Anti-KRAS-g12d t cell receptors

Abstract

To provide methods of detecting the presence of cancer in a mammal, and pharmaceutical compositions for treating or preventing cancer in a mammal.SOLUTION: A pharmaceutical composition for inducing an immune response against cancer in a mammal comprising a KRAS G12D-specific TCR is provided.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to Related Applications This patent application claims the benefit of U.S. Provisional Patent Application No. 62 / 369,883, filed on August 2, 2016, which is hereby incorporated by reference in its entirety.

[0002] Incorporation by Reference of Electronically Submitted Materials A computer - readable nucleotide / amino acid sequence listing, submitted simultaneously with this specification and identified as follows, is hereby incorporated by reference in its entirety: one 60,828 - byte ASCII (text ) file named "728242_ST25.txt" dated July 26, 2017.

Background Art

[0003] Background of the Invention For some cancers, especially when the cancer is metastatic and inoperable, treatment options can be extremely limited. For example, despite advances in treatments such as surgery, chemotherapy, and radiation therapy, the prognosis for many cancers, such as pancreatic, colorectal, lung, endometrial, ovarian, and prostate cancers, can be poor. Thus, there are unmet needs for additional treatments for cancer.

Summary of the Invention

[0004] Summary of the Invention Embodiments of the present invention provide an isolated or purified TCR comprising the amino acid sequence of (a) SEQ ID NOs: 9 - 14; (b) SEQ ID NOs: 17 - 22; (c) SEQ ID NOs: 25 - 30; or (d) SEQ ID NOs: 33 - 38.

[0005] Another embodiment of the present invention provides (a) SEQ ID NOs: 9 - 14; (b) SEQ ID NOs: 17 - 22; (c) ​(d) an isolated or purified amino acid sequence comprising the amino acid sequence of SEQ ID NO: 33 to 38; Polypeptides are provided.

[0006] According to another embodiment of the present invention, a first antibody having the amino acid sequence of SEQ ID NO: 9 to 11 is provided. and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 12 to 14; (b) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 17 to 19 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 20 to 22; (c) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 25 to 27 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 28 to 30; or (d) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 33 to 35 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 36 to 38.

[0007] The invention further provides related nucleic acids, recombinant expression vectors, host cells, cell populations, and pharmaceutical compositions related to the TCRs, polypeptides and proteins of the invention.

[0008] The invention further provides methods for detecting the presence of cancer in a mammal, and methods for treating or preventing cancer in a mammal. [Brief description of the drawings]

[0009] A brief description of some views of the drawing(s)

Figure 1

Figure 2

[0010] [Chemical formula]

[0011] And ND was not detected (<0.0002%). A (TRAV4 / TRBV5-6(A)). B (TRAV12-2 / TRBV10-2). C (TRAV4 / TRBV5-6(B)). D (TRAV4 / TRBV5-6 (C)).

Figure 3

Figure 4

Figure 5

Mode for Carrying Out the Invention

[0012] Detailed Description of the Invention The GTPase KRas, also referred to as V-Ki-Ras2 Kirsten rat sarcoma viral oncogene or KRAS2 is a member of the low molecular weight (small) GTPase superfamily. There are two transcript variants of KRAS (small) GTPase superfamily. There are two transcript variants of KRAS Variants: There are KRAS variant A and KRAS variant B. Hereinafter, unless otherwise specified, references to "KRAS" (mutated or unmutated) refer to both variant A and variant B. Without being bound by a particular theory or mechanism, when mutated, KRAS is thought to be involved in signal transduction in the early stages of carcinogenesis of many human cancers. A single amino acid substitution can activate the protein. When activated, mutant KRAS binds to guanosine-5'-triphosphate (GTP) and converts GTP to guanosine 5'-diphosphate (GDP). The mutant KRAS protein product can be constitutively activated and can be expressed in various human cancers, such as, for example, the pancreas (e.g., pancreatic cancer) , colorectal, lung (e.g., lung adenocarcinoma), endometrium, ovary (e.g., epithelial ovarian cancer) and prostate cancer, etc and can be expressed in any of them.

[0013] One embodiment of the present invention provides an isolated or purified TCR that has antigen specificity for human mutant KRAS (hereinafter, "mutant KRAS"). Hereinafter, unless otherwise specified, references to "TCR" also refer to functional parts and functional variants of the TCR. The TCR of the present invention can have antigen specificity for any KRAS (protein, polypeptide or peptide) having a G12D mutation. In one embodiment of the present invention, the TCR has antigen specificity for a KRAS protein having a G12D mutation, which comprises or consists of the amino acid sequence of SEQ ID NO: 3 or 4. The amino acid sequence of the mutant KRAS variant A protein of SEQ ID NO: 3 is generally the same as that of the wild-type (WT) KRAS protein variant A of SEQ ID NO: 1 at positions 1 to 189, except that glycine at position 12 is substituted with aspartic acid in SEQ ID NO: 3

[0014] and is not mutated at positions 1 to 189 of the amino acid sequence of the wild-type (WT) KRAS protein variant A of SEQ ID NO: 1 except that glycine at position 12 is substituted with aspartic acid in SEQ ID NO: 3 corresponds. The amino acid sequence of the mutant KRAS variant B protein of SEQ ID NO: 4 is generally mutated except that glycine at position 12 is substituted with aspartic acid in SEQ ID NO: 4 and is not, corresponding to positions 1 to 188 of the amino acid sequence of the WT KRAS protein variant B of SEQ ID NO: 2.

[0015] In one embodiment of the present invention, the TCR has antigen specificity for a KRAS peptide having the above G12D mutation, and the KRAS peptide has an arbitrary length. For example, the TCR may have antigen specificity for a KRAS peptide having the G12D mutation, and the KRAS peptide has a length of about 8 to about 24 amino acid residues, preferably about 9 to about 11 amino acid residues. In one embodiment of the present invention the TCR may have antigen specificity for a KRAS peptide having the G12D mutation, and the KRAS peptide has a length of about 8 amino acid residues, about 9 amino acid residues, about 10 amino acid residues, about 11 amino acid residues, about 12 amino acid residues or about 24 amino acid residues. For example, the TCR is a peptide comprising or consisting of the amino acid sequence of GADGVGKSA (SEQ ID NO: 8) and may have antigen specificity for a KRAS peptide having the G12D mutation. The amino acid sequence of the mutant KRAS peptide of SEQ ID NO: 8 having the G12D mutation is generally not mutated except that glycine at position 3 is substituted with aspartic acid in SEQ ID NO: 8, and corresponds to positions 1 to 9 of the amino acid sequence of the WT KRAS peptide of SEQ ID NO: 7. In yet another embodiment of the present invention, the TCR may have antigen specificity for a KRAS peptide having the G12D mutation, and the mutant KRAS peptide comprises or consists of the amino acid sequence of GADGVGKSA (mutant KRAS 10-18 ; SEQ ID NO: 8); or GADGVGKSAL (mutant KRAS 10-18 ; SEQ ID NO: 6). corresponds to positions 1 to 9 of the amino acid sequence of the peptide.

[0016] In yet another embodiment of the present invention, the TCR may have antigen specificity for a KRAS peptide having the G12D mutation, and the mutant KRAS peptide comprises or consists of the amino acid sequence of GADGVGKSA (mutant KRAS 10-18 ; SEQ ID NO: 8); or GADGVGKSAL (mutant KRAS 10-19 ; SEQ ID NO: 6). . In an exemplary embodiment, the TCR has antigen specificity for a mutant KRAS epitope, and the mutant KRAS epitope is GADGVGKSA (mutant KRAS 10-18 ; SEQ ID NO: 8) or GADGVGKSAL (mutant KRAS 10-19 ; SEQ ID NO: 6), comprising or consisting of the amino acid sequence thereof.

[0017] In one embodiment of the present invention, the TCR of the present invention can recognize mutant KRAS in the context of an HLA-Cw8 molecule . In this regard, it means that when the TCR binds to mutant KRAS in the context of an HLA-Cw8 molecule, it can induce an immune response. The TCR of the present invention can recognize mutant KRAS presented by an HLA-Cw8 molecule and can bind to the HLA-Cw8 molecule in addition to mutant KRAS. In connection, exemplary HLA-Cw8 molecules by which the TCR of the present invention recognizes mutant KRAS include those encoded by the HLA-Cw*0801, HLA-Cw*0802, HLA-Cw*0803, HLA-Cw*0804, HLA-Cw*0805, HLA-Cw*0806, HLA-Cw*0807, HLA-Cw*0808 and HLA-Cw*0809 alleles. In a preferred embodiment, the TCR recognizes mutant KRAS in the context of an HLA-Cw*0802 molecule.

[0018] In one embodiment of the present invention, in addition to having the ability to recognize mutant KRAS within the context of an HLA-Cw8 molecule, one of the TCRs of the present invention (TRAV12-2 / TRBV10-2 (Table 5)) can also recognize mutant KRAS within the context of an HLA-Cw5 molecule. In this regard, the TCR can induce an immune response when binding to mutant KRAS within the context of an HLA-Cw5 molecule. The TCR of the present invention can recognize mutant KRAS presented by an HLA-Cw5 molecule and, in addition to mutant KRAS, can also bind to the HLA-Cw5 molecule . can bind. In the context of the TCR of the present invention recognizing mutant KRAS, exemplary HLA-Cw5 molecules include molecules encoded by the HLA-Cw*0501, HLA-Cw*0502, HLA-Cw*0503, HLA-Cw*0504, HLA-Cw*0505, HLA-Cw*0506, HLA-HLA-Cw*0508, HLA-Cw*0509 and HLA-Cw*0510 alleles. In a preferred embodiment, the TCR recognizes mutant KRAS in the context of the HLA-Cw*0501 molecule. The amino acid sequences of HLA-Cw*0802 and HLA-Cw*0501 differ from each other by only two amino acid residues. Without being bound by a particular theory or mechanism, it is believed that the TRAV12-2 / TRBV10-2 TCR may also recognize mutant KRAS presented by other HLA molecules that are similar to one or both of HLA-Cw*0802 and HLA-Cw*0501. differ from each other. Without being bound by a particular theory or mechanism, it is believed that the TRAV12-2 / TRBV10-2 TCR may also recognize mutant KRAS presented by other HLA molecules that are similar to one or both of HLA-Cw*0802 and HLA-Cw*0501.

[0019] The TCRs of the present invention provide many advantages, including when expressed by cells used for adoptive cell transfer. Mutant KRAS is expressed by cancer cells and not by healthy non-cancer cells. Without being bound by a particular theory or mechanism, the TCRs of the present invention are thought to minimize or eliminate the destruction of healthy non-cancer cells, thereby reducing toxicity, for example, by minimizing or eliminating toxicity, while advantageously targeting the destruction of cancer cells. Furthermore, the TCRs of the present invention can advantageously and successfully treat or prevent mutant KRAS-positive cancers that do not respond to other types of treatments, such as, for example, chemotherapy, surgery or radiation therapy. Additionally, the TCRs of the present invention provide a highly avid recognition of mutant KRAS, which is the ability to recognize unmanipulated tumor cells ( e.g., tumor cells not treated with interferon (IFN)γ, not transfected with a vector encoding one or both of mutant KRAS and HLA-Cw*0802, not pulsed with a KRAS peptide having a G12D mutation, or combinations thereof). e.g., tumor cells not treated with interferon (IFN)γ, not transfected with a vector encoding one or both of mutant KRAS and HLA-Cw*0802, not pulsed with a KRAS peptide having a G12D mutation, or combinations thereof). Without being bound by a particular theory or mechanism, the TCRs of the present invention are thought to minimize or eliminate the destruction of healthy non-cancer cells, thereby reducing toxicity, for example, by minimizing or eliminating toxicity, while advantageously targeting the destruction of cancer cells. Furthermore, the TCRs of the present invention can advantageously and successfully treat or prevent mutant KRAS-positive cancers that do not respond to other types of treatments, such as, for example, chemotherapy, surgery or radiation therapy. Additionally, the TCRs of the present invention provide a highly avid recognition of mutant KRAS, which is the ability to recognize unmanipulated tumor cells ( e.g., tumor cells not treated with interferon (IFN)γ, not transfected with a vector encoding one or both of mutant KRAS and HLA-Cw*0802, not pulsed with a KRAS peptide having a G12D mutation, or combinations thereof). can be provided. Furthermore, the HLA-Cw*0802 allele is expressed at up to approximately 8% and up to approximately 11% in each of the Caucasian and African American ethnic groups. Therefore, the TCRs of the present invention can increase the number of cancer patients eligible for immunotherapy, including patients expressing the HLA-Cw*0802 allele, which may not be suitable for immunotherapy using TCRs that recognize antigens in the context of other MHC molecules. .

[0020] As used herein, the expression "antigen specificity" means that the TCR can specifically bind to and immunologically recognize mutant KRAS with high avidity. For example, (a) pulsed with low concentrations of mutant KRAS 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 a range defined by any two of said values) antigen-negative HLA-Cw*0802 + target cells, or (b) antigen-negative HLA-Cw*0802 target cells into which a nucleotide sequence encoding mutant KRAS has been introduced such that the target cells express mutant KRAS, + when co-cultured with, about 1×10 4 to about 1×10 5 T cells expressing the TCR secrete 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 said values) of IFN-γ , the TCR can be considered to have "antigen specificity" for mutant KRAS. Cells expressing the TCRs of the present invention can also be antigen-negative HLA-Cw*0802 pulsed with higher concentrations of mutant KRAS peptide. + ​​​When co-cultured with target cells, it can secrete IFN-γ.

[0021] Alternatively or additionally, (a) antigen-negative HLA-Cw*0802 pulsed with a low concentration of mutant KRAS peptide + target cells, or (b) antigen-negative HLA-Cw*0802 into which a nucleotide sequence encoding mutant KRAS has been introduced so that the target cells express mutant KRAS + target cells, when co-cultured with, TCR If the T cells expressing TCR secrete at least twice as much IFN-γ as the amount of IFN-γ expressed by the negative control, the TCR can be considered to have "antigen specificity" for mutant KRAS. Negative controls are, for example, (i) (a) antigen-negative HLA-Cw*0802 pulsed with the same concentration of irrelevant peptides (e.g., several other peptides having sequences different from the mutant KRAS peptide) + target cells, or alternatively (b) antigen-negative HLA-Cw*0802 into which a nucleotide sequence encoding an irrelevant peptide has been introduced so that the target cells express the irrelevant peptide target cells, co-cultured with T cells expressing TCR, or (ii) (a) antigen-negative HLA-Cw*0802 pulsed with the same concentration of mutant KRAS peptide + target cells, or (b) antigen-negative HLA-Cw*0802 into which a nucleotide sequence encoding mutant KRAS has been introduced so that the target cells express mutant KRAS target cells, co-cultured with untransduced T cells (e.g., derived from PBMCs that do not express TCR). IFN-γ secretion can be + measured by methods known in the art such as, for example, enzyme-linked immunosorbent assay (ELISA). target cells, co-cultured with. IFN-γ secretion can be + measured by methods known in the art such as, for example, enzyme-linked immunosorbent assay (ELISA). For example, it can be measured by methods known in the art such as, for example, enzyme-linked immunosorbent assay (ELISA).

[0022] Alternatively or additionally, (a) antigen-negative HLA-Cw*0802 pulsed with a low concentration of mutant KRAS peptide +either (a) the target cells, or (b) antigen-negative HLA-Cw*0802 into which a nucleotide sequence encoding mutant KRAS has been introduced such that the target cells express mutant KRAS + When co-cultured with the target cells, the number of T cells expressing a TCR that secretes IFN-γ is less than the number of negative control T cells that secrete IFN-γ If it is at least 2-fold, the TCR can be considered to have "antigen specificity" for mutant KRAS. The peptide concentration and negative control can be those described in the specification with respect to other aspects of the present invention. The number of cells secreting IFN-γ can be measured, for example, by methods known in the art such as, for example, ELISPOT as known methods in the art.

