (TILs), memory T cells (such as central memory T cells and effector memory T cells), naive T cells, etc.

[0115] The present invention also provides a cell colony comprising at least one host cell as described herein.Cell colony can be a heterogeneous colony comprising a host cell containing any recombinant expression vector in addition to at least one other cell, and the other cells are, for example, host cells (such as T cells) or cells other than T cells that do not include any recombinant expression vector, such as B cells, macrophages, neutrophils, erythrocytes, hepatocytes, endothelial cells, epithelial cells, myocytes, brain cells, etc. Alternatively, the cell colony can be a substantially homogeneous colony, wherein the colony mainly comprises a host cell containing a recombinant expression vector (such as substantially composed of it). Colony can also be a cloned cell colony, wherein all cells of the colony are clones of a single host cell containing a recombinant expression vector, so that all cells of the colony include a recombinant expression vector. In one embodiment of the present invention, the cell colony is a clonal colony comprising a host cell containing a recombinant expression vector as described herein.

[0116] In an embodiment of the present invention, the number of cells in the population can be rapidly expanded. The expansion of the number of T cells can be achieved by any of the many methods known in the art described in, for example, U.S. Patent 8,034,334; U.S. Patent 8,383,099; U.S. Patent Application Publication No. 2012 / 0244133; Dudley et al., J. Immunother., 26:332-42 (2003); and Riddell et al., J. Immunol. Methods, 128:189-201 (1990). In an embodiment, the number of T cells is expanded by culturing T cells with OKT3 antibody, IL-2 and feeder PBMC (e.g., irradiated allogeneic PBMC).

[0117] The TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors and host cells (including populations thereof) of the present invention may be isolated and / or purified. The term "isolated" as used herein means taken out from its natural environment. The term "purified" as used herein means having an increased purity, wherein "purity" is a relative term and need not be interpreted as absolute purity. For example, the purity may be at least about 50%, may be greater than about 60%, about 70%, about 80%, about 90%, about 95%, or may be about 100%.

[0118] The TCR, polypeptide, protein, nucleic acid, recombinant expression vector and host cell (including its population) of the present invention (all of which are hereinafter referred to as "TCR materials of the present invention") can be formulated into a composition, such as a pharmaceutical composition. In this regard, the present invention provides a pharmaceutical composition comprising any of the TCR, polypeptide, protein, nucleic acid, expression vector and host cell (including its population) described herein and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present invention containing any TCR material of the present invention may include more than one TCR material of the present invention (e.g., polypeptide and nucleic acid) or two or more different TCRs. Alternatively, a pharmaceutical composition may comprise a TCR material of the invention in combination with another pharmaceutically active agent or drug, such as a chemotherapeutic agent, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, and the like.

[0119] Preferably, the carrier is a pharmaceutically acceptable carrier. For pharmaceutical compositions, the carrier may be any of those conventionally used for the specific TCR material of the present invention under consideration. Methods for preparing administrable compositions are known to or apparent to those skilled in the art and are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, 22nd edition, Pharmaceutical Press (2012). Preferably, a pharmaceutically acceptable carrier is a carrier that has no harmful side effects or toxicity under the conditions of use.

[0120] The choice of carrier depends in part on the specific TCR material of the present invention and the specific method for administering the TCR material of the present invention. Therefore, there are a variety of suitable formulations of the pharmaceutical composition of the present invention. Suitable formulations may include any of those formulations for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, intratumoral or intraperitoneal administration. More than one approach can be used to administer the TCR material of the present invention, and in some cases, a specific approach can provide a more direct and more effective response than another approach.

[0121] Preferably, the TCR material of the present invention is administered intravenously by injection, for example. When the TCR material of the present invention is a host cell (or a population thereof) expressing the TCR of the present invention, the pharmaceutically acceptable carrier for the cells for injection may comprise any isotonic carrier, such as, for example, saline (about 0.90% w / v NaCl dissolved in water, about 300mOsm / L NaCl dissolved in water, or about 9.0g NaCl / liter water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose dissolved in water, or Ringer's lactate. In an embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumin.

[0122] For the purposes of the present invention, the amount or dosage of the TCR material of the present invention administered (e.g., the number of cells when the TCR material of the present invention is one or more cells) should be sufficient to cause, for example, a therapeutic or preventive response in an individual or animal within a reasonable time frame. For example, the dosage of the TCR material of the present invention should be sufficient to bind to a cancer antigen (e.g., a mutated RAS) or detect, treat, or prevent cancer within a period of about 2 hours or longer (e.g., 12 to 24 or more hours) from the time of administration. In certain embodiments, the time period may even be longer. The dosage will be determined by the efficacy of the specific TCR material of the present invention and the condition of the animal (e.g., human) and the weight of the animal (e.g., human) to be treated.

