HLA class i restricted t cell receptors against mutated ras

Abstract

To provide pharmaceutical compositions for further cancer treatment.SOLUTION: Disclosed is a pharmaceutical composition that elicits immune response to cancer in a mammal comprising: (a) a T cell receptor (TCR) comprising, (i) an α chain variable region comprising α chain CDR1, α chain CDR2 and α chain CDR3 each having a specific amino acid sequence, and (ii) a β chain variable region comprising β chain CDR1, and β chain CDR2 and β chain CDR3 each having a specific amino acid sequence, and the TCR having antigen specificity to a mutant human RAS amino acid sequence presented by human leukocyte antigen (HLA)-A*11:01; (b) a nucleic acid comprising a nucleotide sequence encoding the TCR; (c) a recombinant expression vector comprising the nucleic acid; (d) a host cell comprising the recombinant expression vector; or (e) a cell population comprising the host cells.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of U.S. Provisional Patent Application No. 62 / 594,244, filed December 4, 2017, which is incorporated by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with federal support by the National Cancer Institute, National Institutes of Health under Project No. BC010985. The federal government has certain rights in this invention.

[0003] Incorporation by Reference of Electronically Filed Materials The computer readable nucleotide / amino acid sequence listing, which has been submitted contemporaneously herewith and is identified as follows, is hereby incorporated by reference in its entirety: One 37,098 byte ASCII (text) file entitled "741039_ST25.txt", dated December 3, 2018. [Background technology]

[0004] 2. Background of the Invention Certain cancers may have very limited treatment options, especially when the cancer becomes metastatic and unresectable. Despite advances in treatment, such as surgery, chemotherapy, and radiation therapy, the prognosis for many cancers, such as pancreatic, colorectal, lung, endometrial, ovarian, and prostate cancer, may be poor. Thus, there is an unmet need for additional treatments for cancer. Summary of the Invention

[0005] One embodiment of the present invention provides an isolated or purified T cell receptor (TCR) comprising the amino acid sequence of (i) SEQ ID NO:1 to 3, (ii) SEQ ID NO:4 to 6, or (iii) SEQ ID NO:1 to 6, wherein the TCR has antigen specificity for a mutant human RAS amino acid sequence presented by a human leukocyte antigen (HLA) class I molecule, and the mutant human RAS amino acid sequence is the amino acid sequence of a mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS), a mutant human Harvey rat sarcoma viral oncogene homolog (HRAS), or a mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS).

[0006] Another embodiment of the present invention provides an isolated or purified polypeptide comprising a functional portion of a TCR of the present invention, wherein the functional portion comprises the amino acid sequence of (a) all of SEQ ID NOs: 1 to 3, (b) all of SEQ ID NOs: 4 to 6, or (c) all of SEQ ID NOs: 1 to 6.

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

[0008] Further embodiments of the invention provide nucleic acids, recombinant expression vectors, host cells, cell populations and pharmaceutical compositions relating to the TCRs, polypeptides and proteins of the invention.

[0009] Further provided in accordance with embodiments of the present invention are methods of detecting the presence of cancer in a mammal, and methods of treating or preventing cancer in a mammal. [Brief description of the drawings]

[0010] [Figure 1A] Figure 1A depicts a graph illustrating CD137 expression by peripheral blood CD8+ T cells after co-culture with autologous DCs pulsed with dimethylsulfoxide (DMSO) or KRAS WT or KRAS G12V 24-mer peptides. Boxed numbers indicate the percentage of T cells expressing CD137 in each co-culture condition. [Figure 1B] Figure IB is a graph showing the number of IFN-γ spots per 2x104 cells after co-culture of T cells with autologous DCs pulsed with DMSO, KRAS WT 24-mer peptide, or KRAS G12V 24-mer peptide. T cells cultured with PMA:ionomycin and T cells cultured with DMSO-pulsed DCs served as controls. [Diagram 2] Figure 2 shows a graph depicting experimental data (dot plots) illustrating the percentage of CD8+ peripheral blood cells that have previously received IVS with KRAS G12V 24-mer peptide bound to KRAS G12V tetramer-allophycocyanin (APC) or KRAS G12V tetramer-phycoerythrin (PE) (right plot). The left plot shows T cells engineered to express a TCR that recognizes the KRAS G12D 10-mer in the context of HLA-A*11:01 bound to KRAS G12V tetramer-APC or KRAS G12V tetramer-PE. [Diagram 3] Figure 3 depicts a graph showing expression of TCR on the surface of allogeneic T cells from first and second donors (left and right panels, respectively) transduced with the TCR of Example 2, as measured by staining for murine TCR beta chain constant region (mTCR beta) expression by flow cytometry. Boxed numbers indicate the percentage of cells that were positive for mTCR beta expression. [Figure 4] Figure 4 is a graph showing IFN-γ production and 4-1BB expression (%mTCRβ+CD137+) measured by ELISPOT assay (IFN-γ spots per 2E4 cells) by T cells from two different donors (donor 1 and donor 2) transduced with the TCR of Example 2 after overnight co-culture with cancer cell lines expressing different KRASG12 mutations. Each target cancer cell line was either untransduced or transduced with HLA-A*11:01. ">" indicates more than 500 spots. [Diagram 5]Figure 5A is a graph showing the concentration (pg / mL) of IFN-γ secreted by T cells transduced with the TCR of Example 2 after co-culture with target cells pulsed with the KRAS G12V 9-mer peptide (filled circles) or the KRAS WT 9-mer peptide (open circles) at the indicated concentrations (ng / mL). Figure 5B is a graph showing the concentration (pg / mL) of IFN-γ secreted by T cells transduced with the TCR of Example 2 after co-culture with target cells pulsed with the KRAS G12V 10-mer peptide (filled circles) or the KRAS WT 10-mer peptide (open circles) at the indicated concentrations (ng / mL). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] RAS family proteins belong to a large family of small GTPases. Without being bound to a particular theory or mechanism, it is believed that when mutated, RAS proteins may be involved in signal transduction in the early stages of carcinogenesis in many human cancers. A single amino acid substitution may activate the protein. Mutant RAS protein products may be constitutively activated. Mutant RAS proteins may be expressed in any of a variety of human cancers, such as, for example, pancreatic cancer (e.g., pancreatic carcinoma), colorectal cancer, lung cancer (e.g., lung adenocarcinoma), endometrial cancer, ovarian cancer (e.g., epithelial ovarian cancer), and prostate cancer. Human RAS family proteins include Kirsten rat sarcoma viral oncogene homolog (KRAS), Harvey rat sarcoma viral oncogene homolog (HRAS), and neuroblastoma rat sarcoma viral oncogene homolog (NRAS).

[0012] KRAS is also referred to as GTPase KRas, V-Ki-Ras2 Kirsten Rat Sarcoma Viral Oncogene, or KRAS2. There are two KRAS transcript variants: 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. Hereinafter, references to "KRAS" (mutated or non-mutated (WT)) refer to both variant A and variant B, unless otherwise indicated. When activated, mutant KRAS binds guanosine 5'-triphosphate (GTP) and converts GTP to guanosine 5'-diphosphate (GDP).

[0013] HRAS is another member of the RAS protein family. HRAS is also called Harvey rat sarcoma virus oncoprotein, V-Ha-Ras Harvey rat sarcoma virus oncogene homolog, or Ras family small GTP-binding protein H-Ras. WT HRAS has the amino acid sequence of SEQ ID NO: 11.

[0014] NRAS is yet another member of the RAS protein family. NRAS is also called GTPase NRas, V-Ras neuroblastoma RAS viral oncogene homolog, or NRAS1. WT NRAS has the amino acid sequence of SEQ ID NO: 12.

[0015] One embodiment of the present invention provides an isolated or purified TCR having antigen specificity for a mutated human RAS amino acid sequence (hereinafter "mutated RAS") presented by a human leukocyte antigen (HLA) class I molecule, the mutated human RAS amino acid sequence being a mutated human KRAS, a mutated human HRAS, or a mutated human NRAS amino acid sequence. Hereinafter, references to "TCR" also refer to functional portions and functional variants of the TCR, unless otherwise specified.

[0016] The TCR of the present invention may have antigen specificity for any mutant human RAS protein, polypeptide or peptide amino acid sequence. In one embodiment of the present invention, the mutant human RAS amino acid sequence is a mutant human KRAS amino acid sequence, a mutant human HRAS amino acid sequence, or a mutant human NRAS amino acid sequence. The amino acid sequences of the 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 the WT human NRAS protein is 86.8% identical to that of the WT human KRAS protein. The amino acid residues 1-86 of the WT human NRAS protein and the WT human KRAS protein are 100% identical. The amino acid sequence of the WT human HRAS protein is 86.3% identical to that of the WT human KRAS protein. The amino acid residues 1-94 of the WT human HRAS protein and the WT human KRAS protein are 100% identical. Hereinafter, references to "RAS" (mutated or non-mutated (WT)) refer collectively to KRAS, HRAS, and NRAS, unless otherwise specified.

[0017] In one embodiment of the present invention, the mutant human RAS amino acid sequence comprises a WT RAS amino acid sequence comprising a substitution of glycine at position 12, where position 12 is defined with respect to the WT RAS protein. 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, so the WT RAS protein may be any of the WT KRAS protein (SEQ ID NO: 9 or 10), the WT HRAS protein (SEQ ID NO: 11), or the WT NRAS protein (SEQ ID NO: 12). Thus, the amino acid residue at position 12 of each of the WT KRAS, WT HRAS, and WT NRAS proteins is identical, i.e., glycine.

[0018] The glycine at position 12 of the WT RAS amino acid sequence may be substituted with any amino acid residue other than glycine. In one embodiment of the invention, the substitution is a substitution of valine for the glycine at position 12 of the WT RAS amino acid sequence. In this regard, embodiments of the invention provide TCRs with antigen specificity for any WT RAS protein, polypeptide or peptide amino acid sequence that contains a G12V mutation.