[0023] Alternatively or additionally, if the T cells expressing the TCR upregulate the expression of one or more T cell activation markers, measured by flow cytometry, for example, after stimulation with target cells expressing mutant KRAS, the TCR can be considered to have "antigen specificity" for mutant KRAS . Examples of T cell activation markers include 4-1BB, OX40, CD107a, CD69, and cytokines upregulated by antigen stimulation (e.g., tumor necrosis factor (TNF), interleukin (IL)-2, etc ).

[0024] The present invention provides a TCR comprising two polypeptides (i.e., polypeptide chains), such as, for example, the alpha (α) chain of the TCR, the beta (β) chain of the TCR, the gamma (γ) chain of the TCR, the delta (δ) chain of the TCR, or a combination thereof. The polypeptides of the TCR of the present invention can comprise any amino acid sequence, provided that the TCR has antigen specificity for mutant KRAS .

[0025] In one embodiment of the present invention, the TCR comprises two polypeptide chains, each comprising a variable region comprising complementarity-determining regions (CDRs) 1, 2, and 3 of the TCR. In one embodiment of the present invention , The TCR includes a first polypeptide chain containing a CDR1 (CDR1 of the α chain) with the amino acid sequence of SEQ ID NO: 9, a CDR2 (CDR2 of the α chain) with the amino acid sequence of SEQ ID NO: 10, and a CDR3 (CDR3 of the α chain) with the amino acid sequence of SEQ ID NO: 11, and a CDR1 (CDR1 of the β chain) with the amino acid sequence of SEQ ID NO: 12 , a CDR2 (CDR2 of the β chain) with the amino acid sequence of SEQ ID NO: 13, and a second polypeptide chain containing a CDR3 (CDR3 of the β chain) with the amino acid sequence of SEQ ID NO: 14.

[0026] In another embodiment of the present invention, the TCR includes a first polypeptide chain containing a CDR1 (CDR1 of the α chain) with the amino acid sequence of SEQ ID NO: 17, a CDR2 (CDR2 of the α chain) with the amino acid sequence of SEQ ID NO: 18, and a CDR3 (CDR3 of the α chain) with the amino acid sequence of SEQ ID NO: 19, and a CDR1 (CDR1 of the β chain) with the amino acid sequence of SEQ ID NO: 20 , a CDR2 (CDR2 of the β chain) with the amino acid sequence of SEQ ID NO: 21, and a second polypeptide chain containing a CDR3 (CDR3 of the β chain) with the amino acid sequence of SEQ ID NO: 22.

[0027] In another embodiment of the present invention, the TCR includes a first polypeptide chain containing a CDR1 (CDR1 of the α chain) with the amino acid sequence of SEQ ID NO: 25, a CDR2 (CDR2 of the α chain) with the amino acid sequence of SEQ ID NO: 26, and a CDR3 (CDR3 of the α chain) with the amino acid sequence of SEQ ID NO: 27, and a CDR1 (CDR1 of the β chain) with the amino acid sequence of SEQ ID NO: 28 , a CDR2 (CDR2 of the β chain) with the amino acid sequence of SEQ ID NO: 29, and a second polypeptide chain containing a CDR3 (CDR3 of the β chain) with the amino acid sequence of SEQ ID NO: 30.

[0028] ​​​​​​​In another embodiment of the present invention, the TCR comprises a CDR1 (CDR1 of the α-chain) comprising the amino acid sequence of SEQ ID NO: 33, a CDR2 (CDR2 of the α-chain) comprising the amino acid sequence of SEQ ID NO: 34, and a CDR3 (CDR3 of the α-chain) comprising the amino acid sequence of SEQ ID NO: 35, and a first polypeptide chain, and a CDR1 (CDR1 of the β-chain) comprising the amino acid sequence of SEQ ID NO: 36, a CDR2 (CDR2 of the β-chain) comprising the amino acid sequence of SEQ ID NO: 37, and a CDR3 (CDR3 of the β-chain) comprising the amino acid sequence of SEQ ID NO: 38, and a second polypeptide chain. 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: 9-14, 17-22, 25-30 and 33-38. In one embodiment of the present invention the TCR comprises the following: (i) SEQ ID NOs: 9-11; (ii) SEQ ID NOs: 12-14; (iii) SEQ ID NOs: 17-19; (iv) SEQ ID NOs: 20-22; (v) SEQ ID NOs: 25-27; (vi) SEQ ID NOs: 28-30; (vii) SEQ ID NOs: 33-35; or (viii) SEQ ID NOs: 36-38. In a particularly preferred embodiment the TCR comprises the following: (a) all of SEQ ID NOs: 9-14; (b) all of SEQ ID NOs: 17-22; (c) all of SEQ ID NOs: 25-30; or (d) all of SEQ ID NOs: 33-38.

[0029] In one embodiment of the present invention, the TCR comprises the amino acid sequence of the variable region of the TCR comprising the above CDRs. In this regard, the TCR comprises the following: SEQ ID NO: 15 (variable region of the α-chain); SEQ ID NO: 23 (variable region of the α-chain); SEQ ID NO: 31 (variable region of the α-chain); SEQ ID NO: 39 (variable region of the α-chain); SEQ ID NO: 16 (variable region of the β-chain); SEQ ID NO: 24 (variable region of the β-chain); SEQ ID NO: 32 (variable region of the β-chain) ; SEQ ID NO: 40 (variable region of the β-chain); both SEQ ID NO: 15 and 16; both SEQ ID NO: 23 and 24;

[0030] It may contain the amino acid sequences of both SEQ ID NOs: 31 and 32; or both SEQ ID NOs: 39 and 40. Preferably, the TCR of the present invention contains (i) both of SEQ ID NOs: 15 to 16; (ii) both of SEQ ID NOs: 23 to 24; (iii) both of SEQ ID NOs: 31 to 32; or (iv) both of SEQ ID NOs: 39 to 40.

[0031] The TCR of the present invention may further contain an α-chain constant region and a β-chain constant region. The constant region may be derived from any suitable species such as, for example , for example, a human or a mouse. In one embodiment of the present invention, the TCR further contains a mouse α-chain and β-chain constant region, or a human α-chain and β-chain constant region . As used herein, the terms "mouse" or "human" refer to a TCR (or any component thereof) derived from a mouse or a human, respectively, i.e., a TCR (or its component) originating from or once expressed in a mouse T cell or a human T cell when referring to the TCR or any component of the TCR described herein (e.g., complementarity-determining region (CDR), variable region, constant region , α-chain and / or β-chain).

[0032] In one embodiment of the present invention, the TCR further contains a human α-chain and β-chain constant region. In this regard, the TCR may contain the amino acid sequence of SEQ ID NO: 41 (constant region of the human α-chain), SEQ ID NO: 42 (constant region of the human β-chain), SEQ ID NO: 43 (constant region of the human β-chain), both of SEQ ID NOs: 41 and 42, or both of SEQ ID NOs: 41 and 43, where X at position 1 is any naturally occurring amino acid residue. α-chain), SEQ ID NO: 42 (constant region of the human β-chain), SEQ ID NO: 43 (constant region of the human β-chain), both of SEQ ID NOs: 41 and 42, or both of SEQ ID NOs: 41 and 43, where X at position 1 is any naturally occurring amino acid residue. In one embodiment of the present invention, the TCR contains any of the human constant regions described herein in combination with any of the CDR regions described herein. In this regard, the TCR is as follows: (a) all of SEQ ID NOs: 9 to 14, 41 and 42; (b) all of SEQ ID NOs: 17 to 22, 41 and 42; (c) as follows: (a) all of SEQ ID NOs: 9 to 14, 41 and 42; (b) all of SEQ ID NOs: 17 to 22, 41 and 42; (c) All of SEQ ID NOs: 25-30, 41 and 42; (d) All of SEQ ID NOs: 33-38, 41 and 42; (e) All of SEQ ID NOs: 9-14, 41 and 43; (f) All of SEQ ID NOs: 17-22, 41 and 43; (g) All of SEQ ID NOs: 25-30, 41 and 43; or (h) All of SEQ ID NOs: 33-38, 41 and 43 may be included. In one embodiment of the present invention, the TCR comprises any of the variable regions described herein in combination with any of the human constant regions described herein. In this regard, the TCR may comprise the following: (i) All of SEQ ID NOs: 15-16, 41 and 42; (ii) All of SEQ ID NOs: 23-24, 41 and 42; (iii) All of SEQ ID NOs: 31-32, 41 and 42; (iv) All of SEQ ID NOs: 39-42; (v) All of SEQ ID NOs: 15-16, 41 and 43; (vi) All of SEQ ID NOs: 23-24, 41 and 43; (vii) All of SEQ ID NOs: 31-32, 41 and 43; or (viii) All of SEQ ID NOs: 39-40, 41 and 43. All of SEQ ID NOs: 9-14, 41 and 43; (f) All of SEQ ID NOs: 17-22, 41 and 43; (g) All of SEQ ID NOs: 25-30, 41 and 43; or (h) All of SEQ ID NOs: 33-38, 41 and 43 may be included. In one embodiment of the present invention, the TCR comprises any of the variable regions described herein in combination with any of the human constant regions described herein. In this regard, the TCR may comprise the following: (i) All of SEQ ID NOs: 15-16, 41 and 42; (ii) All of SEQ ID NOs: 23-24, 41 and 42; (iii) All of SEQ ID NOs: 31-32, 41 and 42; (iv) All of SEQ ID NOs: 39-42; (v) All of SEQ ID NOs: 15-16, 41 and 43; (vi) All of SEQ ID NOs: 23-24, 41 and 43; (vii) All of SEQ ID NOs: 31-32, 41 and 43; or (viii) All of SEQ ID NOs: 39-40, 41 and 43. In one embodiment of the present invention, the TCR comprises any of the variable regions described herein in combination with any of the human constant regions described herein. In this regard, the TCR may comprise the following: (i) All of SEQ ID NOs: 15-16, 41 and 42; (ii) All of SEQ ID NOs: 23-24, 41 and 42; (iii) All of SEQ ID NOs: 31-32, 41 and 42; (iv) All of SEQ ID NOs: 39-42; (v) All of SEQ ID NOs: 15-16, 41 and 43; (vi) All of SEQ ID NOs: 23-24, 41 and 43; (vii) All of SEQ ID NOs: 31-32, 41 and 43; or (viii) All of SEQ ID NOs: 39-40, 41 and 43. In this regard, the TCR may comprise the following: (i) All of SEQ ID NOs: 15-16, 41 and 42; (ii) All of SEQ ID NOs: 23-24, 41 and 42; (iii) All of SEQ ID NOs: 31-32, 41 and 42; (iv) All of SEQ ID NOs: 39-42; (v) All of SEQ ID NOs: 15-16, 41 and 43; (vi) All of SEQ ID NOs: 23-24, 41 and 43; (vii) All of SEQ ID NOs: 31-32, 41 and 43; or (viii) All of SEQ ID NOs: 39-40, 41 and 43. In this regard, the TCR may comprise the following: (i) All of SEQ ID NOs: 15-16, 41 and 42; (ii) All of SEQ ID NOs: 23-24, 41 and 42; (iii) All of SEQ ID NOs: 31-32, 41 and 42; (iv) All of SEQ ID NOs: 39-42; (v) All of SEQ ID NOs: 15-16, 41 and 43; (vi) All of SEQ ID NOs: 23-24, 41 and 43; (vii) All of SEQ ID NOs: 31-32, 41 and 43; or (viii) All of SEQ ID NOs: 39-40, 41 and 43. In this regard, the TCR may comprise the following: (i) All of SEQ ID NOs: 15-16, 41 and 42; (ii) All of SEQ ID NOs: 23-24, 41 and 42; (iii) All of SEQ ID NOs: 31-32, 41 and 42; (iv) All of SEQ ID NOs: 39-42; (v) All of SEQ ID NOs: 15-16, 41 and 43; (vi) All of SEQ ID NOs: 23-24, 41 and 43; (vii) All of SEQ ID NOs: 31-32, 41 and 43; or (viii) All of SEQ ID NOs: 39-40, 41 and 43. In this regard, the TCR may comprise the following: (i) All of SEQ ID NOs: 15-16, 41 and 42; (ii) All of SEQ ID NOs: 23-24, 41 and 42; (iii) All of SEQ ID NOs: 31-32, 41 and 42; (iv) All of SEQ ID NOs: 39-42; (v) All of SEQ ID NOs: 15-16, 41 and 43; (vi) All of SEQ ID NOs: 23-24, 41 and 43; (vii) All of SEQ ID NOs: 31-32, 41 and 43; or (viii) All of SEQ ID NOs: 39-40, 41 and 43. In this regard, the TCR may comprise the following: (i) All of SEQ ID NOs: 15-16, 41 and 42; (ii) All of SEQ ID NOs: 23-24, 41 and 42; (iii) All of SEQ ID NOs: 31-32, 41 and 42; (iv) All of SEQ ID NOs: 39-42; (v) All of SEQ ID NOs: 15-16, 41 and 43; (vi) All of SEQ ID NOs: 23-24, 41 and 43; (vii) All of SEQ ID NOs: 31-32, 41 and 43; or (viii) All of SEQ ID NOs: 39-40, 41 and 43. In this regard, the TCR may comprise the following: (i) All of SEQ ID NOs: 15-16, 41 and 42; (ii) All of SEQ ID NOs: 23-24, 41 and 42; (iii) All of SEQ ID NOs: 31-32, 41 and 42; (iv) All of SEQ ID NOs: 39-42; (v) All of SEQ ID NOs: 15-16, 41 and 43; (vi) All of SEQ ID NOs: 23-24, 41 and 43; (vii) All of SEQ ID NOs: 31-32, 41 and 43; or (viii) All of SEQ ID NOs: 39-40, 41 and 43.