[0123] Many assays for determining the dosage to be administered are known in the art. For purposes of the present invention, the following assay can be used to determine the starting dose to be administered to a mammal, the assay being included in a group of mammals to which different doses of T cells are each administered, when a given dose of T cells expressing the present invention, polypeptides or proteins is administered to a mammal, the extent of target cell lysis or IFN-γ secretion by such T cells is compared. The extent of target cell lysis or IFN-γ secretion when a certain dose is administered can be determined by methods known in the art.

[0124] The dosage of the TCR material of the invention also depends on the presence, nature and extent of any adverse side effects that may accompany the administration of a particular TCR material of the invention. Typically, the attending physician will consider a variety of factors to determine the dosage of the TCR material of the invention to treat each individual patient, such as age, weight, general health, diet, sex, the TCR material of the invention to be administered, the route of administration, and the severity of the cancer being treated. In embodiments where the TCR material of the invention is a cell population, the number of cells administered per infusion may vary, for example, about 1×10 6 About 1×10 12In some embodiments, less than 1×10 6 cells.

[0125] It will be readily understood by those of ordinary skill in the art that the TCR material of the present invention can be modified in a variety of ways so that the therapeutic or preventive efficacy of the TCR material of the present invention is increased by modification. For example, the TCR material of the present invention may be conjugated to a chemotherapeutic agent directly or indirectly via a bridge. The practice of conjugating a compound to a chemotherapeutic agent is known in the art. It will be appreciated by those of ordinary skill in the art that the sites on the TCR material of the present invention that are not required for the function of the TCR material of the present invention are suitable sites for connecting bridges and / or chemotherapeutic agents, provided that the bridges and / or chemotherapeutic agents do not interfere with the function of the TCR material of the present invention after being connected to the TCR material of the present invention, i.e., the ability to bind to mutated RAS or to detect, treat or prevent cancer.

[0126] It is expected that the pharmaceutical compositions, TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, and cell populations of the present invention can be used in methods for treating or preventing cancer. Without being bound by a particular theory, it is believed that the TCR of the present invention can specifically bind to mutated RAS, so that the TCR (or related polypeptides or proteins of the present invention) can mediate an immune response to target cells expressing mutated RAS when expressed by cells. In this regard, the present invention provides a method for treating or preventing cancer in a mammal, comprising administering to the mammal any of the following substances in an amount effective for treating or preventing cancer in the mammal: any pharmaceutical composition, TCR, polypeptide, or protein described herein; any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any TCR, polypeptide, or protein described herein; or any host cell or cell population comprising a recombinant vector encoding any TCR, polypeptide, or protein described herein.

[0127] Embodiments of the present invention provide any pharmaceutical composition, TCR, polypeptide or protein described herein; any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any TCR, polypeptide, protein described herein; or any host cell or cell population comprising a recombinant vector encoding any TCR, polypeptide or protein described herein, for use in treating or preventing cancer in mammals.

[0128] The terms "treatment" and "prevention" used herein and words derived therefrom do not necessarily mean 100% or complete treatment or prevention. On the contrary, there are different degrees of treatment or prevention, which are considered by those of ordinary skill in the art to have potential benefits or therapeutic effects. In this regard, the methods of the present invention can provide treatment or prevention of cancer in mammals at any level of any amount. In addition, the treatment or prevention provided by the methods of the present invention may include the treatment or prevention of one or more conditions or symptoms of the cancer being treated or prevented. For example, treatment or prevention may include promoting tumor regression. In addition, for the purposes of this article, "prevention" may cover delaying the onset of cancer or its symptoms or conditions. Alternatively or additionally, "prevention" may cover preventing or delaying the recurrence of cancer or its symptoms or conditions.

[0129] Also provided is a method for detecting the presence of cancer in a mammal. The method comprises: (i) contacting a sample comprising one or more cells from a mammal with any of the TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, cell population, or pharmaceutical composition of the invention described herein, thereby forming a complex; and (ii) detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

[0130] Regarding the methods of the present invention for detecting cancer in a mammal, the cell sample may be a sample comprising whole cells, a lysate thereof, or a fraction of a whole cell lysate (eg, a nuclear or cytoplasmic fraction, a whole protein fraction, or a nucleic acid fraction).