[0019] RAS mutations and substitutions are defined herein with reference to the amino acid sequence of the WT RAS protein. Thus, RAS mutations and substitutions are described herein with reference to the amino acid residue present at a particular position in the WT RAS protein, then the position number, then the amino acid residue to which that residue is replaced in the particular mutation or substitution under consideration. A RAS amino acid sequence (e.g., a RAS peptide) may contain less than all of the amino acid residues in the full-length WT RAS protein. Thus, position 12 is defined herein with reference to the WT full-length RAS protein (i.e., any one of SEQ ID NOs: 9-12), with the understanding that the actual position of the corresponding residue in a particular example of a RAS amino acid sequence may vary. When a position is as defined in any one of SEQ ID NOs: 9-12, the term "G12" refers to the glycine normally present at position 12 in any one of SEQ ID NOs: 9-12, and "G12V" indicates that the glycine normally present at position 12 in any one of SEQ ID NOs: 9-12 has been replaced with a valine. For example, a particular example of a RAS amino acid sequence may be, e.g.,

[0020] [ka]

[0021] (SEQ ID NO:28) (An exemplary WT corresponding to consecutive amino acid residues 2 to 24 of SEQ ID NO:9) KRAS peptide), "G12V" refers to the substitution of the underlined glycine of SEQ ID NO:28 with a valine, even though the actual position of the underlined glycine of SEQ ID NO:28 is 11.

[0022] Examples of full-length RAS proteins containing the G12V mutation are shown in Table 1 below.

[0023] [Table 1]

[0024] In one embodiment of the present invention, the TCR has antigen specificity for a RAS peptide comprising the G12V mutation described above, and the mutant RAS peptide has any length. In one embodiment of the present invention, the mutant RAS peptide has any length suitable for binding to any of the HLA class I molecules described herein. For example, the TCR may have antigen specificity for a RAS peptide comprising the G12V mutation, and the RAS peptide has a length of about 9 to about 10 amino acid residues. The mutant RAS peptide may comprise any consecutive amino acid residues of a mutant RAS protein comprising the G12V mutation. In one embodiment of the present invention, the TCR has antigen specificity for a RAS peptide comprising the G12V mutation. and the mutant RAS peptide has a length of about 9 amino acid residues or about 10 amino acid residues. Examples of specific peptides each containing a G12V mutation that can be recognized by the G12V TCR of the present invention are the 9-mer VVGAVGVGK (SEQ ID NO: 29) and the 10-mer VVVGAVGVGK.

[0025] In one embodiment of the present invention, the TCR of the present invention can recognize mutant RAS presented by HLA class I molecules. In this regard, the TCR can induce an immune response upon binding to mutant RAS within the context of HLA class I molecules. The TCR of the present invention can bind to HLA class I molecules in addition to mutant RAS.

[0026] In one embodiment of the present invention, the HLA class I molecule is an HLA-A molecule. The HLA-A molecule is a heterodimer of an alpha chain and beta2 microglobulin. The HLA-A alpha chain may be encoded by the HLA-A gene. The beta2 microglobulin non-covalently binds to the alpha1, alpha2, and alpha3 domains of the alpha chain to form the HLA-A complex. The HLA-A molecule may be any HLA-A molecule. In one embodiment of the present invention, the HLA class I molecule is an HLA-A11 molecule. The HLA-A11 molecule may be any HLA-A11 molecule. An example of an HLA-A11 molecule is HLA-A * 11:01, HLA-A * 11:02, HLA-A * 11:03, or HLA-A * Preferably, the HLA class I molecule is HLA-A 11:04. * 11:01 molecule.

[0027] The TCRs of the present invention may provide any one or more of a variety of advantages, including when expressed by cells used for adoptive cell transfer. Mutant RAS is expressed by cancer cells and not by healthy non-cancer cells. Without being bound to a particular theory or mechanism, it is believed that the TCRs of the present invention advantageously target the destruction of cancer cells while minimizing or eliminating the destruction of healthy non-cancer cells, thereby reducing toxicity, e.g., by minimizing or eliminating it. Furthermore, the TCRs of the present invention may successfully and advantageously treat or prevent mutant RAS-positive cancers that do not respond to other types of treatment, such as chemotherapy, surgery, or radiation therapy. RAS G12The mutation is one of the most common hotspot mutations found in many cancer types. For example, the KRAS G12V mutation occurs in about 27% and about 9% of pancreatic and colorectal cancer patients, respectively. Furthermore, members of the RAS family share the G12 hotspot mutation in various types of cancer (e.g., NRAS in melanoma). Additionally, the TCRs of the present invention provide high affinity recognition of mutant RAS, which is consistent with the recognition of unengineered tumor cells (e.g., untreated with interferon (IFN) gamma, mutant RAS and HLA-A * 11:01, or both, or pulsed with a RAS peptide containing the G12V mutation, or a combination thereof). * The 11:01 allele is expressed in approximately 14% and 9% of Caucasian and Hispanic individuals, respectively. * The 11:01 allele is expressed in up to about 45% of Asian people in the United States. Therefore, the TCRs of the present invention may not be suitable for immunotherapy using TCRs that recognize RAS presented by other MHC molecules. * This may increase the number of cancer patients eligible for immunotherapy, including those who express the 11:01 allele. Additionally, the TCRs, polypeptides and proteins of the present invention comprise human amino acid sequences, which may reduce the risk of rejection by the human immune system, as compared to, e.g., TCRs, polypeptides and proteins that comprise mouse amino acid sequences.

[0028] As used herein, the phrase "antigen specificity" means that the TCR can specifically bind to and immunologically recognize the mutant RAS with high avidity, for example, (a) a low concentration of the mutant RAS peptide (e.g., about 0.05 ng / mL to about 10 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 8 ng / mL, 10 ng / mL, or any of the above values); or (b) antigen-negative HLA class I molecule-positive target cells into which a nucleotide sequence encoding a mutant RAS has been introduced such that the target cells express the mutant RAS. 4 ~Approx. 1×10 5 A TCR may be considered to have "antigen specificity" for a mutant RAS if the T cells secrete IFN-γ 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 preceding values). Cells expressing the TCR of the invention may also secrete IFN-γ upon co-culture with antigen-negative HLA class I molecule-positive target cells pulsed with higher concentrations of mutant RAS peptide. The HLA class I molecule may be any of the HLA class I molecules described herein (e.g., HLA-A * 11:01 molecules).

[0029] Alternatively or additionally, a TCR may be considered to have "antigen specificity" for a mutant RAS if, upon co-culture with (a) antigen-negative HLA class I molecule-positive target cells pulsed with a low concentration of a mutant RAS peptide, or (b) antigen-negative HLA class I molecule-positive target cells into which a nucleotide sequence encoding a mutant RAS has been introduced such that the target cells express the mutant RAS, T cells expressing the TCR secrete at least twice as much IFN-γ as the amount of IFN-γ expressed by a negative control. A negative control can be, for example, (i) a T cell expressing a TCR co-cultured with (a) an antigen-negative HLA class I molecule-positive target cell pulsed with the same concentration of an irrelevant peptide (e.g., some other peptide that includes a different sequence than the mutant RAS peptide), or (b) an antigen-negative HLA class I molecule-positive target cell into which a nucleotide sequence encoding an irrelevant peptide has been introduced such that the target cell expresses the irrelevant peptide, or (ii) a non-transduced T cell (e.g., from a PBMC that does not express a TCR) co-cultured with (a) an antigen-negative HLA class I molecule-positive target cell pulsed with the same concentration of the mutant RAS peptide, or (b) an antigen-negative HLA class I molecule-positive target cell into which a nucleotide sequence encoding a mutant RAS has been introduced such that the target cell expresses the mutant RAS. The HLA class I molecule expressed by the negative control target cell is the same as the HLA class I molecule expressed by the target cell co-cultured with the T cell being tested. The HLA class I molecule can be any of the HLA class I molecules described herein (e.g., 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).

[0030] Alternatively or additionally, a TCR may be considered to have "antigen specificity" for a mutant RAS if, upon co-culture with (a) antigen-negative HLA class I molecule-positive target cells pulsed with a low concentration of mutant RAS peptide, or (b) antigen-negative HLA class I molecule-positive target cells into which a nucleotide sequence encoding a mutant RAS has been introduced such that the target cells express the mutant RAS, a number of T cells expressing the TCR secrete IFN-γ that is at least twice as high as the number of negative control T cells secreting IFN-γ. The HLA class I molecule, the concentration of peptide, and the 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.

[0031] Alternatively or additionally, a TCR has "antigen specificity" for a mutant RAS if a T cell expressing the TCR upregulates expression of one or more T cell activation markers, e.g., as measured by flow cytometry following stimulation with a target cell expressing a 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.).

[0032] One embodiment of the present invention provides a TCR comprising two polypeptides (i.e., polypeptide chains), such as a TCR alpha (α) chain, a TCR beta (β) chain, a TCR gamma (γ) chain, a TCR delta (δ) chain, or a combination thereof. The polypeptides of the TCRs of the present invention may comprise any amino acid sequence, provided that the TCR has antigen specificity for a mutated RAS.

[0033] In one embodiment of the present invention, the TCR comprises two polypeptide chains, each of which comprises a variable region comprising TCR complementarity determining regions (CDRs) 1, CDR2, and CDR3. In one embodiment of the present invention, the TCR comprises a first polypeptide chain comprising CDR1 (CDR1 of the α chain) comprising the amino acid sequence of SEQ ID NO: 1, CDR2 (CDR2 of the α chain) comprising the amino acid sequence of SEQ ID NO: 2, and CDR3 (CDR3 of the α chain) comprising the amino acid sequence of SEQ ID NO: 3, and a second polypeptide chain comprising CDR1 (CDR1 of the β chain) comprising the amino acid sequence of SEQ ID NO: 4, CDR2 (CDR2 of the β chain) comprising the amino acid sequence of SEQ ID NO: 5, and CDR3 (CDR3 of the β chain) comprising the amino acid sequence of SEQ ID NO: 6. In this regard, the TCR of the present invention may comprise any one or more of the amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 6. In one embodiment of the present invention, the TCR comprises the amino acid sequences of (a) all of SEQ ID NOs: 1 to 3, (b) all of SEQ ID NOs: 4 to 6, or (c) all of SEQ ID NOs: 1 to 6. In a particularly preferred embodiment, the TCR comprises all of the amino acid sequences of SEQ ID NOs:1-6.

