Anti-KRAS-G12D T cell receptor

Abstract

To provide pharmaceutical compositions and methods for treating cancer.SOLUTION: Disclosed is an isolated or purified T cell receptor (TCR) having antigenic specificity for mutated Kirsten rat sarcoma viral oncogene homolog (KRAS) presented in the context of an HLA-Cw*0802 molecule. Related polypeptides and proteins, as well as related nucleic acids, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions are also provided. Also disclosed are methods of detecting the presence of cancer in a mammal and methods of treating or preventing cancer in a mammal.SELECTED DRAWING: 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 Aug. 2, 2016, each of which is incorporated by reference in its entirety.

[0002] Incorporation by Reference of Electronically Submitted Materials The computer readable nucleotide / amino acid sequence listing, which was submitted contemporaneously herewith and is identified as follows, is incorporated herein by reference in its entirety: )1 file. [Background technology]

[0003] 2. Background of the Invention For some cancers, especially when the cancer becomes metastatic and unresectable, treatment options can be very limited. Despite advances in treatment, such as surgery, chemotherapy, and radiation therapy, the prognosis for many cancers, such as pancreatic, colorectal, lung, endometrial, ovarian, and prostate cancer, can be poor. Thus, there is an unmet need for additional treatments for cancer. Summary of the Invention

[0004] Summary of the Invention According to an embodiment of the present invention, the following are provided: (a) SEQ ID NOs: 9 to 14; (b) SEQ ID NOs: 17 to 22; (c) SEQ ID NOs: (d) an isolated or purified TCR comprising the amino acid sequences of SEQ ID NOs: 33 to 38; is provided.

[0005] According to another embodiment of the present invention, the following are provided: (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 1A is a graph showing IFN-γ production (spots / 2e4 cells) determined by ELISPOT assay of 24 individual TIL cultures after coculture with autologous dendritic cells transfected with unrelated tandem minigene (TMG) RNA (filled circles) or the indicated TMG constructs (TMG-1 (stars), TMG-2 (△), TMG-3 (▽), TMG-4 (white diamonds), TMG-5 (black diamonds)) encoding 61 mutations identified by whole exome and transcriptome sequencing. FIG. 1B is a graph showing IFN-γ production (spots / 2e4 cells) determined by ELISPOT assay of TIL culture #6 after co-culture with dendritic cells (DCs) transfected with irrelevant TMG RNA or TMG-1, or overnight incubation with a long mutant peptide encoded by TMG-1 (left axis; unshaded bars), and flow cytometry analysis of 4-1BB expression on CD8+ T cells (%) (right axis; shaded bars). FIG. 1C is a graph showing IFN-γ production (left axis; unshaded bars) determined by ELISPOT assay of infusion products (TIL culture no. 6 after undergoing rapid expansion at clinical scale) after co-culture with DCs transfected with the indicated TMG RNA or overnight incubation with the 24-AA long KRAS-wild type (WT) or KRASG12D peptides, and flow cytometric analysis of 4-1BB expression on CD8+ T cells (%) (right axis; shaded bars). [Diagram 2] 2A-2D are graphs showing the results of TCR-Vβ deep sequencing analysis quantifying the frequency of each of the four identified KRASG12D-reactive T cell clones in the infusion product (Rx1, black bars), three metastatic lung samples before cell transfer (Tu-1, diamonds; Tu-2A, squares; and Tu-2B, triangles), one progressive lesion after cell transfer (Tu-Pro, inverted triangles), and the patient's peripheral blood (circles) at various time points before and after cell infusion. Numbers in parentheses indicate the rank of the TCR sequence in a given sample.

[0010] [ka]