[0131] For the purposes of the methods of the present invention for detecting cancer, contacting can occur in vitro or in vivo in a mammal. Preferably, contacting is in vitro.

[0132] In addition, the complex can be detected by a variety of methods known in the art. For example, the TCR of the present invention, polypeptide, protein, nucleic acid, recombinant expression vector, host cell or cell colony described herein can be labeled with a detectable marker, such as a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase) and an element particle (e.g., gold particle).

[0133] For the purposes of the methods of the invention wherein a host cell or cell population is administered, the cell may be allogeneic or autologous to the mammal. Preferably, the cell is autologous to the mammal.

[0134] For the methods of the present invention, the cancer can be any cancer, including any of the following: acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, anal canal cancer or rectal anal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer or pleural cancer, nasal cancer, nasal cancer or middle ear cancer, oral cancer, vaginal cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumors, glioma, Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal carcinoma, non-Hodgkin lymphoma Preferably, the cancer is pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer, or prostate cancer. Preferably, the lung cancer is lung adenocarcinoma, the ovarian cancer is epithelial ovarian cancer, and the pancreatic cancer is pancreatic adenocarcinoma. In an embodiment of the present invention, the cancer expresses a mutated human RAS amino acid sequence, wherein the mutated human RAS amino acid sequence is a mutated human KRAS, a mutated human HRAS, or a mutated human NRAS amino acid sequence. The mutated human KRAS, mutated human HRAS, and mutated human NRAS expressed by the cancer may be as described herein for other aspects of the present invention.

[0135] The mammal mentioned in the method of the present invention can be any mammal. As used herein, the term "mammal" refers to any mammal, including but not limited to: rodent mammals (e.g., mice and hamsters) and lagomorpha mammals (e.g., rabbits). Preferably, the mammal is from the order Carnivora, including felines (cats) and canines (dogs). More preferably, the mammal is from the order Artiodactyla (including cattle (cows) and swine (pigs)) or the order Perssodactyla (including horses (horses)). Most preferably, the mammal is a primate, a ceboid, a simian (monkey), or anthropoid (human and ape). A particularly preferred mammal is a human.

[0136] The following examples further illustrate the present invention but, of course, should not be construed as limiting the scope of the invention in any way.

[0137] Example 1

[0138] This example shows the isolation of a TCR with antigenic specificity for human KRAS with a G12V mutation, where the mutant KRAS is presented by an HLA-A*11:01 molecule.

[0139] TCRs were identified and isolated from the peripheral blood of an endometrial cancer patient after in vitro sensitization (IVS). The patient's cancer cells were found to express the KRAS G12V mutation. Briefly, autologous DCs were pulsed with the mutant peptide (MTEYKLVVVGAVGVGKSALTIQLI) (SEQ ID NO: 26) and co-cultured with CD8 T cells sorted from the patient's peripheral blood for 10 days.

[0140] Then, T cells were tested against a 24-mer peptide containing the KRAS G12V mutation (MTEYKLVVVGA V GVGKSALTIQLI) (SEQ ID NO: 26) or the corresponding WT peptide (MTEYKLVVVGA G GVGKSALTIQLI) (SEQ ID NO: 27) pulsed autologous DC. The peptide was resuspended in DMSO. T cells cultured with DC pulsed with DMSO were used as negative controls. T cell reactivity was measured based on CD137 expression and IFN-γ production (as measured by ELISPOT assay). Reactive T cells were then sorted based on CD137 expression, the number of sorted T cells was expanded, and the sorted expanded number of cells was tested again as described in this example. The results are shown in Figure 1A and 1B In. Figure 1A and 1B As shown, T cells upregulated CD137 expression only after co-culture with DCs pulsed with KRAS G12V peptide ( Figure 1A ) and increase the production of IFN-γ ( Figure 1B ).

[0141] Since the patient was HLA-A*11:01 positive, the KRAS G12V 9-mer and 10-mer peptides were predicted to bind to this allele with high affinity. Therefore, in the case of HLA-A*11:01, tetramer staining was used to test whether T cells were responsive to KRAS G12V Reactive. Using tetramer (9-mer VVGA V GVGK (SEQ ID NO: 29) or 10-mer VVVGA VGVGK (SEQ ID NO: 30)) was used to stain peripheral blood CD8+ T cells that had been subjected to IVS with the KRAS G12V 24-mer peptide. Both PE and APC are fluorophores. Tetramers were conjugated to one or the other of these fluorophores. Staining cells with both tetramers increased confidence in specificity. The results are shown in Figure 2 As shown (right). T cells modified to express a TCR that recognizes KRAS G12D 10-mer in the context of HLA-A*11:01 were used as negative controls. The results of the negative control are shown in Figure 2 Middle (left). Figure 2 As shown, CD8 T cells that had been subjected to IVS with the KRAS G12V 24-mer peptide were KRAS G12V -HLA-A*11:01 tetramer staining, while negative control T cells were not stained.