[0034] In one embodiment of the present invention, the TCR comprises the amino acid sequence of a variable region of the TCR comprising the above CDRs. The TCR may comprise a human variable region, e.g., a human α chain variable region and a human β chain variable region. In this regard, the TCR may comprise the amino acid sequence of: SEQ ID NO: 7 (variable region of the α chain); SEQ ID NO: 8 (variable region of the β chain); or both SEQ ID NOs: 7 and 8. Preferably, the TCR comprises the amino acid sequence of both SEQ ID NOs: 7 and 8.

[0035] The TCRs of the invention may further comprise an alpha chain constant region and a beta chain constant region. The constant regions may be derived from any suitable species, e.g., human or mouse. In one embodiment of the invention, the TCR further comprises mouse alpha and beta chain constant regions or human alpha and beta chain constant regions. As used herein, the terms "mouse" or "human", when referring to a TCR or any component of a TCR described herein (e.g., complementarity determining regions (CDRs), variable regions, constant regions, alpha chains, and / or beta chains), refer to a TCR (or component thereof) derived from a mouse or human, respectively, i.e., a TCR (or component thereof) that originates from or was once expressed in a mouse T cell or human T cell, respectively.

[0036] One embodiment of the present invention provides a chimeric TCR comprising a human variable region and a mouse constant region, the TCR having antigen specificity for a mutated human RAS amino acid sequence presented by an HLA class I molecule. The mouse constant region may provide any one or more advantages. For example, the mouse constant region may reduce mispairing of the TCR of the present invention with the endogenous TCR of the host cell into which the TCR of the present invention is introduced. Alternatively or additionally, the mouse constant region may increase expression of the TCR of the present invention compared to the same TCR comprising a human constant region. The chimeric TCR may comprise the amino acid sequence of SEQ ID NO: 19 (wild type (WT) mouse α chain constant region), SEQ ID NO: 20 (WT mouse β chain constant region), or both SEQ ID NO: 19 and 20. Preferably, the TCR of the present invention comprises the amino acid sequence of both SEQ ID NO: 19 and 20. The chimeric TCR may comprise any of the mouse constant regions described herein in combination with any of the CDR regions as described herein with respect to other aspects of the invention. In this regard, In accordance with the present invention, the TCR may comprise the amino acid sequence of: (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 with respect to other aspects of the invention. In this regard, the TCR may comprise the amino acid sequence of: (i) both of SEQ ID NOs: 7 and 19; (ii) both of SEQ ID NOs: 8 and 20; or (iii) all of SEQ ID NOs: 7-8 and 19-20.

[0037] In another embodiment of the invention, the TCR comprises the amino acid sequence(s) of: SEQ ID NO:23 (alpha chain comprising a WT mouse constant region), SEQ ID NO:24 (beta chain comprising a WT mouse constant region), or both SEQ ID NOs:23-24.

[0038] In one embodiment of the invention, the TCR comprises a substituted constant region. In this regard, the TCR may comprise the amino acid sequence of any of the TCRs described herein, comprising one, two, three, or four amino acid substitution(s) in one or both of the α and β chain constant regions. Preferably, the TCR comprises a murine constant region comprising one, two, three, or four amino acid substitution(s) in one or both of the α and β chain murine constant regions. In a particularly preferred embodiment, the TCR comprises a murine constant region comprising one, two, three, or four amino acid substitution(s) in the murine constant region of the α chain and one amino acid substitution in the murine constant region of the β chain. In some embodiments, the TCR comprising the substituted constant region advantageously exhibits a mutant RAS mutation as compared to a parent TCR comprising an unsubstituted (wild type) constant region. +and / or increased target recognition, increased expression by host cells, decreased mispairing with endogenous TCRs, and increased anti-tumor activity. In general, the substituted amino acid sequences of the murine constant regions of the TCR alpha and beta chains, SEQ ID NOs: 17 and 18, respectively, correspond to all or a portion of the unsubstituted murine constant region amino acid sequence, with SEQ ID NO: 17 having 1, 2, 3, or 4 amino acid substitution(s) when compared to SEQ ID NO: 19, and SEQ ID NO: 18 having 1 amino acid substitution when compared to SEQ ID NO: 20. In this regard, one embodiment of the present invention provides a TCR comprising the amino acid sequence of: (a) SEQ ID NO: 17 (constant region of the α chain); (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) where X at position 57 is Ser or Cys; or (c) both SEQ ID NOs: 17 and 18. In one embodiment of the present invention, the TCR comprising SEQ ID NO: 17 does not comprise SEQ ID NO: 19 (unsubstituted mouse constant region of the α chain). In one embodiment of the invention, the TCR comprising SEQ ID NO: 18 does not comprise SEQ ID NO: 20 (unsubstituted murine constant region of the β chain).

[0039] In one 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 α chain comprising the amino acid sequence of SEQ ID NO:21, wherein: (i) the X at position 185 of SEQ ID NO:21 is Thr or Cys; (ii) the X at position 249 of SEQ ID NO:21 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) the X at position 251 of SEQ ID NO:21 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) the X at position 252 of SEQ ID NO:21 is GIy, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) a β chain comprising the amino acid sequence of SEQ ID NO:22, wherein the X at position 190 of SEQ ID NO:22 is Ser or Cys; or (c) both (a) and (b). In one embodiment of the invention, a TCR comprising SEQ ID NO:21 does not comprise SEQ ID NO:23 (unsubstituted α chain). In one embodiment of the invention, a TCR comprising SEQ ID NO:22 does not comprise SEQ ID NO:24 (unsubstituted β chain). Does not include.

[0040] In one embodiment of the invention, the replaced constant region comprises a cysteine ​​substitution in one or both of the constant regions of the α and β chains to provide a cysteine ​​replaced TCR. The opposing cysteines of the α and β chains provide a disulfide bond interconnecting the constant regions of the α and β chains of the replaced TCR that is not present in a TCR comprising an unsubstituted mouse constant region. In this regard, the TCR may be a cysteine ​​replaced TCR in which one or both of the native Thr at position 48 of SEQ ID NO: 19 (Thr48) and the native Ser at position 57 of SEQ ID NO: 20 (Ser57) may be replaced with Cys. Preferably, both the native Thr48 of SEQ ID NO: 19 and the native Ser57 of SEQ ID NO: 20 are replaced with Cys. Examples of cysteine ​​replaced TCR constant region sequences are shown in Table 2. In one embodiment of the invention the cysteine ​​replaced TCR comprises (i) SEQ ID NO: 17, (ii) SEQ ID NO: 18, or (iii) both SEQ ID NOs: 17 and 18, both of which are as defined in Table 2. The cysteine ​​replaced TCRs of the invention may comprise a replaced constant region in addition to any of the CDRs or variable regions described herein.

[0041] In one embodiment of the invention, the cysteine-substituted chimeric TCR comprises a full-length alpha chain and a full-length beta chain. Exemplary sequences of the alpha and beta chains of cysteine-substituted chimeric TCRs are shown in Table 2. In one embodiment of the invention, the TCR comprises (i) SEQ ID NO: 21, (ii) SEQ ID NO: 22, or (iii) both SEQ ID NOs: 21 and 22, where SEQ ID NOs: 21-22 are as defined in Table 2.

[0042] [Table 2]

[0043] In one embodiment of the invention, the substituted amino acid sequence comprises the substitution of one, two or three amino acids in the transmembrane (TM) domain of one or both of the α and β chain constant regions with hydrophobic amino acids to provide a hydrophobic amino acid substituted TCR (also referred to herein as an "LVL modified TCR"). The hydrophobic amino acid substitution(s) in the TM domain of the TCR may increase the hydrophobicity of the TM domain of the TCR compared to a TCR lacking the hydrophobic amino acid substitution(s) in the TM domain. In this regard, the TCR is an LVL modified TCR, in which one, two or three of the native Ser112, Met114 and Gly115 of SEQ ID NO: 19 are independently replaced by Ala, Val, Leu, Ile, Pro, Phosphate, or the like. Preferably, all three of the native Ser112, Met114, and Gly115 of SEQ ID NO: 19 may be independently substituted with Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; preferably with Leu, Ile, or Val. In one embodiment of the invention, the LVL modified TCR comprises (i) SEQ ID NO: 17, (ii) SEQ ID NO: 18, or (iii) both SEQ ID NOs: 17 and 18, both of which are as defined in Table 3. The LVL modified TCRs of the invention may comprise a substituted constant region in addition to any of the CDRs or variable regions described herein.

[0044] In one embodiment of the invention, the LVL modified TCR comprises a full length alpha chain and a full length beta chain. Exemplary sequences of the alpha and beta chains of LVL modified TCRs are shown in Table 3. In one embodiment of the invention, the LVL modified TCR comprises (i) SEQ ID NO: 21, (ii) SEQ ID NO: 22, or (iii) both SEQ ID NOs: 21 and 22, where SEQ ID NOs: 21-22 are as defined in Table 3.

[0045] [Table 3]

[0046] In one embodiment of the invention, the substituted amino acid sequence comprises a cysteine ​​substitution in one or both of the α and β chain constant regions in combination with a hydrophobic amino acid substitution(s) of one, two or three amino acids in the transmembrane (TM) domains of one or both of the α and β chain constant regions (also referred to herein as a "cysteine-substituted, LVL-modified TCR"). In this regard, the TCR is a cysteine-substituted, LVL-modified, chimeric TCR in which native Thr48 of SEQ ID NO: 19 is replaced with Cys. One, two or three of native Ser112, Met114 and Gly115 of SEQ ID NO: 19 are independently replaced with Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; preferably with Leu, Ile or Val; and native Ser57 of SEQ ID NO: 20 is replaced with Cys. Preferably, all three of the native Ser112, Met114, and Gly115 of SEQ ID NO: 19 are independently selected from Ala, Val, Leu, and preferably, Leu, Ile, or Val. In one embodiment of the invention, the cysteine ​​substituted, LVL modified TCR comprises (i) SEQ ID NO: 17, (ii) SEQ ID NO: 18, or (iii) both SEQ ID NOs: 17 and 18, both of which are as defined in Table 4. The cysteine ​​substituted, LVL modified TCRs of the invention may comprise a substituted constant region in addition to any of the CDRs or variable regions described herein.