[0011] and ND were not detected (< 0.0002%). A (TRAV4 / TRBV5-6(A)). B (TRAV12-2 / TRBV10-2). C (TRAV4 / TRBV5-6(B)). D (TRAV4 / TRBV5-6 (C)). [Diagram 3] 3A-3D show the results of the immunohistochemical analysis of autologous PBMCs incubated with titrating amounts of KRAS wild-type (WT) 9-mer (open circles), G12D mutant KRAS 9-mer (closed circles), KRAS WT 10-mer (open triangles) or KRAS G12D mutant KRAS 10-mer peptide (closed triangle) after overnight co-culture with autologous PBMCs incubated with titrating amounts of KRAS wild-type (WT) 9-mer (open circles), G12D mutant KRAS 9-mer (closed circles), KRAS WT 10-mer (open triangles) or KRAS G12D mutant KRAS 10-mer peptide (closed triangles) followed by immunohistochemical analysis of the ... FIG. 3C is a graph showing expression of the T cell activation marker 4-1BB on T cells engineered with a TCR comprising the amino acid sequence of (FIG. 3D). [Figure 4]4A and 4B are graphs showing IFN-γ production (spots / 2e4 cells) (A) and 4-1BB expression (B) of T cells genetically modified with the indicated TCRs after overnight co-culture with two KRASG12D-positive pancreatic cancer cell lines not expressing (mock) or expressing the HLA-C*08:02 allele. TRBV5-6(A) TCR (unshaded bars); TRBV10-02 TCR (shaded bars); TRBV5-6(B) TCR (horizontal stripes); TRBV5-6(C) TCR (diagonal stripes). MD, MDA-Panc48; HP, HPAC. Flow cytometry data gated on CD8+ KRASG12D-specific TCR+ cells. ">", >500 spots (not exact). [Diagram 5] 5A-5D are graphs showing 4-1BB expression (%) of T cells genetically modified with the indicated TCRs after overnight co-culture with target COS cells transduced with full-length wild-type (wt) KRAS (unshaded bars) or KRAS-G12D genes (shaded bars) and the indicated HLA alleles: TRBV5-6(A) TCR (FIG. 5A); TRBV10-02 TCR (FIG. 5B); TRBV5-6(B) TCR (FIG. 5C); TRBV5-6(C) TCR (FIG. 5D). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Detailed Description of the Invention GTPase KRas, also known as V-Ki-Ras2 Kirsten rat sarcoma viral oncogene or KRAS2 Kirsten rat sarcoma viral oncogene homolog (KRAS), also referred to as low molecular weight KRAS is a member of the (small)GTPase superfamily. It has two transcriptional alterations Variants: KRAS mutant A and KRAS mutant B. Hereinafter, unless otherwise specified, reference to "KRAS" (mutated or unmutated) refers to both mutant A and mutant B. Without being bound to a particular theory or mechanism, it is believed that when mutated, KRAS may be involved in signal transduction early in the carcinogenesis of many human cancers. A single amino acid substitution can render the protein active. When activated, mutant KRAS binds guanosine-5'-triphosphate (GTP) and converts GTP to guanosine 5'-diphosphate (GDP). The mutant KRAS protein product is constitutively activated. Mutant KRAS proteins can be found in a variety of human cancers, including pancreatic (e.g., pancreatic cancer). , colorectal, lung (e.g., lung adenocarcinoma), endometrial, ovarian (e.g., epithelial ovarian cancer), and prostate cancer, etc. The vector may be expressed in any of the following locations:

[0013] One embodiment of the present invention is a method for producing a human KRAS mutant KRAS antibody having antigen specificity for human mutant KRAS (hereinafter, "mutant KRAS"). The present invention provides an isolated or purified TCR having a G12D mutation. Hereinafter, unless otherwise specified, reference to "TCR" also refers to functional parts and functional variants of the TCR. The TCR of the present invention may have antigen specificity for any KRAS (protein, polypeptide or peptide) having a G12D mutation.

[0014] In one embodiment of the invention, the TCR comprises the amino acid sequence of SEQ ID NO: 3 or 4, or The amino acid sequence of the mutant KRAS mutant A protein of SEQ ID NO: 3 is generally the same as that of the mutant KRAS mutant A protein of SEQ ID NO: 3, except that the glycine at position 12 in SEQ ID NO: 3 is replaced with aspartic acid. 1 to 189 of the amino acid sequence of wild-type (WT) KRAS protein mutant A of SEQ ID NO: 1 The amino acid sequence of the mutant KRAS mutant B protein of SEQ ID NO: 4 generally corresponds to the mutated KRAS mutant B protein of SEQ ID NO: 4, except that the glycine at position 12 is replaced with aspartic acid. and corresponds to positions 1 to 188 of the amino acid sequence of WT KRAS protein mutant B of SEQ ID NO:2.