[0033] One embodiment of the present invention is a chimeric TCR comprising a human variable region and a mouse constant region, wherein the TCR has antigen specificity for mutant KRAS presented in the context of an HLA-Cw8 molecule. The mouse constant region may provide one or more optional benefits. For example, the mouse constant region may reduce mispairing between the endogenous TCR of the host cell into which the TCR of the present invention is introduced and the TCR of the present invention. Alternatively or additionally, the mouse constant region may increase the expression of the TCR of the present invention compared to the same TCR with a human constant region. The chimeric TCR may comprise the amino acid sequence of SEQ ID NO: 44 (wild-type (WT) mouse alpha chain constant region), SEQ ID NO: 45 (WT mouse beta chain constant region), or both SEQ ID NOs: 44 and 45. Preferably, the TCR of the present invention comprises SEQ ID NOs: 44 and 45. One embodiment of the present invention is a chimeric TCR comprising a human variable region and a mouse constant region, wherein the TCR has antigen specificity for mutant KRAS presented in the context of an HLA-Cw8 molecule. The mouse constant region may provide one or more optional benefits. For example, the mouse constant region may reduce mispairing between the endogenous TCR of the host cell into which the TCR of the present invention is introduced and the TCR of the present invention. Alternatively or additionally, the mouse constant region may increase the expression of the TCR of the present invention compared to the same TCR with a human constant region. The chimeric TCR may comprise the amino acid sequence of SEQ ID NO: 44 (wild-type (WT) mouse alpha chain constant region), SEQ ID NO: 45 (WT mouse beta chain constant region), or both SEQ ID NOs: 44 and 45. Preferably, the TCR of the present invention comprises SEQ ID NOs: 44 and 45. One embodiment of the present invention is a chimeric TCR comprising a human variable region and a mouse constant region, wherein the TCR has antigen specificity for mutant KRAS presented in the context of an HLA-Cw8 molecule. The mouse constant region may provide one or more optional benefits. For example, the mouse constant region may reduce mispairing between the endogenous TCR of the host cell into which the TCR of the present invention is introduced and the TCR of the present invention. Alternatively or additionally, the mouse constant region may increase the expression of the TCR of the present invention compared to the same TCR with a human constant region. The chimeric TCR may comprise the amino acid sequence of SEQ ID NO: 44 (wild-type (WT) mouse alpha chain constant region), SEQ ID NO: 45 (WT mouse beta chain constant region), or both SEQ ID NOs: 44 and 45. Preferably, the TCR of the present invention comprises SEQ ID NOs: 44 and 45. One embodiment of the present invention is a chimeric TCR comprising a human variable region and a mouse constant region, wherein the TCR has antigen specificity for mutant KRAS presented in the context of an HLA-Cw8 molecule. The mouse constant region may provide one or more optional benefits. For example, the mouse constant region may reduce mispairing between the endogenous TCR of the host cell into which the TCR of the present invention is introduced and the TCR of the present invention. Alternatively or additionally, the mouse constant region may increase the expression of the TCR of the present invention compared to the same TCR with a human constant region. The chimeric TCR may comprise the amino acid sequence of SEQ ID NO: 44 (wild-type (WT) mouse alpha chain constant region), SEQ ID NO: 45 (WT mouse beta chain constant region), or both SEQ ID NOs: 44 and 45. Preferably, the TCR of the present invention comprises SEQ ID NOs: 44 and 45. It includes the amino acid sequences of both. The chimeric TCR, with respect to other aspects of the present invention, can be combined with any of the CDR regions described herein and can include any of the mouse constant regions described herein. In this regard, the TCR can include the amino acid sequences of all of the following: (a) all of SEQ ID NOs: 9 - 14, 44, and 45; (b) all of SEQ ID NOs: 17 - 22, 44, and 45; (c) all of SEQ ID NOs: 25 - 30, 44, and 45; or (d) all of SEQ ID NOs: 33 - 38, 44, and 45. In another embodiment of the present invention, the chimeric TCR, with respect to other aspects of the present invention, can be combined with any of the variable regions described herein and can include any of the mouse constant regions described herein. In this regard, the TCR can include the amino acid sequences of the following: (i) SEQ ID NOs: 15 - 16, 44, and 45; (ii) SEQ ID NOs: 23 - 24, 44, and 45; (iii) SEQ ID NOs: 31 - 32, 44, and 45; or (iv) SEQ ID NOs: 39 - 40, 44, and 45. In one embodiment of the present invention, the TCR includes a substituted constant region. In this regard, the TCR can include the amino acid sequence of any of the TCRs described herein having 1, 2, 3, or 4 amino acid substitutions (s) in the constant region of one or both of the α and β chains. Preferably, the TCR includes a mouse constant region having 1, 2, 3, or 4 amino acid substitutions (s) in the mouse constant region of one or both of the α and β chains. In a particularly preferred embodiment, the TCR includes a mouse constant region having 1, 2, 3, or 4 amino acid substitutions (s) in the mouse constant region of the α chain and one amino acid substitution in the mouse constant region of the β chain. In some embodiments, the TCR including a substituted constant region, compared to the parental TCR including an unsubstituted (wild - type) constant region, has a mutant KRAS

[0034] (s). + ​​​​​​​​​​Advantageously provide one or more of increased recognition of the target, increased expression by host cells, abatement of mispairing with the endogenous TCR, and increased antitumor activity. Generally, the substituted amino acids of the mouse constant regions of the TCR α and β chains amino acid sequences (SEQ ID NOs: 46 and 47, respectively) correspond to all or part of the amino acid sequences of the unsubstituted mouse constant regions (SEQ ID NOs: 44 and 45, respectively), SEQ ID NO: 46 has one, two, three or four amino acid substitutions (multiple possible) compared to SEQ ID NO: 44, and SEQ ID NO: 47 has one amino acid substitution compared to SEQ ID NO: 45. In this regard, one embodiment of the present invention is (a) SEQ ID NO: 46 (constant region of the α chain), wherein (i) X at position 48 is Thr or Cys ; (ii) X at position 112 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 114 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 115 is Gly , Ala, Val, Leu, Ile, Pro, Phe, Met or Trp of SEQ ID NO: 46; and (b) SEQ ID NO: 47 (constant region of the β chain), wherein X at position 57 is Ser or Cys of SEQ ID NO: 47, and provides a TCR comprising the amino acid sequence. In one embodiment of the present invention, the TCR comprising SEQ ID NO: 46 does not include SEQ ID NO: 44 (unsubstituted mouse constant region of the α chain). In one embodiment of the present invention is that the TCR comprising SEQ ID NO: 47 does not include SEQ ID NO: 45 (unsubstituted mouse constant region of the β chain).

[0035] In one embodiment of the present invention, the substituted constant region includes cysteine substitutions in the constant region of one or both of the α and β chains, and provides a TCR substituted with cysteine. Opposing cysteines in the α and β chains are in the constant regions of the α and β chains of the substituted TCR . The TCR provides a disulfide bond linking the native Thr at position 48 of SEQ ID NO:44 to the TCR that is not present in the TCR that contains the unsubstituted mouse constant region. The TCR may be a cysteine-substituted TCR, in which one or both of native Ser at position 57 of SEQ ID NO: 45 (Thr48) and native Ser at position 57 of SEQ ID NO: 45 (Ser57) may be substituted with Cys. Preferably, both native Thr48 of SEQ ID NO: 44 and native Ser57 of SEQ ID NO: 45 are substituted with Cys. In one embodiment, the cysteine-substituted TCR comprises an alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 46, in which X at position 48 is Cys, X at position 112 is native Ser, and X at position 114 is native Met, and a region in which X at position 115 is a native Gly, and a β The cysteine-substituted TCR of the present invention comprises a chain constant region, wherein X at position 57 is Cys. In addition to any of the CDRs or variable regions described herein, a substituted constant region is It may include.

[0036] In one embodiment of the present invention, the amino acid sequence that is substituted includes the membrane domain of one or both of the constant regions of the α and β chains to provide a TCR that is substituted with a hydrophobic amino acid. The hydrophobic amino acid substitution(s) in the TM domain of a TCR may include substitution(s) of one, two or three amino acids with hydrophobic amino acids in the TM domain. The hydrophobicity of the TM domain of the TCR may be increased relative to a TCR lacking the hydrophobic amino acid substitution(s). In this regard, the TCR may be modified to include the native Ser112, Met114 and Gly115 of SEQ ID NO:44. one, two or three of which are independently Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; preferably It is a hydrophobic amino acid substitution TCR that can be substituted with Leu, Ile or Val. Preferably, all three of the native Ser112, Met114 and Gly115 of SEQ ID NO: 44 are independently Ala, Val, Leu, Ile , Pro, Phe, Met or Trp; preferably it can be substituted with Leu, Ile or Val. In one embodiment the hydrophobic amino acid substitution TCR is an α-chain constant region containing the amino acid sequence of SEQ ID NO: 46 wherein X at position 48 is the native Thr, X at position 112 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp, X at position 114 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp, and X at position 115 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp, and a β-chain constant region containing the amino acid sequence of SEQ ID NO: 47, wherein X at position 57 is the native Ser (the hydrophobic amino acid substitution TCR containing SEQ ID NO: 46 does not contain SEQ ID NO: 44 (the unsubstituted mouse constant region of the α-chain)). In a preferred embodiment, the hydrophobic amino acid substitution TCR is an α-chain constant region containing the amino acid sequence of SEQ ID NO: 46, wherein X at position 48 is the native Thr, X at position 112 is Leu, X at position 114 is Ile, and X at position 115 is Val, and a β-chain constant region containing the amino acid sequence of SEQ ID NO: 47, and also contains a region where X at position 57 is the native Ser. The hydrophobic amino acid substitution TCR of the present invention may include a substituted constant region in addition to any of the CDRs or variable regions described herein.

[0037] ​​In one embodiment of the present invention, the substituted amino acid sequence includes cysteine substitution in the constant region of one or both of the α and β chains in combination with substitution(s) of one, two, or three amino acids in the transmembrane (TM) domain of the constant region of one or both of the α and β chains with hydrophobic amino acids (also referred to herein as "cysteine substitution / hydrophobic amino acid substitution TCR"). In this regard, for the TCR, the native Thr48 of SEQ ID NO: 46 is substituted with Cys; one, two, or three of the native Ser112, Met114, and Gly115 of SEQ ID NO: 46 are independently substituted with Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; preferably Leu, Ile, or Val; and the native Ser57 of SEQ ID NO: 47 is substituted with Cys, which is a cysteine substitution / hydrophobic amino acid substitution TCR. Preferably, all three of the native Ser112, Met114, and Gly115 of SEQ ID NO: 46 can be independently substituted with Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; preferably Leu, Ile, or Val. In one embodiment, the cysteine substitution / hydrophobic amino acid substitution TCR includes an α chain comprising the amino acid sequence of SEQ ID NO: 46, wherein X at position 48 is Cys, X at position 112 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp, X at position 114 is Met, Ala, Val , Leu, Ile, Pro, Phe, or Trp, and X at position 115 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp, and a β chain comprising the amino acid sequence of SEQ ID NO: 47, wherein X at position 57 is Cys (SEQ ID NO: 46 does not include SEQ ID NO: 44 (unsubstituted α chain), and SEQ ID NO: 47 does not include SEQ ID NO: 45 (unsubstituted β chain)). Preferably, the cysteine substitution / hydrophobic amino acid substitution TCR includes an α chain comprising the amino acid sequence of SEQ ID NO: 46, wherein at position 48 where X at position 112 is Cys, X at position 114 is Ile, and X at position 115 is Val, and an α chain, and a β chain comprising the amino acid sequence of SEQ ID NO: 47, wherein X at position 57 is Cys. In this regard, the cysteine-substituted / hydrophobic amino acid-substituted TCR comprises the amino acid sequence of SEQ ID NO: 48 for the α-chain constant region and the amino acid sequence of SEQ ID NO: 49 for the β-chain constant region. The cysteine-substituted / hydrophobic amino acid-substituted TCR of the present invention may include a substituted constant region in addition to any of the CDRs or variable regions described herein.

[0038] In one embodiment of the present invention, the cysteine-substituted / hydrophobic amino acid-substituted TCR of the present invention may include the α-chain and the β-chain of the TCR. Each of the α-chain and β-chain of the TCR of the present invention may independently include any amino acid sequence. In this regard, the α-chain of the TCR of the present invention may include the amino acid sequence of SEQ ID NO: 50, 52 , 54 or 56. This type of α-chain may pair with any β-chain of the TCR . In this regard, the β-chain of the TCR of the present invention may include the amino acid sequence of SEQ ID NO: 51, 53, 55 or 57. Therefore, the TCR of the present invention may include the amino acid sequences of SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, both SEQ ID NO: 50 and 51, the sequence both SEQ ID NO: 52 and 53, both SEQ ID NO: 54 and 55, or both SEQ ID NO: 56 and 57. Preferably, the TCR of the present invention comprises (1) both SEQ ID NOs: 50-51; (2) the sequence both SEQ ID NOs: 52-53; (3) both SEQ ID NOs: 54-55; or (4) both SEQ ID NOs: 56-57 of the amino acid sequence.

[0039] Furthermore, according to the present invention, a polypeptide comprising any functional part of the TCR 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.

[0040] In relation to the polypeptides of the invention, a functional portion may be any portion comprising consecutive amino acids that are part of a TCR, provided that the functional portion specifically binds to mutant KRAS. When used in reference to a TCR, the term "functional portion" refers to any portion or flag of a TCR of the invention. "functional portion" refers to a portion or fragment of a TCR that retains the biological activity of the TCR of which it is a part (the parent TCR). A functional portion includes, for example, a portion of a TCR that retains the ability to specifically bind to mutant KRAS (e.g., within the context of an HLA-Cw*0802 molecule), or the ability to detect, treat, or prevent cancer to a similar, equal, or greater extent than the parent TCR. With reference to the parent TCR, a functional portion may, for example, retain about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95% of the parent TCR. It may include the above.

[0041] A functional portion is an amino acid sequence of a portion in which additional amino acids are not found in the amino acid sequence of the parent TCR. Additional amino acids may be included at the terminus or carboxy terminus, or both. Desirably, the additional amino acids are associated with the biological function of the functional moiety, e.g., the ability to specifically bind to mutant KRAS; and / or to detect, treat, or prevent cancer. More preferably, the additional amino acids do not interfere with the biological activity of the parent TCR. enhances the biological activity of

[0042] The polypeptide may be a functional portion of either or both of the α and β chains of the TCR of the invention, e.g. , may include a functional portion etc. including one or more of CDR1, CDR2, and CDR3 of the variable region(s) of the α-chain and / or β-chain of the TCR of the present invention. In one embodiment of the present invention, the polypeptide has SEQ ID NO: 9 (CDR1 of the α-chain), SEQ ID NO: 10 (CDR2 of the α-chain), SEQ ID NO: 11 (CDR3 of the α-chain), SEQ ID NO: 12 (CDR1 of the β-chain), SEQ ID NO: 13 (CDR2 of the β-chain), SEQ ID NO: 14 (CDR3 of the β-chain) or a combination thereof The amino acid sequence of may be included. In another embodiment of the present invention, the polypeptide may include the amino acid sequence of SEQ ID NO: 17 (CDR1 of the α-chain), SEQ ID NO: 18 (CDR2 of the α-chain), SEQ ID NO: 19 (CDR3 of the α-chain), SEQ ID NO: 20 (CDR1 of the β-chain), SEQ ID NO: 21 (CDR2 of the β-chain), SEQ ID NO: 22 (CDR3 of the β-chain), or a combination thereof. In another embodiment of the present invention, the polypeptide has SEQ ID NO: 25 (CDR1 of the α-chain), SEQ ID NO: 26 (CDR2 of the α-chain), SEQ ID NO: 27 (CDR3 of the α-chain), SEQ ID NO: 28 (CDR1 of the β-chain), SEQ ID NO: 29 (CDR2 of the β-chain), SEQ ID NO: 30 (CDR3 of the β-chain), or a combination thereof The amino acid sequence of may be included. In another embodiment of the present invention, the polypeptide may include the amino acid sequence of SEQ ID NO: 33 (CDR1 of the α-chain), SEQ ID NO: 34 (CDR2 of the α-chain), SEQ ID NO: 35 (CDR3 of the α-chain), SEQ NO: 36 (CDR1 of the β-chain), SEQ ID NO: 37 (CDR2 of the β-chain), SEQ ID NO: 38 (CDR3 of the β-chain), or a combination thereof Preferably, the polypeptide includes the amino acid sequences of both (a) SEQ ID NO: 9 to 14; (b) both SEQ ID NO: 17 to 22; (c) both SEQ ID NO: 25 to 30; or (d) both SEQ ID NO: 33 to 38.

[0043] In one embodiment of the present invention, the polypeptide of the present invention may include, for example, the variable region of the TCR of the present invention including the above-described combination of CDR regions. In this regard, the polypeptide has a sequence including the above-described combination of CDR regions. In this regard, the polypeptide has a sequence It may include the amino acid sequences of SEQ ID NO: 15 (variable region of the α chain), SEQ ID NO: 16 (variable region of the β chain), both SEQ ID NOs: 15 and 16, SEQ ID NO: 23 (variable region of the α chain), SEQ ID NO: 24 (variable region of the β chain), both SEQ ID NOs: 23 and 24, SEQ ID NO: 31 (variable region of the α chain), SEQ ID NO: 32 (variable region of the β chain), both SEQ ID NOs: 31 and 32, SEQ ID NO: 39 (variable region of the α chain), SEQ ID NO: 40 (variable region of the β chain), or both SEQ ID NOs: 39 and 40. Preferably, the polypeptide includes the amino acid sequences of (i) both SEQ ID NOs: 15 and 16, (ii) both SEQ ID NOs: 23 and 24, (iii) both SEQ ID NOs: 31 and 32, or (iv) both SEQ ID NOs: 39 and 40.