[0047] In one embodiment, the cysteine ​​substituted, LVL modified TCR comprises a full length alpha chain and a full length beta chain. In one 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 NOs: 21 and 22, where SEQ ID NOs: 21-22 are as defined in Table 4.

[0048] [Table 4]

[0049] Also provided according to one embodiment of the present invention is a polypeptide comprising a functional portion of any of the TCRs described herein. As used herein, the term "polypeptide" includes oligopeptides and refers to a single chain of amino acids linked by one or more peptide bonds.

[0050] In relation to the polypeptides of the invention, a functional portion can be any portion comprising consecutive amino acids that are part of a TCR, provided that the functional portion specifically binds to a mutant RAS. When used in relation to a TCR, the term "functional portion" refers to any portion or fragment of a TCR of the invention, which portion or fragment retains the biological activity of the TCR of which it is a part (the parent TCR). A functional portion may, for example, possess the ability to specifically bind to a mutant RAS (e.g., HLA-A * 11:01 molecule) or the ability to detect, treat or prevent cancer to a similar, equal or greater extent than the parent TCR. In the context of the parent TCR, a functional portion can include, for example, about 10%, about 25%, about 30%, about 50%, about 70%, about 80%, about 90%, about 95% or more of the parent TCR.

[0051] A functional portion may include additional amino acids at the amino or carboxy terminus of the portion, or at both termini. The additional amino acids are not found in the amino acid sequence of the parent TCR. Desirably, the additional amino acids do not interfere with the biological function of the functional portion, e.g., specifically binding to mutant RAS; and / or having the ability to detect, treat or prevent cancer. More desirably, the additional amino acids enhance the biological activity of the functional portion compared to the biological activity of the parent TCR.

[0052] The polypeptide may comprise a functional portion of either or both of the alpha and beta chains of the TCR of the invention, such as a functional portion comprising one or more of the CDR1, CDR2 and CDR3 of the variable region(s) of the alpha and / or beta chain of the TCR of the invention. In one embodiment of the invention, the polypeptide may comprise the amino acid sequence of SEQ ID NO:1 (alpha chain CDR1), SEQ ID NO:2 (alpha chain CDR2), SEQ ID NO:3 (alpha chain CDR3), SEQ ID NO:4 (beta chain CDR1), SEQ ID NO:5 (beta chain CDR2), SEQ ID NO:6 (beta chain CDR3) or a combination thereof.

[0053] In this regard, the polypeptide of the present invention may comprise any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 6. In one embodiment of the present invention, the TCR comprises (a) all of SEQ ID NOs: 1 to 3, (b) all of SEQ ID NOs: 4 to 6, or (c) all of the amino acid sequences of SEQ ID NOs: 1 to 6. In a preferred embodiment, the polypeptide comprises all of the amino acid sequences of SEQ ID NOs: 1 to 6.

[0054] In one 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 described above. In this regard, the polypeptide may comprise the amino acid sequence of (i) SEQ ID NO: 7 (variable region of the α chain), (ii) SEQ ID NO: 8 (variable region of the β chain), or (iii) both SEQ ID NOs: 7 and 8. Preferably, the polypeptide comprises the amino acid sequence of both SEQ ID NOs: 7 and 8.

[0055] In one embodiment of the invention, the polypeptide of the invention may further comprise the constant region of a TCR of the invention as described above. In this regard, the polypeptide may further comprise the amino acid sequence of SEQ ID NO: 19 (WT mouse constant region of the alpha chain), SEQ ID NO: 20 (WT mouse constant region of the beta chain), SEQ ID NO: 17 (substituted mouse constant region of the alpha chain), SEQ ID NO: 18 (substituted mouse constant region of the beta chain), both SEQ ID NO: 19 and 20, or both SEQ ID NO: 17 and 18. Preferably, the polypeptide further comprises the amino acid sequence of both SEQ ID NO: 19 and 20, or both SEQ ID NO: 17 and 18, in combination with any of the CDR regions or variable regions described herein with respect to other aspects of the invention.

[0056] In one embodiment of the invention, the polypeptide has: (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; and (iii) X at position 114 of SEQ ID NO:17 is Met, Al. and (iv) the amino acid sequence of SEQ ID NO: 17, wherein X at position 115 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 of (a) and (b). In one 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.

[0057] In one embodiment of the invention, the polypeptide of the invention may comprise the full length of the α or β chain of a TCR as described herein. In this regard, the polypeptide of the invention may comprise the amino acid sequence of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, both SEQ ID NO:21 and 22, or both SEQ ID NO:23 and 24. Preferably, the polypeptide comprises the amino acid sequence of both SEQ ID NO:21 and 22, or both SEQ ID NO:23 and 24.

[0058] In one embodiment of the invention, 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, lie, Pro, Phe, Met, or Trp; (iii) X at position 251 of SEQ ID NO:21 is Met, Ala, Val, Leu, lie, Pro, Phe, or Trp; and (iv) X at position 252 of SEQ ID NO:21 is GIy, Ala, Val, Leu, lie, 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; or (c) both (a) and (b). In one embodiment of the invention, the polypeptide has one or more of SEQ ID NOs: 21-22 as defined in any one of Tables 2-4.

[0059] An embodiment of the present invention further provides a protein comprising at least one polypeptide as described herein. By "protein" is meant a molecule comprising one or more polypeptide chains.

[0060] In one embodiment, the protein of the present invention may comprise a first polypeptide chain comprising the amino acid sequence of any of SEQ ID NOs: 1-3 and a second polypeptide chain comprising the amino acid sequence of any of SEQ ID NOs: 4-6.

[0061] In another embodiment of the invention, a 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.

[0062] The proteins of the invention may further comprise any of the constant regions described herein with respect to other aspects of the invention. In this regard, in one embodiment of the invention, the first polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 17 and the second polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 18. In one embodiment of the invention, the first polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 19 and the second polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 20.

[0063] In one embodiment of the invention, the protein is: (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 first polypeptide chain comprising the amino acid sequence of SEQ ID NO:17, or (c) both of (a) and (b). In one embodiment of the invention, one or both of SEQ ID NOs: 17 and 18 of the protein are as defined in any one of Tables 2-4.

[0064] Alternatively or additionally, a protein of an embodiment of the invention may comprise: (a) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:21, wherein (i) the X at position 185 of SEQ ID NO:21 is Thr or Cys; (ii) the X at position 249 of SEQ ID NO:21 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) the X at position 251 of SEQ ID NO:21 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) the X at position 252 of SEQ ID NO:21 is GIy, 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 the X at position 190 of SEQ ID NO:22 is Ser or Cys; or (c) both (a) and (b). In one embodiment of the 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 one embodiment of the invention, one or both of SEQ ID NOs: 21-22 are as defined in any one of Tables 2-4.

[0065] The protein of the invention may be a TCR. Alternatively, the protein of the invention may be a fusion protein, for example, when the protein comprises a single polypeptide chain comprising the amino acid sequences of both SEQ ID NOs: 21 and 22, both SEQ ID NOs: 23 and 24, or when the first and / or second polypeptide chain(s) of the protein further comprises an amino acid sequence encoding another amino acid sequence, e.g., an immunoglobulin or a portion thereof. In this regard, one embodiment of the invention also provides a fusion protein comprising at least one of the polypeptides of the invention described herein, together with at least one other polypeptide. The other polypeptide may be present as a separate polypeptide of the fusion protein, or as a polypeptide expressed in frame (in tandem) with one of the polypeptides of the invention described herein. The other polypeptide may encode any peptidic or proteinaceous molecule, or a portion thereof, including, but not limited to, an immunoglobulin, CD3, CD4, CD8, an MHC molecule, a CD1 molecule, e.g., CD1a, CD1b, CD1c, CD1d, etc.

[0066] A fusion protein may contain one or more copies of a polypeptide of the invention and / or one or more copies of another polypeptide. For example, a fusion protein may contain 1, 2, 3, 4, 5 or more copies of a polypeptide of the invention and / or another polypeptide. Suitable methods for making fusion proteins are known in the art and include, for example, recombinant methods.

[0067] In some embodiments of the invention, the TCRs, polypeptides and proteins of the invention may be expressed as a single protein comprising a linker peptide linking the alpha and beta chains. In this regard, the TCRs, polypeptides and proteins of the invention may further comprise a linker peptide. The linker peptide may conveniently facilitate expression of the recombinant TCRs, polypeptides and / or proteins in a host cell. The linker peptide may comprise any suitable amino acid sequence. For example, the linker peptide may be a furin-SGSG-P2A linker comprising the amino acid sequence of SEQ ID NO: 25. Upon expression of the construct comprising the linker peptide by the host cell, the linker peptide may be cleaved, resulting in separate alpha and beta chains. In one embodiment of the invention, the TCRs, polypeptides or proteins may comprise an amino acid sequence comprising a full-length alpha chain, a full-length beta chain and a linker peptide located between the alpha and beta chains.

[0068] The protein of the invention may be a recombinant antibody or antigen-binding portion thereof comprising at least one of the polypeptides of the invention described herein. As used herein, "recombinant antibody" refers to a recombinant (e.g., genetically engineered) protein comprising at least one of the polypeptides of the invention, antibody polypeptide chains or antigen-binding portions thereof. The antibody polypeptide or antigen-binding portion thereof may be an antibody heavy chain, light chain, variable or constant region of the heavy or light chain, a single chain variable fragment (scFv), or an Fc, Fab or F(ab) polypeptide. 2 The antibody polypeptide chain or antigen-binding portion thereof may be present as a separate polypeptide of a recombinant antibody. Alternatively, the antibody polypeptide chain or antigen-binding portion thereof may be present as a polypeptide expressed in-frame (in tandem) with a polypeptide of the invention. The antibody polypeptide or antigen-binding portion thereof may be the polypeptide of any antibody or any antibody fragment, including any of the antibodies and antibody fragments described herein.