[0015] In one embodiment of the present invention, the TCR is directed against the KRAS peptide having the G12D mutation. The KRAS peptide has antigen specificity and can be of any length. For example, the TCR has a G12D mutation. The KRAS peptide may have antigen specificity for a KRAS peptide having about 8 to about 24 amino acids. In one embodiment of the present invention, the length of the ribozyme is about 9 to about 11 amino acid residues. The TCR may have antigen specificity for a KRAS peptide having a 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), which is a KRAS with a G12D mutation. 10-18 The amino acid sequence of the mutant KRAS peptide of SEQ ID NO: 8 having the G12D mutation is generally the same as that of the unmutated WT KRAS peptide of SEQ ID NO: 7, except that the glycine at position 3 in SEQ ID NO: 8 is replaced with aspartic acid. 10-18 Peptide A It corresponds to positions 1 to 9 of the amino acid sequence.

[0016] In yet another embodiment of the invention, the TCR is directed against a KRAS peptide having a G12D mutation. The mutant KRAS peptide may have antigen specificity, and the mutant KRAS peptide may be GADGVGKSA (mutant KRAS 10-18 ; SEQ ID NO: 8); or GADGVGKSAL (mutated KRAS 10-19 SEQ ID NO: 6) In an exemplary embodiment, the TCR has antigen specificity for a mutant KRAS epitope, The mutant KRAS epitope is GADGVGKSA (mutant KRAS 10-18 ; SEQ ID NO: 8) or GADGVGKSAL (mutated KRAS 10-19 ;SEQ ID NO: 6)

[0017] In one embodiment of the present invention, the TCR of the present invention recognizes mutant KRAS in the context of the HLA-Cw8 molecule. In this regard, it means that the TCR can induce an immune response when it binds to mutant KRAS in the context of HLA-Cw8 molecules. The TCR of the present invention can recognize mutant KRAS presented by HLA-Cw8 molecules and can bind to HLA-Cw8 molecules in addition to mutant KRAS. Thus, exemplary HLA-Cw8 molecules that 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 morphology, the TCR recognizes mutant KRAS in the context of the HLA-Cw*0802 molecule.

[0018] In one embodiment of the present invention, the ability to recognize mutant KRAS in the context of the HLA-Cw8 molecule is In addition to having the HLA-Cw5 subtype, one of the TCRs of the present invention (TRAV12-2 / TRBV10-2 (Table 5)) also has the HLA-Cw5 subtype. In this regard, the TCR can elicit an immune response when it binds to mutant KRAS in 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 recognize mutant KRAS presented by an HLA-Cw5 molecule. Within the context in which the TCRs of the present invention recognize mutant KRAS, exemplary HLA-Cw5 molecules include those encoded by the HLA-Cw*0501, HLA-Cw*0502, HLA-Cw*0503, HLA-Cw*0504, HLA-Cw*0505, HLA-Cw*0506, HLA-Cw*0508, HLA-Cw*0509 and HLA-Cw*0510 alleles. In a preferred embodiment, the TCR recognizes mutant KRAS within the context of the HLA-Cw*0501 molecule. The amino acid sequences of HLA-Cw*0802 and HLA-Cw*0501 are differentiated by only two amino acid residues. Without being bound to 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 may be expressed by cells used for adoptive cell transfer. This provides many advantages. Mutated KRAS is expressed by cancer cells and not by healthy non-cancer cells. Without being bound to a particular theory or mechanism, the TCRs of the present invention: It is believed that the destruction of healthy non-cancerous cells is advantageously targeted, while the destruction of healthy non-cancerous cells is minimized or eliminated, thereby reducing toxicity, for example by minimizing or eliminating toxicity. Furthermore, the TCRs of the present invention may be used in combination with other types of treatment, such as, for example, chemotherapy, surgery or radiation therapy. Advantageously, unresponsive mutant KRAS positive cancers may be successfully treated or prevented. Additionally, the TCRs of the present invention provide highly avid recognition of mutant KRAS, which is consistent with unengineered tumor cells ( (e.g., the ability to recognize tumor cells that have not been treated with interferon (IFN) gamma, that have not been transfected with vectors encoding one or both of mutant KRAS and HLA-Cw*0802, that have not been pulsed with a KRAS peptide carrying the G12D mutation, or a combination thereof) Furthermore, the HLA-Cw*0802 allele is associated with a higher incidence of HIV infection in Caucasians and African Americans. The TCRs of the present invention are expressed in up to about 8% and up to about 11% of the African population, respectively. Thus, the TCRs of the present invention may increase the number of cancer patients eligible for immunotherapy, including patients who express 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 term "antigen specificity" refers to a TCR that has high avidity. For example, (a) a mutant KRAS peptide having a low concentration (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 the above values) can be bound to and immunologically recognized by the mutant KRAS peptide. Rushed antigen negative HLA-Cw*0802 + or (b) causing the target cell to express mutant KRAS. Antigen-negative HLA-Cw*0802, which contains a nucleotide sequence encoding a mutant KRAS + Target cells, expressing TCR when co-cultured with approximately 1 x 10 4 ~Approx. 1×10 5 T cells are 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) IFN-γ (≥ 700 pg / mL, ≥ 1000 pg / mL, ≥ 5,000 pg / mL, ≥ 7,000 pg / mL, ≥ 10,000 pg / mL, ≥ 20,000 pg / mL, or a range defined by any two of the preceding values) If the TCR secretes the mutant KRAS, the TCR can be considered to have "antigen specificity" for the mutant KRAS. Cells expressing the positive TCR also expressed higher levels of the antigen-negative HLA-Cw*0802 T cells pulsed with the mutant KRAS peptide. +Upon co-culture with target cells, it is capable of secreting IFN-γ.