[0142] Sequencing of tetramer sorted cells using a single cell sequencing method showed one β chain and one α chain (Table 6). The nucleotide sequences of the variable regions of the TCR α chain and β chain were SEQ ID NOs: 31 and 32, respectively.

[0143] Table 6

[0144]

[0145] Example 2

[0146] This example shows the preparation of an expression cassette encoding the TCR of Example 1 with a cysteine-substituted, LVL-modified murine constant region and cloning of the expression cassette into a retroviral vector.

[0147] The TCR of Example 1 was cloned into a MSGV1-retroviral vector. A nucleic acid sequence encoding the isolated G12V reactive TCR of Example 1 (comprising the nucleotide sequences of SEQ ID NO:31 and SEQ ID NO:32) and comprising a cysteine-substituted, LVL-modified murine constant region was cloned into a retroviral vector. The α chain murine constant region comprises the amino acid sequence of SEQ ID NO:17, wherein X at position 48 is Cys, X at position 112 is Leu, X at position 114 is Ile, and X at position 115 is Val. The β chain constant region comprises the amino acid sequence of SEQ ID NO:18, wherein X at position 57 is Cys. A P2A linker (Wargo et al., Cancer Immunol. Immunother., 58(3):383-94 (2009)) comprising the amino acid sequence of SEQ ID NO:25 is located between the α chain constant region and the β chain variable region. From the 5' end to the 3' end, the retroviral vector comprises the following nucleotide sequence, which encodes the TCR β chain variable region, followed by a modified β chain murine constant region, followed by a P2A linker sequence, followed by a TCR α chain variable region, followed by a modified α chain murine constant region.

[0148] Example 3

[0149] This example shows that peripheral blood allogeneic T cells transduced with the TCR of Example 2 specifically recognize human KRAS with a G12V mutation, wherein the mutated KRAS is presented by an HLA-A*11:01 molecule.

[0150] A retrovirus encoding the TCR of Example 2 was prepared and used to transduce peripheral blood allogeneic T cells from two donors (method as described in Tran et al., N. Engl. J. Med., 375: 2255-2262 (2016)). The expression of TCR on the cell surface was measured by flow cytometry. The results are shown in Figure 3 In. Figure 3 As shown, expression of the murine TCRβ chain constant region (mTCRβ) was detected on the cell surface.

[0151] Allogeneic cells transduced with the TCR of Example 2 were tested for their responsiveness to target cancer cell lines expressing KRASG12D / KRASG12C or KRASG12V. Figure 4 As shown, the target cancer cell lines were positive or negative for HLA-A11 expression. Reactivity was measured by IFN-γ production (ELISPOT) and CD137 upregulation (flow cytometry). The results are shown in Figure 4 In. Figure 4 As shown, transduced cells were found to express KRAS G12VIt reacts specifically with cancer cells expressing HLA-A*11:01.

[0152] Example 4

[0153] This example shows that the TCR of Example 2 specifically recognizes the KRAS G12V 9-mer peptide and KRAS G12V 10-mer peptideKRAS G12V 10-mer peptide Each of the .

[0154] Allogeneic T cells were transduced with the TCR of Example 2 as described in Example 3. KRAS G12V9-mer peptide was used at various concentrations. KRAS G12V 10-mer peptide COS7 / A11 target cells were pulsed with KRAS WT 9-mer peptide VVGAGGVGK (SEQ ID NO: 33) or KRAS WT 10-mer peptide VVVGAGGVGK (SEQ ID NO: 34). Transduced T cells were co-cultured with pulsed target cells. IFN-γ secretion was measured. Results are shown in Figure 5A (9-mer) and Figure 5B (10-mer). Figure 5A-5B As shown, the TCR specifically recognized each of the KRAS G12V 9-mer peptide and the KRAS G12V 10-mer peptide.