[0069] Included within the scope of the present invention are functional variants of the TCRs, polypeptides or proteins of the present invention as described herein. As used herein, the term "functional variant" refers to a TCR, polypeptide or protein that has substantial or significant sequence identity or similarity to a parent TCR, polypeptide or protein, where the functional variant retains the biological activity of the TCR, polypeptide or protein of which it is a variant. Functional variants encompass variants of the TCRs, polypeptides or proteins described herein (parent TCRs, polypeptides or proteins) that, for example, retain the ability to specifically bind to the mutant RAS to which the parent TCR has antigen specificity or to a similar, equal or greater extent than the parent TCR, polypeptide or protein. In relation to the parent TCR, polypeptide or protein, for example, the functional variant may be at least about 30%, about 50%, about 75%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or more identical in amino acid sequence to the parent TCR, polypeptide or protein, respectively.

[0070] For example, a functional variant may comprise an amino acid sequence of a parent TCR, polypeptide or protein that comprises at least one conservative amino acid substitution. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having particular physical and / or chemical properties is replaced with another amino acid having the same chemical or physical properties. For example, a conservative amino acid substitution may be the substitution of an acidic amino acid for another acidic amino acid (e.g., Asp or Glu), an amino acid comprising a non-polar side chain for another amino acid comprising a non-polar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid for another basic amino acid (Lys, Arg, etc.), an amino acid comprising a polar side chain for another amino acid comprising a polar side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), etc.

[0071] Alternatively or additionally, the functional variant may comprise the amino acid sequence of the parent TCR, polypeptide or protein comprising at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. Preferably, the non-conservative amino acid substitution enhances the biological activity of the functional variant such that the biological activity of the functional variant is increased compared to the parent TCR, polypeptide or protein.

[0072] A TCR, polypeptide or protein may consist essentially of one or more of the specific amino acid sequences or sequences described herein, such that other components of the TCR, polypeptide or protein, e.g., other amino acids, do not substantially alter the biological activity of the TCR, polypeptide or protein. For example, the TCR, polypeptide or protein of the present invention may consist essentially of the amino acid sequence of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, both of SEQ ID NOs:21 to 22, or both of SEQ ID NOs:23 to 24. Also, for example, the TCR, polypeptide or protein of the present invention may consist essentially of the amino acid sequence(s) of (i) SEQ ID NO:7, (ii) SEQ ID NO:8, or (iii) both of SEQ ID NOs:7 and 8. Furthermore, the TCR, polypeptide or protein of the present invention may consist essentially of the amino acid sequence of (a) any one or more of SEQ ID NOs:1 to 6; (b) all of SEQ ID NOs:1 to 3; (c) all of SEQ ID NOs:4 to 6; or (d) all of SEQ ID NOs:1 to 6.

[0073] The TCRs, polypeptides and proteins of the invention can be of any length, i.e., contain any number of amino acids, provided that the TCRs, polypeptides or proteins retain their biological activity, e.g., the ability to specifically bind to mutant RAS; detect cancer in a mammal; or treat or prevent cancer in a mammal. For example, polypeptides can range from about 50 to about 5000 amino acids in length, 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 invention also include oligopeptides.

[0074] The TCRs, polypeptides and proteins of the invention may contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexane carboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine. β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyllysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentane carboxylic acid, α-aminocyclohexane carboxylic acid, α-aminocycloheptane, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid d), homophenylalanine and α-tert-butylglycine.

[0075] The TCRs, polypeptides and proteins of the present invention may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized (e.g., via disulfide bridges), or converted into acid addition salts, and / or dimerized or polymerized, or conjugated, as appropriate.

[0076] 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. Polypeptides and proteins can also be made recombinantly using the nucleic acids described herein, using standard recombinant techniques. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual(4th ed.) Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Alternatively, the TCRs, polypeptides and / or proteins described herein may be commercially synthesized by companies such as Synpep (Dublin, CA), Peptide Technologies Corp. (Gaithersburg, MD) and Multiple Peptide Systems (San Diego, CA). In this regard, the TCRs, polypeptides and proteins of the invention may be synthetic, recombinant, isolated and / or purified.

[0077] The scope of the invention includes conjugates, e.g., bioconjugates, comprising any of the TCRs, polypeptides or proteins of the invention (including any functional portion or variant thereof), nucleic acids, recombinant expression vectors, host cells, host cell populations, or antibodies or antigen-binding portions thereof. Conjugates and methods of synthesizing conjugates are generally known in the art.

[0078] One embodiment of the present invention provides a nucleic acid comprising a nucleotide sequence encoding any of the TCRs, polypeptides or proteins described herein. As used herein, "nucleic acid" includes "polynucleotides", "oligonucleotides" and "nucleic acid molecules" and generally refers to a polymer of DNA or RNA, which may be single-stranded or double-stranded, may contain natural, non-natural or modified nucleotides, and may contain natural, non-natural or modified internucleotide linkages, such as phosphoramidate or phosphorothioate linkages instead of the phosphodiesters found between nucleotides of unmodified oligonucleotides. In one embodiment, the nucleic acid comprises complementary DNA (cDNA). It is generally preferred that the nucleic acid does not contain any insertions, deletions, inversions and / or substitutions. However, as discussed herein, in some instances, it may be preferred that the nucleic acid contains one or more insertions, deletions, inversions and / or substitutions.

[0079] 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 joining a natural or synthetic nucleic acid segment to a nucleic acid molecule capable of replicating in the living cell, or (ii) a molecule resulting from the replication of what is described in (i) above. For purposes herein, the replication can be in vitro replication or in vivo replication.

[0080] 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, supra. For example, nucleic acids can be composed of naturally occurring nucleotides or variously modified nucleotides (e.g., phosphorothioates, phosphate ions, etc.) designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed upon hybridization. Examples of modified nucleotides that can be used to generate nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N-acetylglucosyl ... 6 -Isopentenyladenine (N 6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6 -Substituted adenine (N 6 -substituted adenine), 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6 -Isopentenyladenine (2-methylthio-N 6-isopentenyladenine, uracil-5-oxyacetic acid(v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the present invention can be purchased commercially from companies such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0081] The nucleic acid may comprise any nucleotide sequence encoding any of the TCRs, polypeptides or proteins described herein. In one embodiment of the invention, the nucleic acid may comprise the nucleotide sequence of any one of SEQ ID NOs: 31-32 (Table 5). In one embodiment of the invention, the nucleic acid comprises the nucleotide sequences of both SEQ ID NOs: 31-32.

[0082] [Table 5]

[0083] In one embodiment of the present invention, the nucleic acid comprises a codon-optimized nucleotide sequence encoding any of the TCRs, polypeptides or proteins described herein. Without being bound to any particular theory or mechanism, it is believed that codon optimization of the nucleotide sequence increases the translation efficiency of the mRNA transcript. Codon optimization of the nucleotide sequence may include replacing native codons with alternative codons that code for the same amino acid but that can be translated by tRNAs that are more readily available in the cell, thus increasing translation efficiency. Optimization of the nucleotide sequence may also reduce secondary structures in the mRNA that interfere with translation, thus increasing translation efficiency.

[0084] The present invention also provides nucleic acids comprising a nucleotide sequence that is complementary to, or that hybridizes under stringent conditions to, any of the nucleotide sequences of the nucleic acids described herein.

[0085] Nucleotide sequences that hybridize under stringent conditions preferably hybridize under high stringency conditions. By "high stringency conditions" is meant that a nucleotide sequence specifically hybridizes to a target sequence (any nucleotide sequence of a nucleic acid described herein) in an amount detectably stronger than non-specific hybridization. High stringency conditions include conditions that distinguish polynucleotides with exactly complementary sequences, or polynucleotides containing only scattered mismatches, from random sequences that happen to have a few small regions (e.g., 3-10 bases) that match the nucleotide sequence. Such small complementary regions are more easily melted than full-length complements of 14-17 or more bases, making them easily distinguishable by high stringency hybridization. Relatively high stringency conditions include, for example, low salt and / or high temperature conditions (e.g., provided by about 0.02-0.1 M NaCl or equivalent at a temperature of about 50-70° C.). Such high stringency conditions tolerate little, if any, mismatch between the nucleotide sequence and the template or target strand and are particularly useful for detecting expression of any of the TCRs of the present invention. It is generally understood that conditions can be rendered more stringent by the addition of increasing amounts of formamide.

[0086] The invention also provides nucleic acids comprising a nucleotide sequence that is at least about 70% or more, e.g., about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to any of the nucleic acids described herein. In this regard, the nucleic acid can consist essentially of any of the nucleotide sequences described herein.

[0087] The nucleic acids of the invention may be incorporated into a recombinant expression vector. In this regard, the invention provides a recombinant expression vector comprising any of the nucleic acids of the invention. In one embodiment of the invention, the recombinant expression vector comprises nucleotide sequences encoding the α chain, the β chain and the linker peptide.

[0088] For purposes herein, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that allows for the expression of an mRNA, protein, polypeptide, or peptide by a host cell, where the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with a cell under conditions sufficient to allow expression of the mRNA, protein, polypeptide, or peptide in the cell. The vector of the present invention is not naturally occurring in its entirety. However, portions 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, may be synthetic, or may be derived in part from a natural source, and may contain natural, non-natural, or modified nucleotides. The recombinant expression vector may contain naturally occurring internucleotide bonds, non-naturally occurring internucleotide bonds, or both types of bonds. Preferably, the non-naturally occurring or modified nucleotides or internucleotide bonds do not interfere with the transcription or replication of the vector.