[0021] Alternatively or additionally, (a) antigen-negative HLA-Cw*0802 pulsed with a low concentration of mutant KRAS peptide. + or (b) an antigen-negative HLA-Cw*0802 into which a nucleotide sequence encoding a mutant KRAS has been introduced such that the target cell expresses the mutant KRAS. + When co-cultured with target cells, TCR A TCR may be considered to have "antigen specificity" for mutant KRAS if the expressing T cells secrete at least twice as much IFN-γ as the amount of IFN-γ expressed by the negative control. The gender control may be, for example, (i) (a) an antigen-negative HLA-Cw*0802 antibody pulsed with the same concentration of an irrelevant peptide (e.g., some other peptide having a different sequence than the mutant KRAS peptide). + target cells, young or (b) encoding an irrelevant peptide such that the target cell expresses the irrelevant peptide. Antigen-negative HLA-Cw*0802 into which the nucleotide sequence has been introduced + Target cells, co-cultured with TCR or (ii) antigen-negative HLA-Cw*0802 T cells pulsed with the same concentration of mutant KRAS peptide (a). + or (b) a gene encoding a mutant KRAS gene such that the target cell expresses the mutant KRAS. Antigen-negative HLA-Cw*0802, into which a nucleotide sequence + The target cells can be non-transduced T cells (e.g., from PBMCs that do not express a TCR) co-cultured with the target cells. IFN-γ secretion is For example, it can be measured by methods known in the art, such as enzyme-linked immunosorbent assay (ELISA).

[0022] Alternatively or additionally, (a) antigen-negative HLA-Cw*0802 pulsed with a low concentration of mutant KRAS peptide. +or (b) an antigen-negative HLA-Cw*0802 into which a nucleotide sequence encoding a mutant KRAS has been introduced such that the target cell expresses the mutant KRAS. + When co-cultured with target cells, the number of T cells expressing a TCR that secretes IFN-γ is reduced compared to the number of negative control T cells that secrete IFN-γ. If the TCR is at least twice as strong as the mutant KRAS, the TCR may be considered to have "antigen specificity" for the mutant KRAS. The peptide concentrations and negative controls may be as described herein for other aspects of the invention. The number of cells secreting IFN-γ may be determined using any method known in the art, e.g., ELISPOT. It can be measured by known methods.

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

[0024] The invention provides TCRs comprising two polypeptides (i.e., polypeptide chains), e.g., a TCR alpha (α) chain, a TCR beta (β) chain, a TCR gamma (γ) chain, a TCR delta (δ) chain, or a combination thereof. The TCR polypeptides of the invention are intended to provide a method for determining whether the TCR is capable of activating an antigen specificity for a mutant KRAS. It may comprise any amino acid sequence, provided that it has the following structure:

[0025] In one embodiment of the invention, the TCR comprises two polypeptide chains, each comprising a variable region comprising complementarity determining region (CDR) 1, CDR2 and CDR3 of the TCR. The TCR comprises a CDR1 (α chain CDR1) comprising the amino acid sequence of SEQ ID NO: 9, an amino acid sequence of SEQ ID NO: 10, a first polypeptide chain comprising a CDR2 (CDR2 of the alpha chain) comprising the amino acid sequence of SEQ ID NO:11, and a CDR3 (CDR3 of the alpha chain) comprising the amino acid sequence of SEQ ID NO:12; and a CDR1 (CDR1 of the beta chain) comprising the amino acid sequence of SEQ ID NO:13. 13 (CDR2 of the β chain), and the amino acid sequence of SEQ ID NO: 14 and a second polypeptide chain comprising a CDR3 (CDR3 of the β chain) comprising:

[0026] In another embodiment of the invention, the TCR comprises CDR1 (α CDR1 (α chain CDR1) comprising the amino acid sequence of SEQ ID NO: 18 (α chain CDR2) and the amino acid sequence of SEQ ID NO: 19 a first polypeptide chain comprising a CDR3 (alpha chain CDR3) comprising the amino acid sequence of SEQ ID NO: 20; a second polypeptide comprising a CDR1 (CDR1 of the β chain) comprising the amino acid sequence of SEQ ID NO:21, a CDR2 (CDR2 of the β chain) comprising the amino acid sequence of SEQ ID NO:22, and a CDR3 (CDR3 of the β chain) comprising the amino acid sequence of SEQ ID NO:22; Chains, including.

[0027] In another embodiment of the invention, the TCR comprises CDR1 (α a first polypeptide chain comprising CDR1 (alpha chain CDR1), CDR2 (alpha chain CDR2) comprising the amino acid sequence of SEQ ID NO: 26, and CDR3 (alpha chain CDR3) comprising the amino acid sequence of SEQ ID NO: 27; a second polypeptide comprising a CDR1 (CDR1 of the β chain) comprising the amino acid sequence of SEQ ID NO:29, a CDR2 (CDR2 of the β chain) comprising the amino acid sequence of SEQ ID NO:30, and a CDR3 (CDR3 of the β chain) comprising the amino acid sequence of SEQ ID NO:30; Chains, including.

[0028] In another embodiment of the invention, the TCR comprises CDR1 (α a first polypeptide chain comprising CDR1 (alpha chain CDR1), CDR2 (alpha chain CDR2) comprising the amino acid sequence of SEQ ID NO: 34, and CDR3 (alpha chain CDR3) comprising the amino acid sequence of SEQ ID NO: 35; a second polypeptide comprising a CDR1 (CDR1 of the β chain) comprising the amino acid sequence of SEQ ID NO: 37, 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. Chains, including.

[0029] 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 to 14, 17 to 22, 25 to 30, and 33 to 38. In a particularly preferred embodiment, the TCR comprises the amino acid sequence of: (i) SEQ ID NOs: 9 to 11; (ii) SEQ ID NOs: 12 to 14; (iii) SEQ ID NOs: 17 to 19; (iv) SEQ ID NOs: 20 to 22; (v) SEQ ID NOs: 25 to 27; (vi) SEQ ID NOs: 28 to 30; (vii) SEQ ID NOs: 33 to 35; or (viii) SEQ ID NOs: 36 to 38. The TCR comprises the following amino acid sequences: (a) all of SEQ ID NOs: 9 to 14; (b) all of SEQ ID NOs: 17 to 22; (c) all of SEQ ID NOs: 25 to 30; or (d) all of SEQ ID NOs: 33 to 38.

[0030] In one embodiment of the present invention, the TCR comprises an amino acid sequence of a variable region of the TCR comprising the above-mentioned CDR. In this regard, the TCR comprises the following sequences: 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: 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 beta chain); both SEQ ID NOs:15 and 16; both SEQ ID NOs:23 and 24; It may comprise 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 comprises the amino acid sequences of (i) both SEQ ID NOs: 15 to 16; (ii) both SEQ ID NOs: 23 to 24; (iii) both SEQ ID NOs: 31 to 32; or (iv) both SEQ ID NOs: 39 to 40.

[0031] The TCR of the present invention may further comprise an alpha chain constant region and a beta chain constant region. The TCR may be derived from any suitable species, e.g., human or mouse. In one embodiment of the invention, the TCR further comprises mouse α and β chain constant regions, or human α and β chain constant regions. As used herein, the terms "murine" or "human" refer to the TCR or any component of a TCR described herein (e.g., complementarity determining regions (CDRs), variable regions, constant regions, etc.). When referring to a TCR (or a component thereof) derived from a mouse or a human, respectively, i.e., originating or having originated in a mouse T cell or a human T cell, respectively, it is understood that the TCR (or a component thereof) is a TCR that is derived from a mouse or a human T cell, respectively. "TCR" means a expressed TCR (or a component thereof).