[0155] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0156] The terms "one / a kind of" and "said" and "at least one / one" and similar indicators (particularly in the context of the following claims) used in the context of describing the present invention should be interpreted as covering both the singular and the plural, unless otherwise noted herein or clearly contradictory to the context. The use of one or more enumerations (e.g., "at least one / one of A and B") after the term "at least one / one" should be interpreted as meaning an item (A or B) selected from the listed items or any combination (A and B) of two or more listed items, unless otherwise noted herein or clearly contradictory to the context. The terms "comprising", "having", "including" and "containing" should be interpreted as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise noted. The description of the range of numerical values ​​herein is intended only to be used as a shorthand method for referring to each individual numerical value within the range respectively, unless otherwise noted herein, and each individual numerical value is incorporated into the specification, as if it is described independently herein. All methods described herein can be performed in any suitable order, unless otherwise noted herein or clearly contradictory to the context. The use of any and all examples or exemplary language (such as "such as") provided herein is intended only to better illustrate the invention and will not limit the scope of the invention unless otherwise protected. No language in this specification should be construed to indicate that any unclaimed component is essential to the practice of the invention.

[0157] Preferred embodiments of the present invention are described herein, including the best mode of implementing the invention known to the inventor. Variations of these preferred embodiments may be apparent to those of ordinary skill in the art after reading the above description. The inventor expects those skilled in the art to utilize these variations as appropriate, and the inventor intends to implement the invention in a manner different from that specifically described herein. Therefore, where permitted by applicable law, the present invention includes all variations and equivalents of the subject matter described in the appended claims. Moreover, any combination of the above elements in all possible variations thereof is included in the present invention, unless otherwise stated herein or clearly contradictory to the context.

Claims

1. A method for producing a human cell or a population of human cells expressing a T cell receptor (TCR), the method comprising: The recombinant expression vector is introduced into an isolated or purified human cell, or an isolated or purified human cell population, wherein the recombinant expression vector comprises a nucleotide sequence encoding a TCR, wherein the TCR comprises: (i) an α chain complementary determining region (CDR) 1 having an amino acid sequence as shown in SEQ ID NO: 1, an α chain CDR2 having an amino acid sequence as shown in SEQ ID NO: 2, and an α chain CDR3 having an amino acid sequence as shown in SEQ ID NO: 3, and (ii) a β-chain CDR1 with an amino acid sequence as shown in SEQ ID NO:4, a β-chain CDR2 with an amino acid sequence as shown in SEQ ID NO:5, and a β-chain CDR3 with an amino acid sequence as shown in SEQ ID NO:6, wherein the TCR has antigenic specificity for a mutated human RAS amino acid sequence presented by a human leukocyte antigen (HLA) class I molecule, and Wherein the human cell or human cell population produced by the method expresses the TCR.

2. The method of claim 1, wherein the TCR further comprises a human variable region.

3. The method of claim 1 or 2, wherein the TCR comprises: (i) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 7; (ii) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 8; (iii) an amino acid sequence that is at least 99% identical to amino acids 22-137 of SEQ ID NO:7; (iv) an amino acid sequence that is at least 99% identical to amino acids 22-133 of SEQ ID NO:8; or (v) both (i) and (ii); both (i) and (iv); both (ii) and (iii); or both (iii) and (iv).

4. The method of any one of claims 1 to 3, wherein the TCR comprises: (i) the amino acid sequence of SEQ ID NO: 7; (ii) the amino acid sequence of SEQ ID NO: 8; (iii) amino acids 22-137 of SEQ ID NO:7; (iv) amino acids 22-133 of SEQ ID NO:8; or (v) both (i) and (ii); both (i) and (iv); both (ii) and (iii); or both (iii) and (iv).

5. The method of any one of claims 1 to 4, wherein the TCR further comprises: (a) an α chain constant region, the α chain constant region comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 17, wherein: (i) X at position 48 of SEQ ID NO: 17 is Thr or Cys; (ii) X at position 112 of SEQ ID NO: 17 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 114 of SEQ ID NO: 17 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 115 of SEQ ID NO: 17 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) a beta chain constant region comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 18, wherein X at position 57 of SEQ ID NO: 18 is Ser or Cys; or (c) Both (a) and (b).

6. The method according to any one of claims 1 to 5, wherein the TCR further comprises: (a) an α chain constant region, the α chain constant region comprising the amino acid sequence of SEQ ID NO: 17, wherein: (i) X at position 48 of SEQ ID NO: 17 is Thr or Cys; (ii) X at position 112 of SEQ ID NO: 17 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 114 of SEQ ID NO: 17 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 115 of SEQ ID NO: 17 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) a β chain constant region comprising the amino acid sequence of SEQ ID NO: 18, wherein X at position 57 of SEQ ID NO: 18 is Ser or Cys; or (c) Both (a) and (b).