[0089] The recombinant expression vector of the present invention may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host cell. Suitable vectors include vectors designed for propagation and amplification, or for expression, or both, such as plasmids and viruses. The vector may be selected from the group consisting of pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 may also be used. Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). Preferably, the recombinant expression vector is a viral vector, e.g., a retroviral vector. In a particularly preferred embodiment, the recombinant expression vector is a MSGV1 vector.

[0090] The recombinant expression vectors of the invention can be prepared using standard recombinant DNA techniques, for example as described in Green and Sambrook et al. (supra). Circular or linear expression vector constructs can be prepared to contain a replication system that functions in prokaryotic or eukaryotic host cells. Replication systems can be derived from, for example, ColEl, 2μ plasmid, lambda, SV40, bovine papilloma virus, and the like.

[0091] Desirably, the recombinant expression vector includes regulatory sequences, such as transcriptional and translational initiation and termination codons, specific for the type of host cell (e.g., bacterial, fungal, plant, or animal) into which the vector will be introduced, taking into account whether the vector is DNA- or RNA-based, as appropriate.

[0092] The recombinant expression vector may contain one or more marker genes to allow for the 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 the expression vectors of the present invention include, for example, the neomycin / G418 resistance gene, the hygromycin resistance gene, the histidinol resistance gene, the tetracycline resistance gene and the ampicillin resistance gene.

[0093] The recombinant expression vector may include a native or non-native promoter operably linked to a nucleotide sequence encoding a TCR, polypeptide, or protein, or to a nucleotide sequence that is complementary to or hybridizes to a nucleotide sequence encoding a TCR, polypeptide, or protein. Selection of a promoter, e.g., strong promoters, weak promoters, inducible promoters, tissue-specific promoters, and developmental-specific promoters, is within the ordinary skill of one of ordinary skill in the art. Similarly, combining a nucleotide sequence with a promoter is also within the ordinary skill of one of ordinary skill in the art. The promoter may be a non-viral promoter, or a viral promoter, e.g., a promoter found in the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, and the murine stem cell virus long-terminal repeat.

[0094] The recombinant expression vectors of the invention can be designed for either transient expression, stable expression, or both. The recombinant expression vectors can also be made for constitutive or inducible expression.

[0095] Additionally, the recombinant expression vector can be made to contain a suicide gene. The term "suicide gene" as used herein refers to a gene that causes the death of a cell expressing the suicide gene. A suicide gene can be a gene that confers sensitivity to a substance, e.g., a drug, on a cell expressing the gene, causing the cell to die when the cell comes into contact with or is exposed to the substance. Suicide genes are known in the art and include, for example, the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, nitroreductase, and inducible caspase 9 gene systems.

[0096] Another embodiment of the present invention further provides a host cell comprising any of the recombinant expression vectors described herein. The term "host cell" as used herein refers to any type of cell that may contain a recombinant expression vector of the present invention. The host cell may be a eukaryotic cell, e.g., a plant, an animal, a fungus, or an algae, or a prokaryotic cell, e.g., a bacterium or a protozoan. The host cell may be a cultured cell or a primary cell, i.e., directly isolated from an organism, e.g., a human. The host cell may be an adherent cell or a suspension cell, i.e., a cell that grows in suspension. Suitable host cells are known in the art and include, for example, DH5α E.coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. For purposes of amplifying or replicating a recombinant expression vector, the host cell is preferably a prokaryotic cell, e.g., a DH5α cell. For purposes of producing a recombinant TCR, polypeptide, or protein, the host cell is preferably a mammalian cell. Most preferably, the host cell is a human cell. The host cell may be any type of cell. Although the host cells may be obtained from any type of tissue and may be at any stage of development, the host cells are preferably peripheral blood lymphocytes (PBLs) or peripheral blood mononuclear cells (PBMCs). More preferably, the host cells are T cells.

[0097] For purposes herein, a T cell can be any T cell, such as a cultured T cell (e.g., primary T cell), or a T cell from a cultured T cell line (e.g., Jurkat, SupT1, etc.), or a T cell obtained from a mammal. If obtained from a mammal, the T cells can be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The T cells can also be enriched or purified. Preferably, the T cells are human T cells. The T cells can be any type of T cell and can be at any stage of development (e.g., CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells, e.g., Th 1 and Th 2 cells, CD4 + T cells, CD8 + These include, but are not limited to, T cells (e.g., cytotoxic T cells), tumor infiltrating lymphocytes (TILs), memory T cells (e.g., central memory T cells and effector memory T cells), naive T cells, and the like.

[0098] Also provided by the present invention is a cell population comprising at least one host cell as described herein. The cell population may be a heterogeneous population comprising host cells comprising any of the described recombinant expression vectors in addition to at least one other cell, e.g., host cell (e.g., T cell), or cell other than a T cell, e.g., B cell, macrophage, neutrophil, erythrocyte, hepatocyte, endothelial cell, epithelial cell, muscle cell, brain cell, etc., that does not comprise any recombinant expression vector. Alternatively, the cell population may be a substantially homogeneous population, comprising primarily (e.g., consisting essentially of) host cells comprising the recombinant expression vector. The population may also be a clonal population of cells, where all cells of the population are clones of a single host cell comprising the recombinant expression vector, such that all cells of the population comprise the recombinant expression vector. In one embodiment of the present invention, the cell population is a clonal population comprising host cells comprising a recombinant expression vector as described herein.

[0099] In one embodiment of the invention, the number of cells in a population can be rapidly expanded. Expansion of T cell numbers can be achieved by, for example, expanding the number of T cells as described in U.S. Pat. No. 8,034,334; U.S. Pat. No. 8,383,099; U.S. Patent Application Publication No. 2012 / 0244133; Dudley et al., J. Immunother., 26:332-42 (2003); and Riddell et al. Expansion of T cell numbers can be accomplished by any of a number of methods known in the art, such as those described in (e.g., J. Immunol. Methods, 128:189-201 (1990). In one embodiment, expansion of T cell numbers is achieved by culturing T cells with OKT3 antibody, IL-2, and feeder PBMCs (e.g., irradiated allogeneic PBMCs).

[0100] The TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, and host cells (including populations thereof) of the present invention may be isolated and / or purified. The term "isolated" as used herein means removed from its natural environment. The term "purified" as used herein means increased purity, although "purity" is a relative term and should not necessarily be construed as absolute purity. For example, 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%.

[0101] The TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, and host cells (including populations thereof) of the invention, all of which are hereinafter collectively referred to as the "TCR material of the invention", may be formulated into compositions, such as pharmaceutical compositions. In this regard, the invention provides pharmaceutical compositions comprising any of the TCRs, polypeptides, proteins, nucleic acids, expression vectors, and host cells (including populations thereof) described herein and a pharma- ceutical acceptable carrier. Pharmaceutical compositions of the invention containing any of the TCR materials of the invention may contain more than one TCR material of the invention, e.g., polypeptides and nucleic acids, or two or more different TCRs. Alternatively, the pharmaceutical compositions may contain the TCR material of the invention in combination with another pharma- ceutical active substance(s) or drug(s), such as a chemotherapeutic agent, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.

[0102] Preferably, the carrier is a pharma- ceutically acceptable carrier. For pharmaceutical compositions, the carrier may be any of those conventionally used for the particular TCR material of the present invention under consideration. Methods for preparing administrable compositions will be known or apparent to those skilled in the art, and are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, 22nd Edition, Pharmaceutical Press (2012). Preferably, the pharma-ceutically acceptable carrier is one that has no adverse side effects or toxicity under the conditions of use.

[0103] The choice of carrier is determined in part by the particular TCR material of the present invention and by the particular method used to administer the TCR material of the present invention. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the present invention. Suitable formulations may include any formulation for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, intratumoral and intraperitoneal administration. More than one route may be used to administer the TCR material of the present invention, and in certain instances, a particular route may provide a more rapid and effective response than another route.

[0104] Preferably, the TCR material of the invention is administered by injection, e.g., intravenously. When the TCR material of the invention is a host cell (including a population thereof) expressing a TCR of the invention, the pharma- ceutically acceptable carrier for the cells for injection may include any isotonic carrier, such as normal saline (about 0.90% w / v NaCl in water, about 300 mOsm / L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water, or lactated Ringer's solution, etc. In one embodiment, the pharma-ceutically acceptable carrier is supplemented with human serum albumin.

[0105] For purposes of the present invention, the amount or dose of the TCR material of the invention administered (e.g., number of cells, if the TCR material of the invention is one or more cells) should be sufficient to effect, e.g., a therapeutic or prophylactic response in the subject or animal over a suitable period of time. For example, the dose of the TCR material of the invention should be sufficient to bind a cancer antigen (e.g., mutant RAS) or to detect, treat or prevent cancer for a period of about 2 hours or more, e.g., 12-24 hours or more, from the time of administration. In certain embodiments, the period may be longer. The dose will be determined by the efficacy of the particular TCR material of the invention and the condition of the animal (e.g., human) and the body weight of the animal (e.g., human) to be treated.

[0106] Many assays for determining the dose to be administered are known in the art. For purposes of the present invention, the extent to which target cells are lysed by T cells expressing a TCR, polypeptide, or protein of the invention upon administration of a particular dose of such T cells to one mammal among a set of mammals, each of which receives a different dose of such T cells, is considered. or the extent to which IFN-γ is secreted can be used to determine the starting dose to administer to a mammal. The extent to which target cells are lysed or IFN-γ is secreted upon administration of a given dose can be assayed by methods known in the art.

[0107] The dose of the TCR material of the invention will also be determined by the existence, nature and extent of any adverse side effects that may accompany administration of a particular TCR material of the invention. Typically, the attending physician will determine the dose of the TCR material of the invention for treating each individual patient, taking into account a variety of factors, such as age, weight, general health, dietary habits, 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 will be, for example, about 1×10 6 From about 1×10 12 The number of cells may vary from 1×10 to 1×10 cells or more. 6 Less than 100 cells may be administered.