[0044] In one embodiment of the present invention, the polypeptide of the present invention may further include the constant region of the TCR of the present invention described above. In this regard, the polypeptide may include SEQ ID NO: 41 (human constant region of the α chain), SEQ ID NO: 42 (human constant region of the β chain), SEQ ID NO: 43 (human constant region of the β chain), SEQ ID NO: 44 (WT mouse constant region of the α chain), SEQ ID NO: 45 (WT mouse constant region of the β chain), SEQ ID NO: 46 ( substituted mouse constant region of the α chain), SEQ ID NO: 47 (substituted mouse constant region of the β chain), SEQ ID NO: 48 (mouse constant region with cysteine substitution and hydrophobic amino acid substitution of the α chain), SEQ ID NO: 49 (mouse constant region with cysteine substitution and hydrophobic amino acid substitution of the α chain), both SEQ ID NOs: 44 and 45, both SEQ ID NOs: 46 and 47, or both SEQ ID NOs: 48 and 49, both SEQ ID NOs: 41 and 42, or both SEQ ID NOs: 41 and 43. Preferably, with respect to other aspects of the present invention, the polypeptide may be in combination with any of the CDR regions or variable regions described herein, and further include the amino acid sequences of (i) both SEQ ID NOs: 44 and 45, (ii) both SEQ ID NOs: 46 and 47, (iii) both SEQ ID NOs: 48 and 49, (iv) both SEQ ID NOs: 41 and 42, or (v) both SEQ ID NOs: 41 and 43. ​

[0045] In one embodiment of the present invention, the polypeptide of the present invention may include the full length of the α or β chain of the TCR described herein. In this regard, the polypeptide of the present invention may include the amino acid sequence of SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 or SEQ ID NO: 57. Alternatively, the polypeptide of the present invention may include both chains of the TCR described herein. For example, the polypeptide of the present invention may include both of the amino acid sequences of SEQ ID NO: 50 and 51 , both of SEQ ID NO: 52 and 53, both of SEQ ID NO: 54 and 55, or both of SEQ ID NO: 56 and 57. Preferably, the polypeptide includes (1) both of SEQ ID NO: 50-51; (2) both of SEQ ID NO: 52-53; (3) both of SEQ ID NO: 54-55; or (4) both of the amino acid sequences of SEQ ID NO: 56-57.

[0046] The present invention further provides a protein comprising at least one polypeptide described herein. "Protein" means a molecule comprising one or more polypeptide chains.

[0047] In one embodiment, the protein of the present invention includes (a) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 9-11 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 12-14; (b) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 17-19 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 20-22; (c) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 25-27 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 28-30; or (d) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 33-35 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 36-38.

[0048] ​​​In another embodiment of the present invention, the protein comprises: (i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 15 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 16; (ii) 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; (iii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 31 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 32; or (iv) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 39 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 40. The protein of the present invention may further comprise any of the constant regions described herein with respect to other aspects of the present invention. In this regard, in embodiments of the present invention, the first polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 46, wherein: (i) X at position 48 of SEQ ID NO: 46 is Thr or Cys; (ii) X at position 112 of SEQ ID NO: 46 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 114 of SEQ ID NO: 46 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 115 of SEQ ID NO: 46 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; and (B) the second polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 47, wherein X at position 57 of SEQ ID NO: 47 is Ser or Cys. In another embodiment of the present invention, the first polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 41 (human α-chain constant region), SEQ ID NO: 44 (mouse WT constant region of the α-chain) or SEQ ID NO: 48 (mouse constant region with cysteine substitution and hydrophobic amino acid substitution of the α-chain), and the second polypeptide chain may comprise the amino acid sequence of SEQ ID NO: 42 (human constant region of the β-chain), SEQ ID NO: 43 (human constant region of the β-chain), SEQ ID NO: 45 (mouse

[0049] constant region of the β-chain). In another embodiment of the present invention, the first polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 46, wherein: (i) X at position 48 of SEQ ID NO: 46 is Thr or Cys; (ii) X at position 112 of SEQ ID NO: 46 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 114 of SEQ ID NO: 46 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and (iv) X at position 115 of SEQ ID NO: 46 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; and (B) the second polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 47, wherein X at position 57 of SEQ ID NO: 47 is Ser or Cys. In another embodiment of the present invention, the first polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 41 (human α-chain constant region), SEQ ID NO: 44 (mouse WT constant region of the α-chain) or SEQ ID NO: 48 (mouse constant region with cysteine substitution and hydrophobic amino acid substitution of the α-chain), and the second polypeptide chain may comprise the amino acid sequence of SEQ ID NO: 42 (human constant region of the β-chain), SEQ ID NO: 43 (human constant region of the β-chain), SEQ ID NO: 45 (mouse constant region of the β-chain). In another embodiment of the present invention, the first polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 41 (human α-chain constant region), SEQ ID NO: 44 (mouse WT constant region of the α-chain) or SEQ ID NO: 48 (mouse constant region with cysteine substitution and hydrophobic amino acid substitution of the α-chain), and the second polypeptide chain may comprise the amino acid sequence of SEQ ID NO: 42 (human constant region of the β-chain), SEQ ID NO: 43 (human constant region of the β-chain), SEQ ID NO: 45 (mouse constant region of the β-chain). In another embodiment of the present invention, the first polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 41 (human α-chain constant region), SEQ ID NO: 44 (mouse WT constant region of the α-chain) or SEQ ID NO: 48 (mouse constant region with cysteine substitution and hydrophobic amino acid substitution of the α-chain), and the second polypeptide chain may comprise the amino acid sequence of SEQ ID NO: 42 (human constant region of the β-chain), SEQ ID NO: 43 (human constant region of the β-chain), SEQ ID NO: 45 (mouse the constant region of SWT) or SEQ ID NO: 49 (cystein substitution in the β-chain and hydrophobic amino acid substitution and may further include the amino acid sequence of the mouse constant region).

[0050] Alternatively or additionally, the protein of the present invention may include: (1) a first polypeptide chain including the amino acid sequence of SEQ ID NO: 50 and a second polypeptide chain including the amino acid sequence of SEQ ID NO: 51; (2) a first polypeptide chain including the amino acid sequence of SEQ ID NO: 52 and a second polypeptide chain including the amino acid sequence of SEQ ID NO: 53; (3) a first polypeptide chain including the amino acid sequence of SEQ ID NO: 54 and a second polypeptide chain including the amino acid sequence of SEQ ID NO: 55; or (4) a first polypeptide chain including the amino acid sequence of SEQ ID NO: 56 and a second polypeptide chain including the amino acid sequence of SEQ ID NO: 57. In this example, the protein of the present invention may be a TCR. Alternatively for example, when the protein includes a single polypeptide chain including the amino acid sequences of both SEQ ID NOs: 50 and 51, both SEQ ID NOs: 52 and 53, both SEQ ID NOs: 54 and 55, or both SEQ ID NOs: 55 and 56, or when the first and / or second polypeptide chain(s) of the protein further include amino acid sequences encoding other amino acid sequences, such as immunoglobulins or portions thereof the protein of the present invention may be a fusion protein. In this regard, the present invention also provides a fusion protein including at least one of the polypeptides of the present invention described herein together with at least one other polypeptide. The other polypeptide may be present as another polypeptide of the fusion protein, or may be present as a polypeptide expressed in frame (tandem) with one of the polypeptides of the present invention described herein. The other polypeptide may encode any peptidic or proteinaceous molecule, including but not limited to immunoglobulins, CD3, CD4, CD8, MHC molecules, CD1 molecules, such as CD1a, CD1b, CD1c, CD1d, etc., or portions thereof.

[0051] The fusion protein may contain one or more copies of the polypeptide of the present invention and / or one or more copies of other polypeptides. For example, the fusion protein may contain 1, 2, 3, 4, 5 or more copies of the polypeptide of the present invention and / or other polypeptides. Suitable methods for producing the fusion protein are known in the art and include, for example, recombinant methods. In some embodiments of the present invention, the TCR, polypeptide and protein of the present invention can be expressed as a single protein containing a linker peptide that links the α and β chains. In this regard, the TCR, polypeptide and protein of the present invention may further contain a linker peptide. The linker peptide can advantageously promote the expression of the recombinant TCR, polypeptide and / or protein in the host cell. The linker peptide can contain any suitable amino acid sequence. For example, the linker peptide can contain SEQ ID NO: 58. When the construct containing the linker peptide is expressed by the host cell, the linker peptide can be cleaved, resulting in separate α and β chains. In one embodiment of the present invention, the TCR, polypeptide or protein can contain an amino acid sequence comprising a full-length α chain, a full-length β chain, and a linker peptide located between the α and β chains. / or 1, 2, 3, 4, 5 or more copies of other polypeptides. Suitable methods for making the fusion protein are known in the art and include, for example, recombinant methods. Suitable methods for producing the fusion protein are known in the art and include, for example, recombinant methods.

[0052] In some embodiments of the present invention, the TCR, polypeptide and protein of the present invention , can be expressed as a single protein containing a linker peptide that links the α and β chains. In this regard, the TCR, polypeptide and protein of the present invention may further contain a linker peptide. The linker peptide can advantageously promote the expression of the recombinant TCR, polypeptide and / or protein in the host cell. The linker peptide can contain any suitable amino acid sequence. For example, the linker peptide can contain SEQ ID NO: 58. When the construct containing the linker peptide is expressed by the host cell, the linker peptide can be cleaved, resulting in separate α and β chains. In one embodiment of the present invention, the TCR, polypeptide or protein may further contain a linker peptide. The linker peptide can advantageously promote the expression of the recombinant TCR, polypeptide and / or protein in the host cell. The linker peptide can contain any suitable amino acid sequence. For example, the linker peptide can contain SEQ ID NO: 58. When the construct containing the linker peptide is expressed by the host cell, the linker peptide can be cleaved, resulting in separate α and β chains. In one embodiment of the present invention, the TCR, polypeptide or protein protein can contain an amino acid sequence comprising a full-length α chain, a full-length β chain, and a linker peptide located between the α and β chains. protein may contain an amino acid sequence comprising a full-length α chain, a full-length β chain, and a linker peptide located between the α and β chains.

[0053] The protein of the present invention can be a recombinant antibody or an antigen-binding portion thereof containing at least one of the polypeptides of the present invention described herein. As used herein, "recombinant antibody" refers to a recombinant (e.g., genetically engineered) protein containing at least one of the polypeptides of the present invention, a polypeptide chain of an antibody, or an antigen-binding portion thereof. The polypeptide of the antibody chain or an antigen-binding portion thereof. The peptide or its antigen-binding portion can be the heavy chain, light chain, variable region or constant region of the heavy chain or light chain of an antibody, a single-chain variable fragment (scFv), or an Fc, Fab or F(ab')2 fragment, etc. The polypeptide chain of the antibody or its antigen-binding portion can exist as another polypeptide of a recombinant antibody. Alternatively, the polypeptide chain of the antibody or its antigen-binding portion can exist as a polypeptide expressed in-frame (in tandem) with the polypeptide of the present invention. The polypeptide of the antibody or its antigen-binding portion can be the polypeptide of any antibody or any antibody fragment, including any of the antibodies and antibody fragments described herein.

[0054] Within the scope of the present invention are functional variants of the TCR, polypeptide or protein of the present invention described herein. As used herein, the term "functional variant" means that the functional variant retains the biological activity of the TCR, polypeptide or protein that is the variant, and has substantial or significant sequence identity or similarity to the parental TCR, polypeptide or protein. Functional variants include, for example, variants of the TCR, polypeptide or protein (parental TCR, polypeptide or protein) described herein that retain the ability to specifically bind to an antigen to the same extent, to a similar extent or to a higher extent as the parental TCR has antigen specificity, or as the parental polypeptide or protein specifically binds to mutant KRAS. Consistent with the parental TCR, polypeptide or protein, for example, a functional variant can be at least about 30%, 50%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identical in amino acid sequence to each of the parental TCR, polypeptide or protein.

[0055] ​​​​​​​​​For example, a functional variant may comprise the amino acid sequence of a parent TCR, polypeptide or protein with at least one conservative amino acid substitution. Conservative amino acid substitutions are known in the art and refer to the replacement of a single amino acid with certain physical and / or chemical properties with a similar chemical structure. Conservative amino acid substitutions include those in which an acidic amino acid is replaced with another amino acid that has the same chemical or physical properties as the original amino acid. For example, a conservative amino acid substitution would be one acidic amino acid replaced with another acidic amino acid (e.g., Asp or Glu). Substitutions, replacing amino acids with non-polar side chains with other amino acids with non-polar side chains (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), replacing basic amino acids with other basic amino acids The amino acid may be substituted with an amino acid having a polar side chain (such as Lys or Arg), or an amino acid having a polar side chain may be substituted with another amino acid having a polar side chain (such as Asn, Cys, Gln, Ser, Thr, Tyr, etc.).

[0056] Alternatively or additionally, a functional variant has at least one non-conservative amino acid substitution. In this case, the amino acid sequence of the parent TCR, polypeptide or protein may be Preferably, the non-conservative amino acid substitutions do not interfere with or inhibit the biological activity of the functional variant. Alternatively, non-conservative amino acid substitutions may increase the biological activity of the functional variant, such that the biological activity is increased compared to the protein.

[0057] The TCR, polypeptide or protein may be modified so that other components, e.g., other amino acids, do not substantially alter the biological activity of the TCR, polypeptide or protein. Or proteins may consist essentially of a particular amino acid sequence or sequences described herein. In this regard, for example, the TCRs, polypeptides or proteins of the invention may be those of SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, No. 57, and may essentially consist of the amino acid sequences of (1) both of SEQ ID NOs: 50-51; (2) both of SEQ ID NOs: 52-53; (3) both of SEQ ID NOs: 54-55; or (4) both of SEQ ID NOs: 56-57. Also, for example, the TCR, polypeptide or protein of the present invention may essentially consist of the amino acid sequences of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 39, SEQ ID NO: 40, (i) both of SEQ ID NOs: 15-16; (ii) both of SEQ ID NOs: 23-24; (iii) both of SEQ ID NOs: 31-32; or (iv) both of SEQ ID NOs: 39-40. Further, the TCR, polypeptide or protein of the present invention may essentially consist of the amino acid sequences of (a) all of SEQ ID NOs: 9-14; (b) all of SEQ ID NOs: 17-22; (c) all of SEQ ID NOs: 25-30; or (d) all of SEQ ID NOs: 33-38. The TCR, polypeptide or protein of the present invention may contain any length, i.e., any number of amino acids, provided that the TCR, polypeptide or protein retains their biological activities, such as the ability to specifically bind to mutant KRAS, detect cancer in a mammal, or treat or prevent cancer in a mammal. For example, the polypeptide may be in the range of about 50 to about 5000 amino acids in length, and may be, for example, 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino acids in length. In this regard, the polypeptide of the present invention also includes oligopeptides. The TCR, polypeptide and protein of the present invention may have one or more naturally-occurring amino acids replaced ; (c) all of SEQ ID NOs: 25-30; or (d) all of SEQ ID NOs: 33-38.

[0058] The TCR, polypeptide and protein of the present invention may have one or more naturally-occurring amino acids replaced

[0059] The TCR, polypeptide and protein of the present invention may have one or more naturally-occurring amino acids replaced It may contain synthetic amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino-n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- 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, 6-hydroxylysine, ornithine, α-aminocyclo pentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-dia minopropionic acid, homophenylalanine and α-tert-butylglycine.