[0032] In one embodiment of the invention, the TCR further comprises human α and β chain constant regions. The TCR is SEQ ID NO: 41 (human The amino acid sequence of SEQ ID NO:41 (human α chain constant region), SEQ ID NO:42 (human β chain constant region), SEQ ID NO:43 (human β chain constant region), both SEQ ID NOs:41 and 42, or both SEQ ID NOs:41 and 43. In one embodiment of the present invention, the TCR is a combination of any of the CDR regions described herein. In combination, the TCR comprises any of the human constant regions described herein. The following: (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) (d) all of SEQ ID NOs: 25 to 30, 41 and 42; (e) all of SEQ ID NOs: 33 to 38, 41 and 42; (f) all of SEQ ID NOs: 17 to 22, 41 and 43; (g) all of SEQ ID NOs: 25 to 30 or (h) all of the amino acid sequences of SEQ ID NOs: 33-38, 41 and 43. In one embodiment of the present invention, the TCR may comprise any of the variable regions described herein. In this regard, the TCR (i) all of SEQ ID NOs: 15 to 16, 41, and 42; (ii) all of SEQ ID NOs: 23 to 24, 41, and 42 (iii) all of SEQ ID NOs: 31 to 32, 41 and 42; (iv) all of SEQ ID NOs: 39 to 42; (v) SEQ ID NOs: (vi) all of SEQ ID NOs: 23-24, 41 and 43; (vii) all of SEQ ID NO: 31 or (viii) all of the amino acid sequences of SEQ ID NOs: 39 to 40, 41, and 43. It may include.

[0033] One embodiment of the present invention is a chimeric TCR comprising a human variable region and a mouse constant region, The present invention provides a chimeric TCR, the TCR having 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 provide a mismatch 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 reduce expression of the TCR of the present invention. may be increased 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 α chain constant region), SEQ ID NO: 45 (WT mouse β chain constant region), or both SEQ ID NOs: 44 and 45. Preferably, the TCR of the present invention comprises the amino acid sequence of SEQ ID NOs: 44 and 45. The chimeric TCR comprises both amino acid sequences as described herein with respect to other aspects of the invention. Any of the murine constant regions described herein in combination with any of the CDR regions described herein. In this regard, the TCR may comprise the amino acid sequence of: (a) all of SEQ ID NOs: 9 to 14, 44 and 45; (b) all of SEQ ID NOs: 17 to 22, 44 and 45; (c) all of SEQ ID NOs: 25 to 30, 44 and 45; or (d) all of SEQ ID NOs: 33 to 38, 44 and 45. In another embodiment of the invention, the chimeric TCR may comprise any of the variable regions described herein in relation to other aspects of the invention. In this regard, the TCR may comprise any of the murine constant regions described herein in combination with the amino acid sequences of: (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. It may comprise an array.

[0034] In one embodiment of the invention, the TCR comprises a substituted constant region. The TCR may have one, two, three or four amino acid substitutions in the constant regions of one or both of the α and β chains. The TCR may comprise any of the amino acid sequences of the TCRs described herein having (a) or (b). Preferably, the TCR comprises a murine constant region having one, two, three or four amino acid substitution(s) in one or both of the α and β chain murine constant regions. In this case, the TCR contains one, two, three or four amino acid substitution(s) in the murine constant region of the α chain. and a murine constant region having one amino acid substitution in the murine constant region of the β chain. In some embodiments, the TCR comprising a substituted constant region is an unsubstituted TCR. Compared to the parent TCR containing the mutant (wild-type) constant region, +Advantageously, the antibody provides one or more of the following: increased recognition of the target, increased expression by the host cell, reduced mispairing with endogenous TCR, and increased anti-tumor activity. Generally, the antibody is an antibody that contains a substituted murine constant region of the TCR α and β chains. The amino acid sequences (SEQ ID NOs: 46 and 47, respectively) correspond to all or part of the amino acid sequences of the unsubstituted murine constant regions (SEQ ID NOs: 44 and 45, respectively), with SEQ ID NO: 46 having one, two, three or four amino acid substitution(s) when compared to SEQ ID NO: 44, and SEQ ID NO: 47 having one amino acid substitution when compared to SEQ ID NO: 45. In this regard, the present invention One embodiment of the present invention is a polypeptide comprising (a) SEQ ID NO: 46 (the alpha chain constant region), in which (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; and (b) SEQ ID NO:47 (β chain constant region) in which X at position 57 is Ser or Cys. In one embodiment of the invention, the TCR comprising SEQ ID NO: 46 does not comprise SEQ ID NO: 44 (unsubstituted murine constant region of the α chain). In one embodiment of the invention For example, a TCR comprising SEQ ID NO:47 does not comprise SEQ ID NO:45 (unsubstituted murine constant region of the β chain).