[0108] Those skilled in the art will appreciate that modifications of the inventive TCR materials may be used to enhance the therapeutic or prophylactic efficacy of the inventive TCR materials. It will be readily understood that the TCR material of the invention can be modified in any number of ways. For example, the TCR material of the invention can be conjugated either directly or indirectly via a bridge to a chemotherapeutic agent. The practice of conjugating compounds to chemotherapeutic agents is known in the art. Those skilled in the art will recognize that sites on the TCR material of the invention that are not necessary for the function of the TCR material of the invention are suitable sites for attaching a bridge and / or chemotherapeutic agent (provided that the bridge and / or chemotherapeutic agent do not bind to the TCR material of the invention and interfere with the function of the TCR material of the invention (i.e., ability to bind mutant RAS or ability to detect, treat or prevent cancer)).

[0109] It is contemplated that the pharmaceutical compositions, TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells and cell populations of the invention may be used in methods of treating or preventing cancer. Without being bound to a particular theory, it is believed that the TCRs of the invention specifically bind to mutant RAS, such that when expressed in a cell, the TCR (or the associated polypeptide or protein of the invention) can regulate an immune response against a target cell expressing mutant RAS. In this regard, the invention provides a method of treating or preventing cancer in a mammal, comprising administering to the mammal any of the pharmaceutical compositions, TCRs, polypeptides, or proteins described herein, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein, or any host cell or cell population comprising a recombinant vector encoding any of the TCRs, polypeptides, or proteins described herein, in an amount effective to treat or prevent cancer in the mammal.

[0110] One embodiment of the present invention provides any of the pharmaceutical compositions, TCRs, polypeptides or proteins described herein, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCRs, polypeptides or proteins described herein, or any host cell or population of cells comprising a recombinant vector encoding any of the TCRs, polypeptides or proteins described herein, for use in the treatment or prevention of cancer in a mammal.

[0111] The terms "treat" and "prevent" and their derivatives, as used herein, do not necessarily mean 100% or complete treatment or prevention. Rather, there are various degrees of treatment or prevention that one of skill in the art will recognize as having potential benefit or therapeutic effect. In this regard, the methods of the present invention may provide any amount of treatment or prevention of cancer in a mammal at any level. Moreover, the treatment or prevention provided by the methods of the present invention may be used without limitation to treatment or prevention. may include treatment or prevention of one or more of the cancerous conditions or symptoms being prevented. For example, treatment or prevention may include promoting tumor regression. For purposes herein, "prevention" may also include delaying the onset of cancer, or a symptom or condition thereof. Alternatively or additionally, "prevention" may include preventing or delaying the recurrence of cancer, or a symptom or condition thereof.

[0112] Also provided is a method of detecting the presence of cancer in a mammal, the method comprising: (i) contacting a sample comprising one or more cells from a mammal with any of the TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, cell populations, or pharmaceutical compositions of the invention described herein, thereby forming a complex, and (ii) detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

[0113] For the methods of the present invention for detecting cancer in a mammal, the cellular sample may be a sample comprising whole cells, a lysate thereof, or a fraction of a whole cell lysate, e.g., a nuclear or cytoplasmic fraction, a total protein fraction, or a nucleic acid fraction.

[0114] For purposes of the method of detecting cancer of the present invention, the contacting can be performed in vitro or in vivo with respect to a mammal. Preferably, the contacting is in vitro.

[0115] Detection of the complex may also be by any number of methods known in the art. For example, the TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, or cell populations of the invention described herein may be labeled with a detectable label, such as, for example, radioisotopes, fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

[0116] For purposes of the methods of the invention in which a host cell or cell population is administered, the cells may be allogeneic or autologous to the mammal. Preferably, the cells are autologous to the mammal.

[0117] In the context of the methods of the present invention, the cancer is selected from the group consisting of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal or anorectum, eye cancer, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder or pleura, cancer of the nose, nasal cavity or middle ear, cancer of the oral cavity, cancer of the vagina, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumors, and gliomas. The cancer may be any cancer, including any of the following: Hodgkin's lymphoma, hypopharyngeal cancer, renal cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, oropharynx cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal cancer, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, ureteral cancer, and bladder cancer. Preferred cancers are pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer, or prostate cancer. Preferably, the lung cancer is lung adenocarcinoma, the ovarian cancer is epithelial ovarian cancer, and the pancreatic cancer is pancreatic adenocarcinoma. In one embodiment of the present invention, the cancer expresses a mutant human RAS amino acid sequence, which is a mutant human KRAS, a mutant human HRAS, or a mutant human NRAS amino acid sequence. The mutant human KRAS, mutant human HRAS, and mutant human NRAS expressed by the cancer may be as described herein with respect to other aspects of the invention.

[0118] The mammal referred to in the methods of the present invention may be any mammal. The term "mammal" as used herein includes rodent mammals, such as mice and hamsters; It refers to any mammal, including but not limited to mammals of the order Lagomorpha, such as rabbits and rabbits. Preferably, the mammal is from the order Carnivora, including Felidae (cats) and Canidae (dogs). More preferably, the mammal is from the order Bovidae, including Bovidae (cows) and Suidae (pigs), or the order Perssodactyla, including Equidae (horses). Most preferably, the mammal is from the order Primates, Ceboids, or Simoids (monkeys), or the order Anthropoids (humans and apes). A particularly preferred mammal is the human.

[0119] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. EXAMPLES

[0120] Example 1 This example describes a TCR with antigen specificity for human KRAS containing the G12V mutation, wherein the mutant KRAS is associated with HLA-A * 11: Illustrates the isolation of a TCR presented by the 01 molecule.

[0121] TCR was 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 selected from the patient's peripheral blood for 10 days.

[0122] Next, T cell reactivity was assessed using the KRAS G12V mutation (MTEYKLVVVGA V GVGKSALTIQLI) (SEQ ID NO: 26) or the corresponding WT peptide (MTEYKLVVVGA GThe DCs were tested against autologous DCs pulsed with a 24-mer peptide encompassing KRAS G12V peptide (SEQ ID NO: 27). The peptides were resuspended in DMSO. T cells cultured with DMSO-pulsed DCs were used as a negative control. T cell reactivity was measured based on CD137 expression and IFN-γ production measured by ELISPOT assay. Responsive T cells were then selected based on CD137 expression, the number of selected T cells was expanded, and the number of selected and expanded cells was tested again as described in this example. The results are shown in Figures 1A and 1B. As shown in Figures 1A and 1B, T cells upregulated CD137 expression (Figure 1A) and increased IFN-γ production (Figure 1B) only after co-culture with DCs pulsed with KRAS G12V peptide.

[0123] The patient is HLA-A * Since the KRAS G12V 9-mer and 10-mer peptides were positive for HLA-A 11:01, it was predicted that the KRAS G12V 9-mer and 10-mer peptides would bind to this allele with high affinity. Therefore, using tetramer staining techniques, we were able to confirm that T cells were HLA-A positive. * KRAS in relation to 11:01 G12V We tested whether peripheral blood CD8+ T cells that had undergone IVS with the KRAS G12V 24-mer peptide were reactive to tetramers (9-mer VVGA V GVGK (SEQ ID NO: 29) or 10-mer VVVGA V The cells were stained with PE, APC, and GVGK (SEQ ID NO: 30). Both PE and APC are fluorophores. The tetramers were conjugated to one or the other of these fluorophores. Staining the cells with both tetramers increases confidence in the specificity. The results are shown in Figure 2 (right plot). HLA-A * KRAS G12D in relation to 11:01 T cells engineered to express a TCR that recognizes a 10-mer were used as a negative control. The results for the negative control are shown in Figure 2 (left plot). As shown in Figure 2, CD8 T cells that underwent IVS with the KRAS G12V 24-mer peptide were able to recognize the KRAS G12V -HLA-A *11:01 tetramer staining, whereas negative control T cells did not.

[0124] Tetramer-sorted cells were sequenced using single-cell sequencing, revealing 1 beta chain and 1 alpha chain (Table 6). The nucleotide sequences of the TCR alpha and beta chain variable regions were SEQ ID NOs: 31 and 32, respectively.

[0125] [Table 6]

[0126] Example 2 This example demonstrates the preparation of an expression cassette encoding the TCR of Example 1 containing a cysteine ​​substituted, LVL modified murine constant region and the cloning of the expression cassette into a retroviral vector.

[0127] The TCR of Example 1 was cloned into the 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 including the cysteine-substituted, LVL-modified mouse constant region was cloned into the retroviral vector. The α chain mouse constant region was composed of the amino acid sequence of SEQ ID NO:17, where X at position 48 is Cys, X at position 112 is Leu, X at position 114 is Ile, and X at position 115 is Val. The β chain constant region was composed of the amino acid sequence of SEQ ID NO:18, where 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 was placed between the α chain constant region and the β chain variable region. The retroviral vector contained, from the 5' to the 3' end, a nucleotide sequence encoding a 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.

[0128] Example 3 This example shows that peripheral blood allogeneic T cells transduced with the TCR of Example 2 are human KRAS containing the G12V mutation and are associated with HLA-A * We demonstrate specific recognition of mutant KRAS presented by the 11:01 molecule.

[0129] Retroviruses encoding the TCR of Example 2 were generated 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)). Expression of TCR on the surface of the cells was measured by flow cytometry. The results are shown in Figure 3. As shown in Figure 3, expression of mouse TCR beta chain constant region (mTCR beta) was detected on the surface of the cells.

[0130] Allogeneic cells transduced with the TCR of Example 2 were treated with KRAS G12D, KRAS G The cells were then tested for reactivity against target cancer cell lines expressing KRAS 12C, or KRAS G12V. As shown in FIG. 4, 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 FIG. 4. As shown in FIG. 4, the transduced cells expressed KRAS G12V and HLA-A * Specific reactivity was found against cancer cell lines expressing 11:01.

[0131] Example 4 This example shows the TCR of Example 2 is KRAS G12V 9-mer peptide

[0132] [ka]

[0133] (SEQ ID NO: 29) and KRAS G12V 10-mer peptide (KRAS G12V 10-mer peptide KRAS G12V 10-mer peptide)

[0134] [ka]

[0135] (SEQ ID NO: 30).