7. The method of any one of claims 1 to 6, wherein the TCR comprises: (a) an α chain comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 21, wherein: (i) X at position 185 of SEQ ID NO:21 is Thr or Cys; (ii) X at position 249 of SEQ ID NO:21 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 251 of SEQ ID NO:21 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 252 of SEQ ID NO:21 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) a β chain comprising an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:22, wherein X at position 190 of SEQ ID NO:22 is Ser or Cys; (c) an alpha chain comprising an amino acid sequence that is at least 99% identical to amino acids 22-274 of SEQ ID NO:21, wherein: (i) X at position 185 of SEQ ID NO:21 is Thr or Cys; (ii) X at position 249 of SEQ ID NO:21 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 251 of SEQ ID NO:21 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 252 of SEQ ID NO:21 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (d) a beta chain comprising an amino acid sequence that is at least 99% identical to amino acids 22-306 of SEQ ID NO:22, wherein X at position 190 of SEQ ID NO:22 is Ser or Cys; or (e) Both (a) and (b); Both (a) and (d); Both (b) and (c); or Both (c) and (d).

8. The method of any one of claims 1 to 7, wherein the TCR comprises: (a) an α chain, the α chain comprising the amino acid sequence of SEQ ID NO: 21, wherein: (i) X at position 185 of SEQ ID NO:21 is Thr or Cys; (ii) X at position 249 of SEQ ID NO:21 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 251 of SEQ ID NO:21 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 252 of SEQ ID NO:21 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) a β chain comprising the amino acid sequence of SEQ ID NO: 22, wherein X at position 190 of SEQ ID NO: 22 is Ser or Cys; (c) an alpha chain comprising amino acids 22-274 of SEQ ID NO: 21, wherein: (i) X at position 185 of SEQ ID NO:21 is Thr or Cys; (ii) X at position 249 of SEQ ID NO:21 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 251 of SEQ ID NO:21 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 252 of SEQ ID NO:21 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (d) a β chain comprising amino acids 22-306 of SEQ ID NO:22, wherein X at position 190 of SEQ ID NO:22 is Ser or Cys; or (e) Both (a) and (b); Both (a) and (d); Both (b) and (c); or Both (c) and (d).

9. A method for producing a human cell or a population of human cells expressing a polypeptide, the method comprising: introducing a recombinant expression vector into an isolated or purified human cell, or a population of isolated or purified human cells, wherein the recombinant expression vector comprises a nucleotide sequence encoding a polypeptide comprising a functional portion of a TCR having antigenic specificity for a mutated human RAS amino acid sequence presented by an HLA class I molecule, wherein the functional portion comprises: an α chain CDR1 having an amino acid sequence as shown in SEQ ID NO:1, an α chain CDR2 having an amino acid sequence as shown in SEQ ID NO:2, and an α chain CDR3 having an amino acid sequence as shown in SEQ ID NO:3, and a β chain CDR1 having an amino acid sequence as shown in SEQ ID NO:4, a β chain CDR2 having an amino acid sequence as shown in SEQ ID NO:5, and a β chain CDR3 having an amino acid sequence as shown in SEQ ID NO:6, and Wherein the human cell or human cell population produced by the method expresses the polypeptide.

10. The method of claim 9, wherein the polypeptide further comprises: (i) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 7; (ii) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 8; (iii) an amino acid sequence that is at least 99% identical to amino acids 22-137 of SEQ ID NO:7; (iv) an amino acid sequence that is at least 99% identical to amino acids 22-133 of SEQ ID NO:8; or (v) both (i) and (ii); both (i) and (iv); both (ii) and (iii); or both (iii) and (iv).

11. The method of claim 9 or 10, wherein the polypeptide comprises: (i) the amino acid sequence of SEQ ID NO: 7; (ii) the amino acid sequence of SEQ ID NO: 8; (iii) amino acids 22-137 of SEQ ID NO:7; (iv) amino acids 22-133 of SEQ ID NO:8; or (v) both (i) and (ii); both (i) and (iv); both (ii) and (iii); or both (iii) and (iv).

12. The method of any one of claims 9 to 11, wherein the polypeptide comprises: (a) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 17, wherein: (i) X at position 48 of SEQ ID NO: 17 is Thr or Cys; (ii) X at position 112 of SEQ ID NO: 17 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 114 of SEQ ID NO: 17 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 115 of SEQ ID NO: 17 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 18, wherein X at position 57 of SEQ ID NO: 18 is Ser or Cys; or (c) Both (a) and (b).