[0060] The TCRs, polypeptides and proteins of the present invention can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, for example, via disulfide bridges, or converted to acid addition salts, and / or optionally dimerized or polymerized, or conjugated. The TCRs, polypeptides and / or proteins of the present invention can be obtained, for example, by methods known in the art such as de novo synthesis. Polypeptides and proteins can also be produced recombinantly using the nucleic acids described herein by standard recombinant methods. For example, Green and Sambrook,

[0061] The TCRs, polypeptides and / or proteins of the present invention can be obtained, for example, by methods known in the art such as, for example, de novo synthesis. The polypeptides and proteins can also be produced recombinantly using the nucleic acids described herein by standard recombinant methods. For example, Green and Sambrook, Molecular Cloning:A Laboratory ManualSee (4th Edition) Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Alternatively, The TCRs, polypeptides and / or proteins described herein can be commercially synthesized by, for example, companies such as Synpep (Dublin, CA), Peptide Technologies Corp. (Gaithersburg, MD), and Multiple Peptide Systems (San Diego, CA). In this regard, the TCRs, po lypeptides and proteins of the present invention can be synthesized, recombinant, isolated and / or purified.

[0062] The scope of the present invention includes conjugates, e.g., bioconjugates, comprising any of the TCRs, polypeptides or proteins of the present invention (including any functional portions or variants 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.

[0063] 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 "polynucleotide", "oligonucleotide" and "nucleic acid molecule", and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, and can include natural, non-natural or modified nucleotides, and natural, non-natural or modified internucleotide linkages, e.g., phosphoramidate linkages or phosphorothioate linkages instead of the phosphodiesters found between nucleotides of unmodified oligonucleotides. In one embodiment, the nucleic acid comprises complementary DNA (cDNA). The nucleic acid may have insertions, deletions, inversions and / or substitutions and / or modifications, and may be a recombinant nucleic acid molecule. It is generally preferred that there be no insertions at all. However, as discussed herein, in some instances it may be preferable for the nucleic acid to contain one or more insertions, deletions, inversions, and / or substitutions.

[0064] Preferably, the nucleic acids of the invention are recombinant. As used herein, the term "recombinant" refers to a molecule that is constructed extracellularly by adding (i) a natural or synthetic nucleic acid segment to a nucleic acid molecule capable of replicating in a living cell, or (ii) a molecule obtained by replication of what is described in (i) above. For the purposes of this specification, replication can be in vitro replication or in vivo replication. A nucleic acid can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, for example, Green and Sambrook et al., supra. For example, a nucleic acid can be composed of naturally occurring nucleotides, or various modified nucleotides (e.g., phosphorothioate derivatives and acridine substituted nucleotides) designed to increase the biological stability of the molecule, or to increase the physical stability of the duplex formed upon hybridization. For the purposes of this specification, replication can be in vitro replication or in vivo replication.

[0065] Nucleic acids can be chemically synthesized using, for example, naturally occurring nucleotides or various modified nucleotides (e.g., phosphorothioate derivatives and acridine substituted nucleotides) designed to increase the biological stability of the molecule, or to increase the physical stability of the duplex formed upon hybridization. Examples of modified nucleotides that can be used to generate a nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil A nucleic acid can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, for example, Green and Sambrook et al., supra. ) Hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine osine, N 6 -isopentenylade nine (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, β-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-carboxy propyl)uracil, and 2,6-diaminopurine, but are not limited thereto. Alternatively, one or more of the nucleic acids of the present invention can be purchased from companies such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX). The nucleic acid can include any nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein. In one embodiment of the present invention, the nucleic acid can include any one of the nucleotide sequences of SEQ ID NOs: 63-70 (Table 1). In one embodiment of the present invention, the nucleic acid includes both of the nucleotide sequences of SEQ ID NOs: 63-64, both of the nucleotide sequences of SEQ ID NOs: 65-66, both of the nucleotide sequences of SEQ ID NOs: 67-68, or both of the nucleotide sequences of SEQ ID NOs: 69-70.

[0066]

[0067]

Table 1

[0068] In one embodiment of the present invention, the nucleic acid comprises a codon-optimized nucleotide sequence encoding any one of the TCRs, polypeptides or proteins described herein. Without being bound by any particular theory or mechanism, codon optimization of the nucleotide sequence is thought to increase the translation efficiency of the mRNA transcript. Codon optimization of the nucleotide sequence may involve replacing native codons with other codons that encode the same amino acid but can be translated by tRNAs that are more readily available within the cell, thus increasing translation efficiency. Optimization of the nucleotide sequence may also reduce the secondary structure of the mRNA that interferes with translation, thus increasing translation efficiency.

[0069] The present invention also provides a nucleic acid comprising a nucleotide sequence complementary to the nucleotide sequence of any one of the nucleic acids described herein, or a nucleotide sequence that hybridizes to the nucleotide sequence of any one of the nucleic acids described herein under stringent conditions.

[0070] A nucleotide sequence that hybridizes under stringent conditions preferably hybridizes under high stringency conditions. "High stringency conditions" means that the nucleotide sequence hybridizes specifically to the target sequence (the nucleotide sequence of any one of the nucleic acids described herein) in a detectably greater amount than non-specific hybridization. High stringency conditions include from random sequences that fortuitously have a few small regions (e.g., 3-10 bases) that match the nucleotide sequence, exactly Conditions are included for distinguishing polynucleotides having a complementary array or polynucleotides containing only scattered minor mismatches. Such small regions of complementarity melt more readily than full-length complements of 14 to 17 bases or more, and they can be readily distinguished by high stringency hybridization. Relatively high stringency conditions include, for example, low salt conditions and / or high temperature conditions provided by about 0.02 to 0.1 M NaCl or the equivalent at a temperature of about 50 to 70 °C. Such high stringency conditions are hardly tolerable if there are mismatches between the nucleotide sequence and the template or target strand, and are particularly suitable for detecting the expression of any TCR of the present invention. It is generally understood that the conditions can be made more stringent by increasing the amount of formamide added.

[0071] The present invention also provides a nucleic acid comprising a nucleotide sequence that is at least about 70% or more, such as about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical to any of the nucleic acids described herein. In this regard, the nucleic acid can consist essentially of any of the nucleotide sequences described herein.

[0072] The nucleic acids of the present invention can be incorporated into recombinant expression vectors. In this regard, the present invention provides a recombinant expression vector comprising any of the nucleic acids of the present invention. In one embodiment of the present invention, the recombinant expression vector comprises nucleotide sequences encoding an α chain, a β chain, and a linker peptide.

[0073] For the purposes of this specification, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that contains a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide and enables the expression of the mRNA, protein, polypeptide, or peptide by a host cell when the vector is contacted with the cell under conditions sufficient to effect expression of the mRNA, protein, polypeptide, or peptide within the cell. The vectors of the present invention are not naturally occurring as a whole. However, portions of the vectors may be naturally occurring. The recombinant expression vectors of the present invention can be single-stranded or double-stranded, can be partially synthetic or obtained from natural sources, and can contain natural, non-natural, or modified nucleotides, including, but not limited to, DNA and RNA, and can contain any type of nucleotide that can include naturally occurring nucleotide linkages, non-naturally occurring nucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or modified nucleotides, or nucleotide linkages, do not interfere with transcription or replication of the vector.

[0074] The recombinant expression vectors of the present invention can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host cell. Suitable vectors include, for example, those designed for propagation and amplification (expansion), or expression, or both, such as plasmids and viruses. The vector is of the pUC series or the like. It may be selected from the group consisting of pUC (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 etc. 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-C1, pMAM and pMAMneo (Clontech). Preferably, the recombinant expression vector is a viral vector, such as a retroviral vector. In a particularly preferred embodiment, the recombinant expression vector is the MSGV1 vector.

[0075] The recombinant expression vector of the present invention can be prepared using standard recombinant DNA techniques, for example, as described by Green and Sambrook et al. above. The construct of the expression vector, whether circular or linear, can be prepared to contain a replication system that is functional in prokaryotic or eukaryotic host cells. The replication system can be derived from, for example, ColE1, 2μ plasmid, λ, SV40, bovine papillomavirus, etc. The construct of the expression vector, whether circular or linear, can be prepared to contain a replication system that is functional in prokaryotic or eukaryotic host cells. The replication system can be derived from, for example, ColE1, 2μ plasmid, λ, SV40, bovine papillomavirus, etc.

[0076] Desirably, the recombinant expression vector appropriately contains control sequences specific to the type of host cell (e.g., bacteria, fungi, plants or animals) into which the vector is introduced, such as transcriptional, as well as start and stop codons for translation, considering whether the vector is DNA-based or RNA-based. Desirably, the recombinant expression vector appropriately contains control sequences specific to the type of host cell (e.g., bacteria, fungi, plants or animals) into which the vector is introduced, such as transcriptional, as well as start and stop codons for translation, considering whether the vector is DNA-based or RNA-based.

[0077] The recombinant expression vector may contain one or more marker genes that enable the selection of transformed or transfected host cells. Examples of marker genes include biocide resistance, such as resistance to antibiotics, heavy metals, etc., and complementation in auxotrophic host cells to provide prototrophy. 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.

[0078] The recombinant expression vector may contain a native or non-native promoter operably linked to a nucleotide sequence encoding a TCR, polypeptide, or protein, or a nucleotide sequence complementary to or hybridizing with a nucleotide sequence encoding a TCR, polypeptide, or protein. The selection of the promoter, such as strong, weak, inducible, tissue-specific, and developmental stage-specific, is within the scope of the ordinary skill of those in the art. Similarly, combining the nucleotide sequence and the promoter is also within the scope of the skill of those in the art. The promoter can be a non-viral promoter or a viral promoter, such as the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, and the promoter found in the long terminal repeat of the murine stem cell virus. The recombinant expression vector may contain a native or non-native promoter operably linked to a nucleotide sequence encoding a TCR, polypeptide, or protein, or a nucleotide sequence complementary to or hybridizing with a nucleotide sequence encoding a TCR, polypeptide, or protein. The selection of the promoter, such as strong, weak, inducible, tissue-specific, and developmental stage-specific, is within the scope of the ordinary skill of those in the art. Similarly, combining the nucleotide sequence and the promoter is also within the scope of the skill of those in the art. The promoter can be a non-viral promoter or a viral promoter, such as the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, and the promoter found in the long terminal repeat of the murine stem cell virus. The recombinant expression vector may contain a native or non-native promoter operably linked to a nucleotide sequence encoding a TCR, polypeptide, or protein, or a nucleotide sequence complementary to or hybridizing with a nucleotide sequence encoding a TCR, polypeptide, or protein. The selection of the promoter, such as strong, weak, inducible, tissue-specific, and developmental stage-specific, is within the scope of the ordinary skill of those in the art. Similarly, combining the nucleotide sequence and the promoter is also within the scope of the skill of those in the art. The promoter can be a non-viral promoter or a viral promoter, such as the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, and the promoter found in the long terminal repeat of the murine stem cell virus.

[0079] The recombinant expression vector of the present invention can be designed for either transient expression, stable expression, or both. Also, the recombinant expression vector can be prepared for constitutive expression or inducible expression.

[0080] Furthermore, the recombinant expression vector can be prepared to contain a suicide gene. As used herein, the term "suicide gene" refers to a gene that kills cells expressing the suicide gene. A suicide gene can be a gene that confers sensitivity to a reagent, such as a drug, on cells expressing the gene and kills the cells when the cells come into contact with or are exposed to the reagent. Suicide genes are known in the art and include, for example, the herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, nitroreductase, and the inducible caspase 9 gene system .

[0081] Another embodiment of the present invention further provides a host cell containing any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that can contain the recombinant expression vector of the present invention. The host cell can be a eukaryotic cell, such as a plant, animal, fungus, or alga, or a prokaryotic cell, such as a bacterium or protozoan. The host cell can be a cultured cell or a primary cell, i.e., a cell that can be isolated directly from an organism, such as a human. The host cell can 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 Escherichia coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, etc. For the purpose of amplifying or replicating the recombinant expression vector , the host cell is preferably a prokaryotic cell, such as DH5α cells . For the purpose 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 can be of any cell type, can be derived from any type of tissue, and can be at any developmental stage, while the host cell is preferably a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). More preferably , the host cell is a T cell

[0082] For the purposes of this specification, a T cell can be any T cell, such as a cultured T cell, e.g., a primary T cell, or a cultured T cell derived from a cell line, e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal, etc. When obtained from a mammal, the T cell can be obtained from a number of sources including, but not limited to, blood, bone marrow, lymph nodes, thymus or other tissues or body fluids. The T cell can also be concentrated or purified. Preferably, the T cell is a human T cell. The T cell can include, but is not limited to, + CD4 + / CD8 + double positive T cells, CD4 + helper T cells, e.g., Th1 and Th2 cells, CD4 + T cells, CD8 T cells (e.g., cytotoxic T cells), tumor infiltrating lymphocytes (TIL), memory T cells (e.g., central memory T cells and effector memory T cells), naive T cells, etc., and can be any type of T cell and can be at any stage of development.

[0083] The present invention also provides a cell population comprising at least one host cell described herein . The cell population can be a heterogeneous population that includes at least one other cell, e.g., a host cell (e.g., a T cell) that does not include any of the recombinant expression vectors, or a cell other than a T cell, e.g., a B cell, macrophage, neutrophil, erythrocyte, hepatocyte, endothelial cell, epithelial cell, muscle cell, brain cell, etc., in addition to a host cell that includes any of the described recombinant expression vectors. Alternatively, the cell population can be a substantially homogeneous population that mainly includes (e.g., consists essentially of) host cells that include the recombinant expression vector. The population can also be a clonal population of cells that are clones of a single host cell such that all cells of the population include the recombinant expression vector. In one embodiment of the present invention, the cell population is a clonal population that includes host cells that include the recombinant expression vector described herein.

[0084] ​In one embodiment of the present invention, the number of cells in a population can be rapidly expanded. Breadth can be achieved by any of a number of methods known in the art, for example, 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., J. Immunol. Methods, 128:189-201 (1990). In one embodiment, expansion of T cell numbers is achieved by T cells were then co-injected with OKT3 antibody, IL-2 and feeder PBMCs (e.g., irradiated allogeneic PBMCs). This is carried out by culturing the cells in the culture medium.

[0085] The TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors and host cells of the present invention A population of a particular target organism (including a population thereof) may be isolated and / or purified. As used herein, the term "isolated" means removed from its natural environment. As used herein, the term "purified" means increased in purity, and "purity" is a relative term and need not be interpreted as absolute purity. For example, purity may be at least about 50% or more, may be greater than 60%, 70%, 80%, 90%, 95%, or may be about 100%. do.

[0086] The TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors and host cells of the present invention (including populations thereof) (all of which are hereinafter collectively referred to as the "TCR materials of the invention" ) may be formulated into a composition, e.g., a pharmaceutical composition. In this regard, the invention relates to the TCRs, polypeptides, proteins, nucleic acids, expression vectors and host cells described herein (including The present invention provides a pharmaceutical composition comprising any of the TCR materials of the present invention, including a population of TCR materials of the present invention, and a pharma- ceutical acceptable carrier. Examples may include polypeptides and nucleic acids, or two or more different TCRs. Alternatively, the pharmaceutical composition may be, for example, a chemotherapeutic agent such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil ), gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vi nblastine, vincristine, etc., and may include the TCR material of the present invention in combination with another pharmaceutically active agent(s) or agent(s).