[0136] Allogeneic T cells were transduced with the TCR of Example 2 as described in Example 3. COS7 / A11 target cells were transduced with various concentrations of the KRAS G12V 9-mer peptide.

[0137] [ka]

[0138] (SEQ ID NO:29), KRAS G12V 10-mer peptide

[0139] [ka]

[0140] (SEQ ID NO:30), KRAS WT 9-mer peptide

[0141] [ka]

[0142] (SEQ ID NO: 33), or KRAS WT 10-mer peptide

[0143] [ka]

[0144] (SEQ ID NO: 34). Transduced T cells were co-cultured with pulsed target cells. IFN-γ secretion was measured. The results are shown in Figure 5A (9-mer) and Figure 5B (10-mer). As shown in Figures 5A-5B, TCR specifically recognized KRAS G12V 9-mer and KRAS G12V 10-mer peptides, respectively.

[0145] All references cited in this specification, including publications, patent applications, and patents, are herein incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and to the same extent as if each reference was set forth in its entirety herein.

[0146] In describing the present invention (particularly in relation to the claims which follow), the use of the terms "a" and "an" and "the" and "at least one" and similar referents should be construed to cover both the singular and the plural, unless otherwise stated herein or clearly contradicted by context. The use of the term "at least one" following a list of one or more items (e.g., "at least one of A and B") should be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise stated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise stated. Recitation of ranges of values ​​herein is intended only to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated herein as if it were individually set forth herein. All methods described herein can be performed in any suitable order unless otherwise specified herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary phrases (e.g., "such as") provided herein is intended only to better illustrate the invention and does not impose limitations on the scope of the invention unless specifically claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0147] Preferred embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments may become apparent to those of skill in the art upon reading the foregoing description. The inventors anticipate that those of skill in the art will employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

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

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

3. The TCR of claim 1, wherein the HLA class I molecule is an HLA-A11 molecule.

4. HLA class I molecule is HLA-A ＊ The TCR of claim 1 which is a 11:01 molecule.

5. The TCR of any one of claims 1 to 4, wherein the mutant human RAS amino acid sequence comprises the amino acid sequence of wild-type human KRAS, wild-type human HRAS, or wild-type human NRAS, including a substitution of glycine at position 12, where position 12 is defined relative to the wild-type human KRAS, wild-type human HRAS, or wild-type human NRAS protein, respectively.

6. 6. The TCR of claim 5, wherein the substitution is a substitution of glycine at position 12 with valine.

7. The TCR of any one of claims 1 to 6, comprising a human variable region.

8. (i) SEQ ID NO: 7 (ii) SEQ ID NO:8, or (iii) both of SEQ ID NOs: 7 and 8 The TCR according to any one of claims 1 to 7, comprising an amino acid sequence of:

9. (a) an alpha chain constant region comprising the amino acid sequence of SEQ ID NO:17: (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, He, Pro, Phe, Met, or Trp; (iii) X at position 114 of SEQ ID NO: 17 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an alpha chain constant region, in which X at position 115 of SEQ ID NO: 17 is Gly, Ala, Val, Leu, He, 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) The TCR of any one of claims 1 to 8, further comprising:

10. (a) an alpha chain comprising the amino acid sequence of SEQ ID NO:21: (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, He, Pro, Phe, Met, or Trp; (iii) X at position 251 of SEQ ID NO:21 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an alpha chain, in which X at position 252 of SEQ ID NO:21 is Gly, Ala, Val, Leu, lie, 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; or (c) Both (a) and (b) The isolated or purified TCR of any one of claims 1 to 9, comprising:

11. 11. An isolated or purified polypeptide comprising a functional portion of the TCR of any one of claims 1 to 10, wherein the functional portion comprises: (a) all of SEQ ID NOs: 1 to 3; (b) all of SEQ ID NOs: 4 to 6; or (c) All of SEQ ID NOs: 1 to 6 A polypeptide comprising the amino acid sequence of

12. The functional parts are: (i) SEQ ID NO: 7 (ii) SEQ ID NO:8, or (iii) both of SEQ ID NOs: 7 and 8 12. The isolated or purified polypeptide of claim 11, comprising the amino acid sequence(s) of:

13. (a) an amino acid sequence of SEQ ID NO:17: (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, He, Pro, Phe, Met, or Trp; (iii) X at position 114 of SEQ ID NO: 17 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an amino acid sequence in which X at position 115 of SEQ ID NO: 17 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (b) the amino acid sequence of SEQ ID NO: 18, in which X at position 57 of SEQ ID NO: 18 is Ser or Cys; or (c) Both (a) and (b) 13. The isolated or purified polypeptide of claim 11 or 12, further comprising:

14. (a) an amino acid sequence of SEQ ID NO:21: (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, He, Pro, Phe, Met, or Trp; (iii) X at position 251 of SEQ ID NO:21 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an amino acid sequence in which X at position 252 of SEQ ID NO:21 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (b) the amino acid sequence of SEQ ID NO: 22, in which X at position 190 of SEQ ID NO: 22 is Ser or Cys; or (c) Both (a) and (b) 14. The isolated or purified polypeptide of any one of claims 11 to 13, comprising:

15. An isolated or purified protein comprising at least one of the polypeptides according to any one of claims 11 to 14.

16. 16. The isolated or purified protein of claim 15, comprising a first polypeptide chain comprising an amino acid sequence of SEQ ID NO:1-3 and a second polypeptide chain comprising an amino acid sequence of SEQ ID NO:4-6.

17. 17. The isolated or purified protein of claim 15 or 16, comprising 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.

18. (a) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:17: (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, He, Pro, Phe, Met, or Trp; (iii) X at position 114 of SEQ ID NO: 17 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) a first polypeptide chain, wherein X at position 115 of SEQ ID NO: 17 is Gly, Ala, Val, Leu, He, 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) 18. The isolated or purified protein of any one of claims 15 to 17, further comprising:

19. (a) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:21: (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, He, Pro, Phe, Met, or Trp; (iii) X at position 251 of SEQ ID NO:21 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) a first polypeptide chain, wherein X at position 252 of SEQ ID NO:21 is Gly, Ala, Val, Leu, He, 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) 19. The isolated or purified protein of any one of claims 15 to 18, comprising:

20. An isolated or purified nucleic acid comprising a nucleotide sequence encoding a TCR according to any one of claims 1 to 10, a polypeptide according to any one of claims 11 to 14, or a protein according to any one of claims 15 to 19.

21. A recombinant expression vector comprising the nucleic acid of claim 20.

22. 22. An isolated or purified host cell comprising the recombinant expression vector of claim 21.

23. 23. An isolated or purified cell population comprising the host cell of claim 22.

24. 22. A pharmaceutical composition comprising: (a) a TCR according to any one of claims 1 to 10, a polypeptide according to any one of claims 11 to 14, a protein according to any one of claims 15 to 19, a nucleic acid according to claim 20, a recombinant expression vector according to claim 21, a host cell according to claim 22, or a cell population according to claim 23; and (b) a pharma- ceutical acceptable carrier.

25. 1. A method for detecting the presence of cancer in a mammal, comprising: (a) contacting a sample containing cancer cells with a TCR according to any one of claims 1 to 10, a polypeptide according to any one of claims 11 to 14, a protein according to any one of claims 15 to 19, a nucleic acid according to claim 20, a recombinant expression vector according to claim 21, a host cell according to claim 22, a host cell population according to claim 23, or a pharmaceutical composition according to claim 24, thereby forming a complex; and (b) detecting the complex wherein detection of the complex indicates the presence of cancer in the mammal.

26. 25. A TCR according to any one of claims 1 to 10, a polypeptide according to any one of claims 11 to 14, a protein according to any one of claims 15 to 19, a nucleic acid according to claim 20, a recombinant expression vector according to claim 21, a host cell according to claim 22, a cell population according to claim 23, or a pharmaceutical composition according to claim 24, for use in the treatment or prevention of cancer in a mammal.

27. 27. The method of claim 25 or the TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, cell population, or pharmaceutical composition for use of claim 26, wherein the cancer expresses a mutated human RAS amino acid sequence, and the mutated human RAS amino acid sequence is a mutated human KRAS, a mutated human HRAS, or a mutated human NRAS amino acid sequence.

28. 28. The method of claim 27, or the TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, cell population, or pharmaceutical composition for use of claim 27, wherein the mutant human RAS amino acid sequence comprises the amino acid sequence of wild-type human KRAS, wild-type human HRAS, or wild-type human NRAS comprising a substitution of glycine at position 12, where position 12 is defined with reference to the amino acid sequence of wild-type human KRAS, wild-type human HRAS, or wild-type human NRAS, respectively.

29. 29. The method of claim 28 or the TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, cell population, or pharmaceutical composition for use of claim 28, wherein the substitution is a substitution of glycine at position 12 with valine.

30. 30. The method of any one of claims 27 to 29, or the TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, cell population, or pharmaceutical composition for use of any one of claims 27 to 29, wherein the mutated human RAS amino acid sequence is the amino acid sequence of a mutated human Kirsten rat sarcoma viral oncogene homolog (KRAS).

31. 30. The method of any one of claims 27 to 29, or the TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, cell population, or pharmaceutical composition for use of any one of claims 27 to 29, wherein the mutated human RAS amino acid sequence is the amino acid sequence of a mutated human neuroblastoma rat sarcoma viral oncogene homolog (NRAS).

32. 30. The method of any one of claims 27 to 29, or the TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, cell population, or pharmaceutical composition for use of any one of claims 27 to 29, wherein the mutated human RAS amino acid sequence is a mutated human Harvey rat sarcoma viral oncogene homolog (HRAS) amino acid sequence.

33. The method of any one of claims 25 to 32 or the TCR, polypeptide, protein, nucleic acid, recombinant expression vector for use of any one of claims 26 to 32, wherein the cancer is pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer, or prostate cancer. A host cell, a cell population, or a pharmaceutical composition.

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