13. The method of any one of claims 9 to 12, wherein the polypeptide comprises: (a) the amino acid sequence of SEQ ID NO: 17, wherein: (i) X at position 48 of SEQ ID NO: 17 is Thr or Cys; (ii) X at position 112 of SEQ ID NO: 17 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 114 of SEQ ID NO: 17 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 115 of SEQ ID NO: 17 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) the amino acid sequence of SEQ ID NO: 18, wherein X at position 57 of SEQ ID NO: 18 is Ser or Cys; or (c) Both (a) and (b).

14. The method of any one of claims 9 to 13, wherein the polypeptide comprises: (a) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 21, wherein: (i) X at position 185 of SEQ ID NO:21 is Thr or Cys; (ii) X at position 249 of SEQ ID NO:21 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 251 of SEQ ID NO:21 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 252 of SEQ ID NO:21 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:22, wherein X at position 190 of SEQ ID NO:22 is Ser or Cys; (c) an amino acid sequence that is at least 99% identical to amino acids 22-274 of SEQ ID NO:21, wherein: (i) X at position 185 of SEQ ID NO:21 is Thr or Cys; (ii) X at position 249 of SEQ ID NO:21 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 251 of SEQ ID NO:21 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 252 of SEQ ID NO:21 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (d) an amino acid sequence that is at least 99% identical to amino acids 22-306 of SEQ ID NO:22, wherein X at position 190 of SEQ ID NO:22 is Ser or Cys; or (e) Both (a) and (b); Both (a) and (d); Both (b) and (c); or Both (c) and (d).

15. The method of any one of claims 9 to 14, wherein the polypeptide comprises: (a) the amino acid sequence of SEQ ID NO: 21, wherein: (i) X at position 185 of SEQ ID NO:21 is Thr or Cys; (ii) X at position 249 of SEQ ID NO:21 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 251 of SEQ ID NO:21 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 252 of SEQ ID NO:21 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) the amino acid sequence of SEQ ID NO:22, wherein X at position 190 of SEQ ID NO:22 is Ser or Cys; (c) amino acids 22-274 of SEQ ID NO:21, wherein: (i) X at position 185 of SEQ ID NO:21 is Thr or Cys; (ii) X at position 249 of SEQ ID NO:21 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 251 of SEQ ID NO:21 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 252 of SEQ ID NO:21 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (d) amino acids 22-306 of SEQ ID NO:22, wherein X at position 190 of SEQ ID NO:22 is Ser or Cys; or (e) Both (a) and (b); Both (a) and (d); Both (b) and (c); or Both (c) and (d).

16. A method for producing a human cell or a population of human cells expressing a protein, the method comprising: introducing a recombinant expression vector into an isolated or purified human cell, or a population of isolated or purified human cells, wherein the recombinant expression vector comprises a nucleotide sequence encoding a protein, wherein the protein has antigenic specificity for a mutated human RAS amino acid sequence presented by a human leukocyte antigen (HLA) protein class I molecule, The protein comprises a first polypeptide chain and a second polypeptide chain, the first polypeptide chain comprises an α chain CDR1 with an amino acid sequence as shown in SEQ ID NO: 1, an α chain CDR2 with an amino acid sequence as shown in SEQ ID NO: 2, and an α chain CDR3 with an amino acid sequence as shown in SEQ ID NO: 3; and the second polypeptide chain comprises a β chain CDR1 with an amino acid sequence as shown in SEQ ID NO: 4, a β chain CDR2 with an amino acid sequence as shown in SEQ ID NO: 5, and a β chain CDR3 with an amino acid sequence as shown in SEQ ID NO: 6, and The human cell or human cell population produced by the method expresses the protein.

17. The method of claim 16, wherein: (i) the first polypeptide chain further comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 7; (ii) the second polypeptide chain comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 8; (iii) the first polypeptide chain comprises an amino acid sequence that is at least 99% identical to amino acids 22-137 of SEQ ID NO:7; (iv) the second polypeptide chain further comprises an amino acid sequence that is at least 99% identical to amino acids 22-133 of SEQ ID NO:8; or (v) both (i) and (ii); both (i) and (iv); both (ii) and (iii); or both (iii) and (iv).