[0087] Preferably, the carrier is a pharmaceutically acceptable carrier. For a pharmaceutical composition, the carrier can be any of those customarily considered for use with a particular TCR material of the present invention. Methods for preparing administrable compositions are 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, 22 Ed., Pharmaceutical Press (2012). A pharmaceutically acceptable carrier preferably has no adverse side effects or toxicity under the conditions of use. nd Ed., Pharmaceutical Press (2012).

[0088] ​​The choice of carrier is determined to some extent by the specific TCR material of the present invention and by the specific method used to administer the TCR material of the present invention. Accordingly, there are various suitable formulations of the pharmaceutical composition of the present invention. Suitable formulations may include any of the formulations for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, intratumoral or intraperitoneal administration. More than one route may be used to administer the TCR material of the present invention, and in some cases, a particular route may provide a more rapid and effective response than another route.

[0089] Preferably, the TCR material of the present invention is administered by injection (e.g., intravenous). When the TCR material of the present invention is a host cell expressing the TCR of the present invention, a pharmaceutically acceptable carrier for the injectable cells may include, for example, normal saline (about 0.90% w / v NaCl in water, about 300 mOsm / L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water or Ringer's lactate, or any other isotonic carrier. In one embodiment, the pharmaceutically acceptable carrier is supplemented with human serum albumin.

[0090] For the purposes of the present invention, the amount or dose of the TCR material of the present invention administered (e.g., the number of cells if the TCR material of the present invention is one or more cells) must be sufficient to effect, e.g., over a reasonable period of time, a therapeutic or prophylactic response in a subject or animal. For example, the dose of the TCR material of the present invention must be sufficient to bind to a cancer antigen (e.g., mutant KRAS), or to detect, treat or prevent cancer within about 2 hours or more from the time of administration, e.g., for a period of 12 - 24 hours or more. In certain embodiments, the period may be even longer. The dose is determined by the efficacy of the specific TCR material of the present invention and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) being treated. ​

[0091] Many assays for determining the amount to be administered are known in the art. For the purposes of the present invention, the degree to which target cells are lysed, or different doses of T cells are each administered to one mammal out of a set of mammals, and T cells expressing the TCR, polypeptide or protein of the present invention are administered to that mammal at a given dose An assay method can be used to determine the initial dose to be administered to a mammal, which includes comparing the degree of IFN-γ secreted by T cells expressing the TCR, polypeptide or protein of the present invention. The degree to which target cells are lysed or IFN-γ is secreted upon administration of a certain dose can be assayed by methods known in the art.

[0092] The dose of the TCR material of the present invention is also determined by the presence, nature and degree of any adverse side effects that may accompany the administration of a particular TCR material of the present invention. Typically, the attending physician will consider various factors such as, for example, age, weight, general health, diet, sex, the TCR material of the present invention being administered, the route of administration and the severity of the cancer being treated and determine the dosage of the TCR material of the present invention for treating an individual patient. In embodiments where the TCR material of the present invention is a cell population, the number of cells administered per injection can vary, for example, from about 1×10 6 to about 1×10 12 cells, or more. In certain embodiments, fewer than 1×10 cells can be administered. 6 Less than cells can be administered.

[0093] One skilled in the art can readily understand that the inventive TCR materials of the invention can be modified in any number of ways to enhance the therapeutic or prophylactic effect of the TCR material of the present invention by improvement. For example, the TCR material of the present invention can be used in combination with chemotherapeutic agents in any number of ways to enhance the therapeutic or prophylactic effect of the TCR material of the present invention. For example, the TCR material of the present invention can be used in combination with chemotherapeutic agents It can be directly or indirectly coupled by cross-linking. The practice of coupling a compound to a chemotherapeutic agent is known in the art. One skilled in the art will recognize that when cross-linking and / or a chemotherapeutic agent binds to the TCR material of the present invention, a site of the TCR material of the present invention that is not necessary for the function of the TCR material of the present invention, provided that the cross-linking and / or chemotherapeutic agent does not interfere with the function of the TCR material of the present invention, i.e., the ability to bind to mutant KRAS or detect, treat or prevent cancer, is an ideal site for binding the cross-linking and / or chemotherapeutic agent. When binding to the TCR material of the present invention, provided that the cross-linking and / or chemotherapeutic agent does not interfere with the function of the TCR material of the present invention, i.e., the ability to bind to mutant KRAS or detect, treat or prevent cancer. A site of the TCR material of the present invention that is not necessary for the function of the TCR material of the present invention is an ideal site for binding the cross-linking and / or chemotherapeutic agent.

[0094] The pharmaceutical compositions, TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells and cell populations of the present invention are expected to be used in methods of treating or preventing cancer. Without being bound by a particular theory, it is believed that the TCRs of the present invention specifically bind to mutant KRAS such that when the TCR (or related polypeptides or proteins of the present invention) is expressed by a cell, it can mediate an immune response against target cells expressing mutant KRAS. In this regard, the present invention provides a method of treating or preventing cancer in a mammal, comprising administering to the mammal an effective amount of 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. 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. 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. 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.

[0095] One embodiment of the present invention is for use in the treatment or prevention of cancer in a mammal, any of the pharmaceutical compositions, TCRs, polypeptides or proteins described herein, any nucleic acid encoding any of the TCRs, polypeptides or proteins described herein Any nucleic acid or recombinant expression vector containing a chimeric sequence, or a TCR described herein , provides any host cell or cell population containing a recombinant vector encoding any of the polypeptides or proteins.

[0096] As used herein, the terms "treatment" and "prevention", and terms derived therefrom, do not necessarily mean 100% or complete treatment or prevention. Rather, there are various degrees of treatment or prevention that a person skilled in the art would recognize as having a potential benefit or therapeutic effect. In this regard, the methods of the present invention can provide treatment or prevention of any amount or any level of cancer in a mammal. Furthermore, the treatment or prevention provided by the methods of the present invention can include treatment or prevention of one or more conditions or symptoms of the cancer being treated or prevented. For example, the treatment or prevention can include promoting regression of a tumor. Also, for the purposes of this specification, "prevention" can include delaying the onset of cancer, or its symptoms or conditions. Alternatively or additionally, "prevention" can include preventing or delaying recurrence of cancer, or its symptoms or conditions. provided by the methods of the present invention can include treatment or prevention of one or more conditions or symptoms of the cancer being treated or prevented. For example, the treatment or prevention can include promoting regression of a tumor. Also, for the purposes of this specification, "prevention" can include delaying the onset of cancer, or its symptoms or conditions. Alternatively or additionally, "prevention" can include preventing or delaying recurrence of cancer, or its symptoms or conditions. Furthermore, a method of detecting the presence of cancer in a mammal is provided. The method includes (i) contacting any of the TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, cell populations or pharmaceutical compositions of the present invention described herein with a sample containing one or more cells derived from a mammal, thereby forming a complex, and detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

[0097] Also provided is a method of detecting the presence of cancer in a mammal. The method includes (i) contacting any of the TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, cell populations or pharmaceutical compositions of the present invention described herein with a sample containing one or more cells derived from a mammal, thereby forming a complex, and detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal. For the method of the present invention for detecting cancer in a mammal, the sample of cells can be a sample containing 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. , host cells, cell populations or pharmaceutical compositions of the present invention described herein with a sample containing one or more cells derived from a mammal, thereby forming a complex, and detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

[0098] For the method of the present invention for detecting cancer in a mammal, the sample of cells can be a sample containing 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.

[0099] For the purposes of the detection method of the present invention, the contact can be carried out in vitro or in vivo with respect to a mammal. Preferably, the contact is in vitro.

[0100] Also, the detection of the complex can be carried out by any number of methods known in the art. For example, the TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell or cell population of the present invention described herein can be labeled with a detectable label, such as, for example, a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles), etc.

[0101] For the purposes of the method of the present invention, a host cell or cell population can be administered, and the cell can be allogeneic or autologous to the mammal. Preferably, the cell is autologous to the mammal.

[0102] With respect to the method of the present invention, cancer can be acute lymphoblastic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal, anal canal or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck, gallbladder or pleural cancer, nose, nasal cavity or middle ear cancer, oral cavity cancer, vaginal cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, glioma, Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, oropharyngeal cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal, greater omentum 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, colorectal, lung, endometrial, ovarian 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 another preferred embodiment, the cancer is a cancer that expresses a mutant KRAS amino acid sequence having a G12D mutation.

[0103] The mammal referred to in the method of the present invention can be any mammal. As used herein, the term "mammal" refers to any mammal including, but not limited to, rodents such as mice and hamsters, and lagomorphs such as rabbits. Preferably, the mammal is from the order Carnivora including Felidae (cats) and Canidae (dogs). More preferably, the mammal is from the order Artiodactyla including Bovidae (cows) and Suidae (pigs), or the order Perissodactyla including Equidae (horses). Most preferably, the mammal is a primate, a cercopithecoid or a hominoid (monkey) or a hominid (humans and great apes). A particularly preferred mammal is a human.

[0104] In the following examples, the present invention will be further described, but it should not be construed as limiting its scope in any way.

Examples

[0105] Next-generation sequencing Using the QIAGEN AllPrep DNA / RNA kit (Qiagen, Venlo, Netherlands), genomic DNA (gDNA) and total RNA were purified from various tumors and matched normal adjacent samples according to the manufacturer's recommendations. One sample (Tu-Pri) was formalin-fixed and paraffin-embedded Formalin-fixed paraffin-embedded (FFPE) gDNA was extracted using the Covaris truXTRACTM FFPE DNA Kit according to the manufacturer's instructions (Covaris, Woburn, MA). The Agilent Technologies SureSelectXT Target Enrichment System for paired-end libraries was used in combination with the Human All Exon V6 RNA baits (Agilent Technologies, Santa Clara, CA, USA) to prepare whole-exome library constructs and exon capture of approximately 20,000 protein-coding genes. Subsequently, whole-exome sequencing (WES) libraries were sequenced on a NEXTSEQ 500 desktop sequencer (Illumina, San Diego, CA, USA). According to the manufacturer's protocol, 3 μg of gDNA from fresh tumor tissue samples and 200 ng of gDNA from FFPE tumor samples were used to prepare libraries. Paired-end sequencing was initially performed using the reagent / flow cell kit v1 of the Illumina High Output Flow Cell Kit (300 cycles), and subsequent runs of the same library preparation were continued with the reagent / flow cell kit v2. Using the Illumina TruSeq RNA Strand Library Preparation Kit, according to the manufacturer's protocol RNA-seq libraries were prepared using 2 μg of total RNA. RNA-seq libraries were paired-end sequenced on a NextSeq 500 desktop sequencer (Illumina, San Diego, CA, USA).

[0106] Alignment, processing, and variant calling ​​For WES, alignment against the human genome build hg19 was performed using the novoalignMPI program from Novocraft (Selangor, Malaysia) (novocraft.com / ). Duplicates were marked using the Picard MARKDUPLICATES tool. Indel realignment and base recalibration were performed according to the GATK best practices workflow (broadinstitute.org / gatk / ). After data cleanup, the samtools utility (samtools.sourceforge.net) was used to generate a pileup file, and the Varscan2 platform-independent variant caller (varscan.sourceforge.net) was used to call somatic variants using the following criteria: 10 or more tumor and normal read counts, a variant allele frequency of 10% or more, and 4 or more tumor variant reads. Then, the Annovar software tool (annovar.openbioinformatics.org) was used to annotate these variants.

[0107] For RNA-seq, alignment against the human genome build hg19 was performed using the two-pass method of STAR (github.com / alexdobin / STAR). Duplicates were marked using the Picard MARKDUPLICATES tool. The GATK SplitNTrim tool was used to split and trim the reads. Subsequently, indel realignment and base recalibration were performed using the GATK toolbox. Finally, a pileup file was created using the finally realigned bam file and the samtools mpileup tool. Finally, the VARSCAN2 platform -independent variant caller was used to call variants.

[0108] Whole exome sequencing (WES) and RNA sequencing (RNA-seq) were performed on three metastatic fresh tumor samples (Tu-1, Tu-2A, and Tu-2B). Subsequently, to remove false positive calls likely due to sequencing or mapping errors, the Integrated Genomics Viewer (IGV) tool (Broad Institute, Cambridge, MA) was used to manually curate variants with a minimum exome frequency of 7% and a minimum of three alternative reads. Mutations that were either clonal or potentially representative of a dominant clonal population were targeted, and 61 mutations were selected for further analysis based on detection in two or more tumor samples. One of these 61 mutations was in KRAS (Table 2). These included 29 mutations identified in a minimum of one WES and one RNA-seq library, and 32 mutations identified in WES libraries from two or more metastatic lesions. To focus on mutations that could be representative of a clonal or dominant clonal population, 61 mutations were selected for further analysis based on detection in two or more tumor samples. One of these 61 mutations was in KRAS (Table 2). These included 29 mutations identified in a minimum of one WES and one RNA-seq library, and 32 mutations identified in WES libraries from two or more metastatic lesions. These included 29 mutations identified in a minimum of one WES and one RNA-seq library, and 32 mutations identified in WES libraries from two or more metastatic lesions.

[0109] [Table 2]

[0110] Generation of tumor-infiltrating lymphocytes (TILs), adoptively transferred TILs, and antigen-presenting dendritic cells (DCs) TILs, adoptively transferred TILs, and dendritic cells were generated as described by Tran et al., Science, 350:1387-90 (2015). Briefly, to generate TILs, surgically resected tumors were cut into 24 fragments approximately 1-2 mm in size, and each fragment was placed separately into wells of a 24-well plate containing 2 ml of complete medium (CM) containing high-dose IL-2 (6000 IU / ml, Chiron, Emeryville, CA). CM consisted of 10% human serum (in-house), 2 mM L-glutamine, 25 mM HEPES, and 10 μg / ml gentamicin. The cultures contained RPMI medium supplemented with 5% human AB serum, 3000 IU / ml IL-2 and 30 ng / ml OKT3 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany). TIL fragment culture number 6 was selected for treatment and underwent a rapid expansion process in gas-permeable G-REX100 flasks using irradiated PBMCs at a ratio of 1:100 in 400 ml of 50 / 50 medium supplemented with 5% human AB serum, 3000 IU / ml IL-2 and 30 ng / ml OKT3 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany). 50 / 50 medium contained a 1:1 mixture of CM and AIM-V medium. All cells were cultured at 37°C with 5% CO2.

[0111] Immature DCs were generated from peripheral blood monocytes using the plastic adherence method. Briefly, patient apheresis was thawed, washed, and resuspended in AIM-V medium (Life Technologies, Carlsbad, CA). The cells were then cultured in tissue culture flasks (162 cm) at 7.5-10e6 cells / ml. 2 (surface area) Approximately 1e6 cells / cm 2 The plates were incubated at 5% CO2 and 37°C for 90 min. After that, the non-adherent cells were collected and vigorously washed with AIM-V medium in a flask. The cells were then further incubated with AIM-V medium for 60 minutes. The medium and non-adherent cells were removed. The adherent cells were then washed again vigorously with AIM-V medium in the flask and then cultured in DC medium. They were incubated using the ground. The DC medium contained RPMI with 5% human serum, 100 U / ml penicillin and 100 μg / ml streptomycin, 2 mM L-glutamine, 800 IU / ml GM-CSF (Leukine (sargramostim)) and 200 U / ml IL-4 (Peprotech, Rocky Hill, NJ). On the 2nd to 3rd day, fresh DC medium was added to the culture solution. After the start of the culture, on the 4th or 5th day, the DCs were cryopreserved. Between the 4th and 6th day after the start of the culture, the DCs were used in the experiment.