18. The method of claim 16 or 17, wherein: (i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 7; (ii) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 8; (iii) the first polypeptide chain contains amino acids 22-137 of SEQ ID NO:7; (iv) the second polypeptide chain contains amino acids 22-133 of SEQ ID NO:8; or (v) both (i) and (ii); both (i) and (iv); both (ii) and (iii); or both (iii) and (iv).

19. The method according to any one of claims 16 to 18, wherein: (a) the first polypeptide chain comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 17, wherein: (i) X at position 48 of SEQ ID NO: 17 is Thr or Cys; (ii) X at position 112 of SEQ ID NO: 17 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 114 of SEQ ID NO: 17 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 115 of SEQ ID NO: 17 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) the second polypeptide chain comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 18, wherein X at position 57 of SEQ ID NO: 18 is Ser or Cys; or (c) Both (a) and (b).

20. The method according to any one of claims 16 to 19, wherein: (a) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 17, wherein: (i) X at position 48 of SEQ ID NO: 17 is Thr or Cys; (ii) X at position 112 of SEQ ID NO: 17 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 114 of SEQ ID NO: 17 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 115 of SEQ ID NO: 17 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 18, wherein X at position 57 of SEQ ID NO: 18 is Ser or Cys; or (c) Both (a) and (b).

21. The method according to any one of claims 16 to 20, wherein: (a) the first polypeptide chain comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 21, wherein: (i) X at position 185 of SEQ ID NO:21 is Thr or Cys; (ii) X at position 249 of SEQ ID NO:21 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 251 of SEQ ID NO:21 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 252 of SEQ ID NO:21 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) the second polypeptide chain comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:22, wherein X at position 190 of SEQ ID NO:22 is Ser or Cys; (c) the first polypeptide chain comprises an amino acid sequence that is at least 99% identical to amino acids 22-274 of SEQ ID NO:21, wherein: (i) X at position 185 of SEQ ID NO:21 is Thr or Cys; (ii) X at position 249 of SEQ ID NO:21 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 251 of SEQ ID NO:21 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 252 of SEQ ID NO:21 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (d) the second polypeptide chain comprises an amino acid sequence that is at least 99% identical to amino acids 22-306 of SEQ ID NO:22, wherein X at position 190 of SEQ ID NO:22 is Ser or Cys; or (e) Both (a) and (b); Both (a) and (d); Both (b) and (c); or Both (c) and (d).

22. The method of any one of claims 16 to 21, wherein: (a) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 21, wherein: (i) X at position 185 of SEQ ID NO:21 is Thr or Cys; (ii) X at position 249 of SEQ ID NO:21 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 251 of SEQ ID NO:21 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 252 of SEQ ID NO:21 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (b) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:22, wherein X at position 190 of SEQ ID NO:22 is Ser or Cys; (c) the first polypeptide chain comprises amino acids 22-274 of SEQ ID NO: 21, wherein: (i) X at position 185 of SEQ ID NO:21 is Thr or Cys; (ii) X at position 249 of SEQ ID NO:21 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 251 of SEQ ID NO:21 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 252 of SEQ ID NO:21 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (d) the second polypeptide chain comprises amino acids 22-306 of SEQ ID NO:22, wherein X at position 190 of SEQ ID NO:22 is Ser or Cys; or (e) Both (a) and (b); Both (a) and (d); Both (b) and (c); or Both (c) and (d).

23. The method according to any one of claims 1 to 22, The mutated human RAS amino acid sequence is a mutated human Kirsten rat sarcoma viral oncogene homolog (KRAS), a mutated human Harvey rat sarcoma viral oncogene homolog (HRAS) or a mutated human neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence.

24. The method of claim 23, wherein the amino acid sequence of the mutated human RAS amino acid sequence comprises or consists of SEQ ID NO: 29 or 30.

25. The method of any one of claims 1-24, wherein the HLA class I molecule is an HLA-A molecule.

26. The method of any one of claims 1-24, wherein the HLA class I molecule is an HLA-A11 molecule.

27. The method of any one of claims 1-24, wherein the HLA class I molecule is an HLA-A*11:01 molecule.

28. The method of any one of claims 1-24, wherein the mutant human RAS amino acid sequence comprises a wild-type human KRAS, wild-type human HRAS or wild-type human NRAS amino acid sequence in which glycine at position 12 is substituted, wherein position 12 is defined by reference to wild-type human KRAS, wild-type human HRAS or wild-type human NRAS protein, respectively.

29. The method of claim 28, wherein the substitution is of glycine at position 12 with valine.

Images