[0112] Identification of mutant-reactive T cells and co-culture experiments The detailed method is described in Tran et al., Science, 350:1387-90 (2015). Briefly, 61 mutations were identified by whole exome and transcriptome sequencing. One of these 61 mutations was KRAS (Table 2). For each mutation, a mini-gene encoding the mutation was generated with 12 amino acids flanking either side of the parental protein, synthesized tandemly, and a tandem mini-gene (TMG) construct was created. Five TMGs (TMG1-5) encoding 61 mutations were created, transcribed into RNA in vitro, and made it possible to process and present all the mutations in the context of the patient's own HLA-I and HLA-II molecules (Table 4). They were electroporated into autologous antigen-presenting DCs. The TMGs for wild-type and mutant KRAS are shown in Table 3. The HLA data shown in Table 4 were determined from next-generation sequencing data using the algorithm PHLAT as described in Bai et al., BMC Genomics, 15:325 (2014). Next Twenty-four individual patient-derived TIL cultures were co-cultured with these TMG-expressing DCs, and T cell reactivity was determined by IFN-γ enzyme-linked immunosorbent spot (ELISPOT) assay (Figure 1A), as well as flow cytometric analysis of the T cell activation markers 4-1BB and OX40. Multiple TIL cultures reactive to TMG-1 were identified. To identify which mutant antigens in TMG-1 were recognized by TIL culture 6, peptides encoded in TMG-1 were synthesized (ThermoFisher Scientific (Waltham, MA) and GenScript Inc. (Piscataway Township, NJ)), and then they were pulsed individually overnight onto DCs and co-culture with TIL culture 6 was continued (Figure 1B).

[0113]

Table 3

[0114]

Table 4

[0115] The following HPLC-purified peptides (GenScript Inc.): wild-type (WT)-9-mer: GAGGVGKSA (SEQ ID No. 7); mutant (G12D)-9-mer: GADGVGKSA (SEQ ID No. 8); WT-10-mer: GAGGVGKSAL (SEQ ID No. 5); G12D-10-mer: GADGVGKSAL (SEQ ID No. 6) were used in the peptide titer experiments .

[0116] Intracellular cytokine staining (ICS) and flow cytometry were used to determine expression of cytokines IFN-γ, TNF, and IL-2, and degranulation marker CD107a, as described in Tran et al., Science, 344:641-5 (2014). Briefly, target and effector cells were combined in wells of a 96-well plate, and both GolgiStop and GolgiPlug protein transport inhibitors (both at 1 / 2 the recommended concentration) (BD Biosciences, Franklin Lakes, NJ) were added to the cultures. At t=6 hours after stimulation, cells were stained using a Cytofix / Cytoperm kit (BD Biosciences). Cells were processed according to the manufacturer's instructions on a FACSCantoII flow cytometer. Cells were acquired and data were analyzed using FlowJo software (TreeStar Inc., Ashland, OR). Boolean gate analysis was used to identify the effector functions (cytokines) of the indicated numbers. The percentage of cells expressing IgG4-associated markers (inhibitory and degranulation markers) was determined.

[0117] KRAS G12D Identification of reactive T cell clones Using various methods, four KRAS G12D Reactive TCRs were identified. Prior to rapid expansion, dominant TRBV5-6 (Vβ5.2) clones in the infusion bag were isolated from TIL fragment culture #6. Briefly, TIL culture #6 was stained with anti-Vβ5.2-PE (phycoerythrin) antibody (Beckman Coulter, Schaumburg, IL) and Vβ5.2+ cells were enriched using anti-PE specific antibody conjugated to magnetic microbeads as per the manufacturer's instructions (Miltenyi Biotec). Total RNA was isolated from Vβ5.2+ T cells (RNeasy Mini kit, Qiagen) according to the manufacturer's instructions. Next, 5’RACE was performed using the constant primers of TCR-alpha and beta chains (SMARTER RACE cDNA amplification kit, Clontech). The sequences of the constant primers of the alpha and beta chains were as follows: TCR-alpha, 5’ -GCC ACA GCA CTG TTG CTC TTG AAG TCC-3’ (SEQ ID NO: 59); TCR-beta, 5’ -CAG GCA GTA TCT GGA GTC ATT GAG-3 (SEQ ID NO: 60). For PCR, the extension time was changed (2 minutes instead of 3 minutes), and the kit protocol was used. (SEQ ID NO: 60). For PCR, the extension time was changed (2 minutes instead of 3 minutes), and the kit protocol was used. Lamb 1 was used. Then, the TCR PCR products were isolated by standard agarose gel electrophoresis and gel extraction (zymogen), and then the products were sequenced (Macrogen, Seoul, Korea).

[0118] The second and third ranked TCRs in the infusion bag were also KRAS G12D reactive, long KRAS G12D Peptide-pulsed DCs were used to stimulate the apheresis samples 40 days after cell transfer for the first time. After overnight stimulation, 4-1BB of the T cell activation marker was upregulated in Vβ5.2-positive and -negative CD8+ T cells, which were separately sorted by FACS. As described above, prior to performing 5’RACE, Vβ5.2+ cells were further amplified, followed by TOPO-TA cloning of the TCR PCR products and sequencing of individual colonies to identify the TCR-alpha and beta chains. As described in Pasetto et al., Cancer Immunol. Res., (2016), multiplex PCR of single-cell TCRs was performed on Vβ5.2-negative cells to identify the TCR-alpha and beta chains.

[0119] The fourth KRAS G12DThe reactive TCR (ranked 45th in the infusion bag) is another identified from different TIL fragments (TIL fragment 5) using a single-cell technology approach. Briefly, TIL culture 5 was co-cultured with DC transfected with TMG-1 (encoding G12D KRAS). After 4 hours (h), the TILs were harvested and subjected to the Fluidigm C1 system (Fluidigm, San Francisco, CA) to prepare single-cell RNA-seq samples according to the manufacturer's protocol. Subsequently, the single-cell RNA-seq samples were sequenced by the Illumina MiSeq system and the data were analyzed by an in-house bioinformatics program. TCR-alpha and beta sequences were extracted from samples that showed upregulation of IFN-γ transcripts upon stimulation.

[0120] KRAS G12D In vivo tracking of reactive T cells To determine the frequency of KRAS G12D reactive T cells in the sample, the TCR sequences of the KRAS G12D reactive T cell clones were first identified and these sequences were interrogated against the TCR-Vβ deep sequencing data from the indicated samples. The number of reads of in-frame productive TCRs in the sample ranged from 522,499 to 1,990,345.

[0121] Flow cytometry antibodies The following anti-human flow cytometry antibodies: CD3-AF700 (clone: UCHT1, BioLegend), CD8-PE-Cy7 (clone: SK1, BD Biosciences), CD4-APC-Cy7 (clone: SK3, BioLegend), OX40-FITC (clone: Ber-ACT35, BD Biosciences), 4-1BB-APC (clone: 4B4-1, BioLegend) and Vβ5.2-PE (Beckman Coulter) were used in this report. Anti-mouse TCR-beta constant region antibody (H57-597, eBioscience) conjugated with a fluorescent dye was used to evaluate the transduction efficiency of TCR. The IO Test beta Mark TCR V kit was used to evaluate the repertoire of TCR-Vβ (Beckman Coulter).

[0122] Identification of mutant-reactive T cells and generation of infusion products Using the methods previously described (Lu et al., Clin. Cancer Res., 20:3401-10 (2014); Tran et al., Science, 344:641-5 (2014); Tran et al., Science, 350:1387-90 (2015)), it was tested whether TILs derived from patient 4095 could recognize the somatic mutations expressed in her metastatic lung tumor. TIL culture 6 had the highest frequency of KRAS G12D reactive CD8+ T cells and thus, as described by Tran et al., Science, 344:641-5 (2014), underwent a 2 week rapid expansion process prior to cell infusion.

[0123] KRAS G12D In vivo tracking of KRAS-specific T cell clones T cell receptor (TCR) deep sequencing was performed on gDNA isolated from the patient's infusion product, three separate lung nodules before treatment, the progressive lesion (lesion 3), and peripheral blood at various time points before and after cell infusion (Adaptive Biotechnologies, Seattle WA) to interrogate the frequency of KRAS reactive TCR sequences. G12D

[0124] KRAS G12D Evaluation of the reactivity of KRAS-specific TCR Four KRAS​G12D Reactive TCRs were identified, TCR-alpha and beta chain sequences were synthesized, and then cloned into the MSGV1 retroviral vector (GenScript Inc.). As described in Tran et al., Science, 344:641-5 (2014), retroviral supernatants encoding the TCRs were generated and used to transduce autologous peripheral blood T cells. The TCR-transduced T cells were then co-cultured with autologous peripheral blood mononuclear cells (PBMCs) loaded with serially diluted doses of various KRAS peptides, or with KRAS positive pancreatic cancer cell lines stably expressing or not expressing the restrictive HLA-C*08:02 allele (Tran et al., Science, 350:1387-90 (2015)). T cell reactivity was determined the next day by IFN-γ ELISPOT assay and flow cytometry analysis of the T cell activation markers 4-1BB and OX40 (Tran et al., Science, 350:1387-90 (2015)). G12D positive The reactivity of T cells was determined the next day by IFN-γ ELISPOT assay and flow cytometry analysis of the T cell activation markers 4-1BB and OX40 (Tran et al., Science, 350:1387-90 (2015)). Analysis (Tran et al., Science, 350:1387-90 (2015)) was used to determine the reactivity of T cells the next day.

[0125] Example 1 This example demonstrates the in vivo frequency of KRAS G12D mutation-reactive CD8+ T cells. The patient was a 49-year-old woman with colorectal adenocarcinoma and multiple bilateral lung metastases. She had already received 12 cycles of FOLFOX chemotherapy after sigmoid colectomy and partial cystectomy, followed by 4182 cGy of radiation to the bladder suture line. Immediately thereafter, an increase in the number and size of bilateral lung nodules with FDG avidity occurred in her. Biopsy of the right lower lobe nodule was consistent with metastatic colorectal adenocarcinoma. The biopsy of the right lower lobe nodule was consistent with metastatic colorectal adenocarcinoma.

[0126] A phase II clinical trial (ClinicalTrials.gov number, NCT01174121), approved by the institutional review board and designed to test whether adoptive transfer of ex vivo - expanded tumor - infiltrating lymphocytes (TILs) containing T cells targeting cancer mutations can mediate regression of metastatic solid cancers, enrolled patients. Baseline CT scans revealed lung disease as the only source of cancer progression. Three lung lesions were resected using video - assisted thoracoscopic surgery (VATS), and 24 individual TIL cultures were generated from multiple tumor fragments. To identify the mutations expressed by the tumors, whole - exome and transcriptome sequencing was also performed on the three lesions (Table 2). Each TIL culture was evaluated for reactivity against these mutations. Patient TILs were found to contain CD8+ T cells that specifically recognize KRAS mutations (Figures 1A and B). The TIL culture with the highest frequency of KRAS - reactive CD8+ T cells was selected. The number of selected cells was amplified for treatment (Figures 1B and C). Prior to cell infusion, the patient received a non - myeloablative lymphodepleting chemotherapy regimen (Dudley et al., J. Clin. Oncol., 23:2346 - 57 (2005)) consisting of 60 mg / kg of cyclophosphamide for 2 days, followed by 25 mg / m of fludarabine for 5 days. The patient received a single infusion of 1.48×10 TILs, followed by five administrations of 720,000 IU / kg of interleukin 2 (IL - 2), and was discontinued due to fatigue. The patient tolerated the treatment well and returned home 2 weeks after cell infusion. Approximately 75% of the infused product contained CD8+ T cells that specifically recognize KRAS mutations, and most of these T cells produced multiple effector cytokines (IFN - γ, TNF, and IL - 2) and demonstrated cytotoxic potential. Seven were resected, and 24 individual TIL cultures were generated from multiple tumor fragments. The tumors were expressing mutations. To identify the mutations, whole - exome and transcriptome sequencing was also performed on the three lesions (Table 2). Each TIL culture was evaluated for reactivity against these mutations. Patient TILs were found to contain CD8+ T cells that specifically recognize KRAS mutations (Figures 1A and B). The TIL culture with the highest frequency of KRAS - reactive CD8+ T cells was selected. The number of selected cells was amplified for treatment (Figures 1B and C). Prior to cell infusion, the patient received a non - myeloablative lymphodepleting chemotherapy regimen (Dudley et al., J. Clin. Oncol., 23:2346 - 57 (2005)) consisting of 60 mg / kg of cyclophosphamide for 2 days, followed by 25 mg / m of fludarabine for 5 days. The patient received a single infusion of 1.48×10 TILs, followed by five administrations of 720,000 IU / kg of interleukin 2 (IL - 2), and was discontinued due to fatigue. The patient tolerated the treatment well and returned home 2 weeks after cell infusion. Approximately 75% of the infused product contained CD8+ T cells that specifically recognize KRAS mutations, and most of these T cells produced multiple effector cytokines (IFN - γ, TNF, and IL - 2) and demonstrated cytotoxic potential. Seven were resected, and 24 individual TIL cultures were generated from multiple tumor fragments. The tumors were G12D found to contain CD8+ T cells that specifically recognize KRAS mutations (Figures 1A and B). The TIL culture with the highest frequency of KRAS - reactive CD8+ T cells was selected. The number of selected cells was amplified for treatment (Figures 1B and C). Prior to cell infusion, the patient received a non - myeloablative lymphodepleting chemotherapy regimen (Dudley et al., J. Clin. Oncol., 23:2346 - 57 (2005)) consisting of 60 mg / kg of cyclophosphamide for 2 days, followed by 25 mg / m of fludarabine for 5 days. The patient received a single infusion of 1.48×10 TILs, followed by five administrations of 720,000 IU / kg of interleukin 2 (IL - 2), and was discontinued due to fatigue. The patient tolerated the treatment well and returned home 2 weeks after cell infusion. Approximately 75% of the infused product contained CD8+ T cells that specifically recognize KRAS mutations, and most of these T cells produced multiple effector cytokines (IFN - γ, TNF, and IL - 2) and demonstrated cytotoxic potential. Seven were found to contain CD8+ T cells that specifically recognize KRAS mutations (Figures 1A and B). The TIL culture with the highest frequency of KRAS - reactive CD8+ T cells was selected. The number of selected cells was amplified for treatment (Figures 1B and C). Prior to cell infusion, the patient received a non - myeloablative lymphodepleting chemotherapy regimen (Dudley et al., J. Clin. Oncol., 23:2346 - 57 (2005)) consisting of 60 mg / kg of cyclophosphamide for 2 days, followed by 25 mg / m of fludarabine for 5 days. The patient received a single infusion of 1.48×10 TILs, followed by five administrations of 720,000 IU / kg of interleukin 2 (IL - 2), and was discontinued due to fatigue. The patient tolerated the treatment well and returned home 2 weeks after cell infusion. Approximately 75% of the infused product contained CD8+ T cells that specifically recognize KRAS mutations, and most of these T cells produced multiple effector cytokines (IFN - γ, TNF, and IL - 2) and demonstrated cytotoxic potential. Seven G12D - reactive CD8+ T cells was selected. The number of selected cells was amplified for treatment (Figures 1B and C). Prior to cell infusion, the patient received a non - myeloablative lymphodepleting chemotherapy regimen (Dudley et al., J. Clin. Oncol., 23:2346 - 57 (2005)) consisting of 60 mg / kg of cyclophosphamide for 2 days, followed by 25 mg / m of fludarabine for 5 days. The patient received a single infusion of 1.48×10 TILs, followed by five administrations of 720,000 IU / kg of interleukin 2 (IL - 2), and was discontinued due to fatigue. The patient tolerated the treatment well and returned home 2 weeks after cell infusion. Approximately 75% of the infused product contained CD8+ T cells that specifically recognize KRAS mutations, and most of these T cells produced multiple effector cytokines (IFN - γ, TNF, and IL - 2) and demonstrated cytotoxic potential. Seven 2 of fludarabine for 5 days. The patient received a single infusion of 1.48×10 TILs, followed by five administrations of 720,000 IU / kg of interleukin 2 (IL - 2), and was discontinued due to fatigue. The patient tolerated the treatment well and returned home 2 weeks after cell infusion. Approximately 75% of the infused product contained CD8+ T cells that specifically recognize KRAS mutations, and most of these T cells produced multiple effector cytokines (IFN - γ, TNF, and IL - 2) and demonstrated cytotoxic potential. Seven TILs, followed by five administrations of 720,000 IU / kg of interleukin 2 (IL - 2), and was discontinued due to fatigue. The patient tolerated the treatment well and returned home 2 weeks after cell infusion. Approximately 75% of the infused product contained CD8+ T cells that specifically recognize KRAS mutations, and most of these T cells produced multiple effector cytokines (IFN - γ, TNF, and IL - 2) and demonstrated cytotoxic potential. Seven 11 were resected, and 24 individual TIL cultures were generated from multiple tumor fragments. The tumors were G12D found to contain CD8+ T cells that specifically recognize KRAS mutations (Figures 1A and B). The TIL culture with the highest frequency of KRAS - reactive CD8+ T cells was selected. The number of selected cells was amplified for treatment (Figures 1B and C). Prior to cell infusion, the patient received a non - myeloablative lymphodepleting chemotherapy regimen (Dudley et al., J. Clin. Oncol., 23:2346 - 57 (2005)) consisting of 60 mg / kg of cyclophosphamide for 2 days, followed by 25 mg / m of fludarabine for 5 days. The patient received a single infusion of 1.48×10 TILs, followed by five administrations of 720,000 IU / kg of interleukin 2 (IL - 2), and was discontinued due to fatigue. The patient tolerated the treatment well and returned home 2 weeks after cell infusion. Approximately 75% of the infused product contained CD8+ T cells that specifically recognize KRAS mutations, and most of these T cells produced multiple effector cytokines (IFN - γ, TNF, and IL - 2) and demonstrated cytotoxic potential. Seven effector cytokines (IFN - γ, TNF, and IL - 2) and demonstrated cytotoxic potential. Seven All metastatic lung lesions regressed during the first 40 days of follow-up after cell infusion, and 6 / 7 lesions continued to regress or had a complete response until one lesion (lesion 3) progressed approximately 9 months after treatment. Approximately 9 months after cell infusion, the only progressive lesion (lesion 3), as well as a reactive lesion (lesion 2) that was PET-negative and had no viable tumor cells and was completely necrotic on pathological analysis, were resected by VATS of the left lower lung for excision. The patient remained clinically disease-free 3 months after lung resection.

[0127] The infused TIL product contained at least four KRAS G12D reactive T cell clone types at various frequencies. The three most frequent TCRs in the infused product, which constituted 49.5%, 19.1%, and 6.9% of the infused bag, were reactive to KRAS G12D while the fourth KRAS G12D reactive TCR was at the 45th frequency and was present in only 0.04% of the infused bag (Figures 2A - 2D and Table 5). These KRAS G12D reactive TCRs were not detected in the patient's peripheral blood 1 week before infusion (frequency <0.0002%, Figures 2A - 2D). Dramatic differences were observed in the engraftment of KRAS G12D reactive TCRs after cell infusion. The most dominant clone type of the infused T cells (~7.3×10 10 cells) was not detected in the blood 40 days after cell infusion, while the remaining KRAS reactive T cell clones G12D were detected at this time point (Figures 2A - 2D). The KRAS reactive T cell clones remaining in the peripheral blood were 10.4%, 4.5%, and 0.005% of the total peripheral blood T cells approximately 9 months after cell infusion, G12D and the T cell clone that was most dominant in the peripheral blood at that time was the TRBV10 - 02 mutant KRAS G12DIt was a reactive TCR (Figures 2A-2D and Table 5). In tumors that had progressed compared to peripheral blood, KRAS G12D There did not appear to be enrichment of reactive T cell clones (Figures 2A-2D).

[0128]

Table 5

[0129] Example 2 This example demonstrates the specificity and sensitivity of the KRAS G12D reactive TCR. The nucleotide sequences encoding the TCRs were cloned from each of the four clone types of the KRAS G12D reactive T cells. Each TCR was cloned into an MSGV1-retroviral vector. The amino acid sequences of the alpha and beta chains of each of the four TCRs are shown in Table 6.

[0130]

Table 6

[0131] The nucleotide sequence cloned into the MSGV1-retroviral vector encoded the variable region of the TCR alpha chain (shown in Table 6) and the constant region of the mouse TCR alpha chain, followed by the P2A linker sequence (SEQ ID NO: 58), and the nucleotide sequence encoding the variable region of the TCR beta chain (shown in Table 6) and the constant region of the mouse TCR beta chain. The TCR was further modified to include, in the TM region of the mouse constant region of the alpha chain, substitution(s) of 3 amino acids with hydrophobic amino acids and in combination, cysteine substitutions in the mouse constant regions of both the alpha and beta chains. The full-length amino acid sequences of each of the four TCRs are shown in Table 7. Without being bound by a particular theory or mechanism Although not being constrained, the mouse constant chains of TCR alpha and beta are thought to be able to reduce the mispairing with the endogenous TCR and promote the expression by the host cells of the introduced TCR. Also, the promoted expression and pairing of the introduced TCR alpha chain and beta chain can be achieved by incorporating hydrophobic amino acids into the constant chain of TCR alpha and by introducing a second disulfide bond between the constant regions of the alpha chain and the beta chain.

[0132]

Table 7

[0133] To express one of these four TCRs, autologous peripheral blood T cells were genetically modified. Since the TCR was designed with the constant regions of mouse TCR-alpha and beta, the expression of the introduced TCR on the cell surface was evaluated by flow cytometry analysis for the constant region of mouse TCR-beta (mTCR-beta) 10 days after modification of the TCR gene. Vector-introduced cells were used as a negative control. The data were gated on CD8+ T cells. The percentage of cells expressing the mouse TCR-beta constant region is shown in Table 8.

[0134]

Table 8

[0135] TCR-modified T cells were co-cultured overnight with autologous PBMCs incubated with stepwise amounts of wild-type (WT) or G12D mutant 9-mer or 10-mer peptides of KRAS. The percentage of cells expressing the T cell activation marker 4-1BB was measured. The results are shown in Figures 3A - 3D. ​Well. Three of the four TCRs were selectively reactive against the 9 - amino - acid - long KRAS G12D peptide

[0136]

Chem.

[0137] while one TCR was reactive only against the 10 - amino - acid - long KRAS G12D peptide

[0138]

Chem.

[0139] (Figure 3A - 3D). All TCRs were specific for the mutation and did not recognize wild - type KRAS peptide (Figure 3A - 3D). Peptide titration experiments demonstrated that the TCRs could recognize the peptide at concentrations between 1 - 10 nM when pulsed against autologous PBMC (Figure 3A - 3D).

[0140] TCR - modified T cells were co - cultured overnight with one of two KRAS G12D positive pancreatic cancer cell lines (MDA - Panc48 or HPAC) that either did not express or expressed the HLA - C*08:02 allele. IFN - γ secretion was measured by ELISPOT assay and 4 - 1BB expression was measured by flow cytometry. The results are shown in Figure 4A - 4B. The TCR specifically recognized the pancreatic cancer cell line only when the cell line expressed both the KRAS G12D mutation and the HLA - C*08:02 allele (Figure 4A - 4B).

[0141] Example 3 ​This example demonstrates that cells transduced to express the TRAV12-2 / TRBV10-2 TCR (SEQ ID NOs: 56 and 57) recognize mutant KRAS in the context of HLA alleles C*08:02 or C*05:01. As described in Example 2, autologous peripheral blood T cells were genetically modified to express one of four TCRs. COS7 cells were co-transfected with the full-length wild-type (wt) KRAS or KRAS-G12D gene, and the HLA alleles C*07:01, C*08:02, or C*05:01, and then co-cultured with the indicated KRAS G12D reactive TCR-transduced cells. The next day, cells were analyzed for 4-1BB expression by flow cytometry . The results are shown in FIGS. 5A-5D . Data were gated on TCR-transduced (mouse TCRβ+) CD8+ T cells. HLA-C*07:01 was used as the HLA allele for the negative control.

[0142] All references, including publications, patent applications, and patents cited herein, are hereby incorporated by reference in their entirety to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in full herein.

[0143] The terms "a" and "an" and "the" and "at least one" and similar referents used in the context of describing the invention (in particular, in the context of the following claims) are to be construed to include both the singular and the plural unless otherwise indicated herein or clearly contradicted by context. The use of the term "at least one" following a listing of one or more items (e.g., "at least one of A and B") means, unless otherwise stated herein or clearly contradicted by context, one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B). should be construed. The terms "comprising", "having", "including", and "containing" shall, unless otherwise specified, be construed as open-ended terms (i.e., meaning "including, but not limited to"). The values enumerated in the ranges in this specification are intended to serve simply as a shorthand way of referring individually to each separate value within the range, and each individual value is incorporated into the specification as if it were individually recited herein. All methods described in this specification may be performed in any suitable order, unless otherwise specified herein or unless clearly inconsistent with the context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is merely intended to better explain the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0144] Preferred embodiments of the invention, including the best mode known to the inventors for carrying out the invention, are described herein. Variations of these preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to appropriately use such variations, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, the invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Further, unless otherwise specified herein or unless clearly inconsistent with the context, any combination of the above elements in all possible variations thereof is included in the invention.

Claims

1. 1. An isolated or purified TCR comprising: (a) SEQ ID NOs: 9-14; (b) SEQ ID NOs: 17-22; (c) SEQ ID NOs: 25-30; or (d) SEQ ID NOs: 33 to 38 A TCR comprising the amino acid sequence of

2. below: (i) SEQ ID NO: 15-16; (ii) SEQ ID NOs:23-24; (iii) SEQ ID NOs: 31-32; or (iv) SEQ ID NOs: 39-40 The isolated or purified TCR of claim 1, comprising the amino acid sequence:

3. below: (A) an amino acid sequence of SEQ ID NO:46: (i) X at position 48 of SEQ ID NO:46 is Thr or Cys; (ii) X at position 112 of SEQ ID NO: 46 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 114 of SEQ ID NO: 46 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) the sequence of SEQ ID NO: 46, in which X at position 115 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; and (B) an amino acid sequence of SEQ ID NO: 47, in which X at position 57 of SEQ ID NO: 47 is Ser or Cys; 3. The isolated or purified TCR of claim 1 or 2, further comprising:

4. below: (1) SEQ ID NO:50-51; (2) SEQ ID NOs: 52-53; (3) SEQ ID NO: 54-55; or (4) SEQ ID NOs: 56 to 57 The isolated or purified TCR of any one of claims 1 to 3, comprising the amino acid sequence:

5. 1. An isolated or purified polypeptide comprising: (a) SEQ ID NOs: 9-14; (b) SEQ ID NOs: 17-22; (c) SEQ ID NOs: 25-30; or (d) SEQ ID NOs: 33 to 38 A polypeptide comprising the amino acid sequence of

6. below: (i) SEQ ID NOs: 15-16; (ii) SEQ ID NOs: 23-24; (iii) SEQ ID NOs: 31-32; or (iv) SEQ ID NOs: 39-40 6. The isolated or purified polypeptide of claim 5, comprising the amino acid sequence:

7. below: (A) an amino acid sequence of SEQ ID NO:46: (i) X at position 48 of SEQ ID NO:46 is Thr or Cys; (ii) X at position 112 of SEQ ID NO: 46 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 114 of SEQ ID NO: 46 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) the sequence of SEQ ID NO: 46, in which X at position 115 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; and (B) an amino acid sequence of SEQ ID NO: 47, in which X at position 57 of SEQ ID NO: 47 is Ser or Cys; 7. The isolated or purified polypeptide of claim 5 or 6, further comprising:

8. below: (1) SEQ ID NOs: 50-51; (2) SEQ ID NOs: 52-53; (3) SEQ ID NOs: 54-55; or (4) SEQ ID NOs: 56 to 57 8. The isolated or purified polypeptide according to any one of claims 5 to 7, comprising the amino acid sequence:

9. 1. An isolated or purified protein comprising: (a) a first polypeptide chain comprising an amino acid sequence of SEQ ID NO:9-11 and a second polypeptide chain comprising an amino acid sequence of SEQ ID NO:12-14; (b) a first polypeptide chain comprising an amino acid sequence of SEQ ID NO:17-19 and a second polypeptide chain comprising an amino acid sequence of SEQ ID NO:20-22; (c) a first polypeptide chain comprising an amino acid sequence of SEQ ID NO:25-27 and a second polypeptide chain comprising an amino acid sequence of SEQ ID NO:28-30; or (d) a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 33-35 and a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 36-38. , including protein.

10. below: (i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:15 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:16; (ii) 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; (iii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:31 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:32; or (iv) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 39 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:

40.

10. The isolated or purified protein of claim 9 .

11. below: (A) the first polypeptide chain further comprises the amino acid sequence of SEQ ID NO:46: (i) X at position 48 of SEQ ID NO:46 is Thr or Cys; (ii) X at position 112 of SEQ ID NO: 46 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 114 of SEQ ID NO: 46 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) X at position 115 of SEQ ID NO: 46 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; and (B) the second polypeptide chain further comprises the amino acid sequence of SEQ ID NO:47, wherein X at position 57 of SEQ ID NO:47 is Ser or Cys; 11. An isolated or purified protein according to claim 9 or 10.

12. below: (1) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:50 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:51; (2) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:52 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:53; (3) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:54 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:55; or (4) The first polypeptide chain comprises the amino acid sequence of SEQ ID NO:56, and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:57; 12. An isolated or purified protein according to any one of claims 9 to 11.

13. 13. An isolated or purified nucleic acid comprising a nucleotide sequence encoding a TCR according to any one of claims 1 to 4, a polypeptide according to any one of claims 5 to 8, or a protein according to any one of claims 9 to 12.

14. A recombinant expression vector comprising the nucleic acid of claim 13.

15. 15. An isolated or purified host cell comprising the recombinant expression vector of claim 14.

16. 16. A cell population comprising at least one isolated or purified host cell of claim 15.

17. 17. A pharmaceutical composition comprising a TCR according to any one of claims 1 to 4, a polypeptide according to any one of claims 5 to 8, a protein according to any one of claims 9 to 12, a nucleic acid according to claim 13, a recombinant expression vector according to claim 14, a host cell according to claim 15, or a host cell population according to claim 16, and a pharma- ceutical acceptable carrier.

18. 1. A method for detecting the presence of cancer in a mammal, said method comprising: (a) contacting a sample containing cancer cells with the TCR of any one of claims 1 to 4, the polypeptide of any one of claims 5 to 8, the protein of any one of claims 9 to 12, the nucleic acid of claim 13, the recombinant expression vector of claim 14, the host cell of claim 15, the host cell population of claim 16, or the pharmaceutical composition of claim 17, thereby forming a complex; and (b) detecting the complex. wherein detection of the complex indicates the presence of cancer in the mammal.

19. 19. The method of claim 18, wherein the cancer is pancreatic, colorectal, lung, endometrial, ovarian, or prostate cancer.

20. A TCR according to any one of claims 1 to 4, a polypeptide according to any one of claims 5 to 8, or a polypeptide according to claims 9 to 10 for use in the treatment or prevention of cancer in a mammal.

12. A protein according to any one of claims 12, a nucleic acid according to claim 13, a recombinant expression vector according to claim 14, a host cell according to claim 15, a host cell population according to claim 16, or a pharmaceutical composition according to claim 17.

21. 21. The TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, host cell population, or pharmaceutical composition for use according to claim 20, wherein the cancer is pancreatic, colorectal, lung, endometrial, ovarian, or prostate cancer.

Images