[0035] In one embodiment of the invention, the substituted constant region comprises a cysteine ​​substitution in the constant region of one or both of the α and β chains to provide a cysteine ​​substituted TCR. The opposing cysteines in the α and β chains are located in the constant regions of the α and β chains of the TCR being replaced. 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 is a hydrophobic amino acid substituted TCR, in which the native Ser112, Met114 and Gly115 of SEQ ID NO: 44 may be substituted with Leu, Ile or Val. Preferably, all three of the native Ser112, Met114 and Gly115 of SEQ ID NO: 44 are independently substituted with Ala, Val, Leu, Ile , Pro, Phe, Met, or Trp; preferably Leu, Ile, or Val. In this case, the hydrophobic amino acid substituted TCR is an α chain constant region comprising the amino acid sequence of SEQ ID NO:46. wherein X at position 48 is native Thr, X at position 112 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp, and X at position 114 is Met, Ala, Val, Leu, Ile, Pro, Phe or Trp; and a region in which X at position 115 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp, A β chain constant region comprising the amino acid sequence of SEQ ID NO: 47, in which X at position 57 is the native Ser (a hydrophobic amino acid substituted TCR comprising SEQ ID NO: 46 is SEQ ID NO: 44 (a substituted α chain In a preferred embodiment, the polypeptide comprises a hydrophobic amino acid substitution. The recombinant TCR comprises an α chain constant region comprising the amino acid sequence of SEQ ID NO:46, where X at position 48 is native Thr, X at position 112 is Leu, X at position 114 is He, and X at position 115 is Val, and a β chain constant region comprising the amino acid sequence of SEQ ID NO:47, where X at position 57 is native Thr, X at position 112 is Leu, X at position 114 is He, and X at position 115 is Val. The hydrophobic amino acid substituted TCRs of the present invention may comprise a substituted constant region in addition to any of the CDRs or variable regions described herein.

[0037] In one embodiment of the invention, the substituted amino acid sequence comprises a cysteine ​​substitution in the constant region of one or both of the α and β chains in combination with a hydrophobic amino acid 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 (also referred to herein as a "cysteine-substituted, hydrophobic amino acid substituted TCR"). In this regard, the TCR has native Thr48 of SEQ ID NO:46 substituted with Cys; One, two or three of the native Ser112, Met114 and Gly115 of SEQ ID NO:46 are independently is replaced by Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; preferably by Leu, Ile, or Val. and a cysteine-substituted, hydrophobic amino acid substituted TCR, in which native Ser57 of SEQ ID NO: 47 is substituted with Cys. Preferably, all three of native Ser112, Met114 and Gly115 of SEQ ID NO: 46 may be independently substituted with Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; preferably Leu, Ile or Val. In one embodiment, the cysteine-substituted, hydrophobic amino acid substituted TCR The recombinant TCR is an α chain comprising the amino acid sequence of SEQ ID NO: 46, in which X at position 48 is Cys, X at position 112 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp, and 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 Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp. (SEQ ID NO: 46 does not include SEQ ID NO: 44 (unsubstituted alpha chain), and SEQ ID NO: 47 does not include SEQ ID NO: 45 (unsubstituted beta chain).) Preferably, The substituted / hydrophobic amino acid substituted TCR is an α chain comprising the amino acid sequence of SEQ ID NO: 46, X is Cys, X at position 112 is Leu, X at position 114 is Ile, and X at position 115 is Val and a β-chain comprising the amino acid sequence of SEQ ID NO: 47, where X at position 57 is Cys. In this regard, a cysteine-substituted, hydrophobic amino acid-substituted TCR comprises the amino acid sequence of SEQ ID NO: 48. and a β chain constant region comprising the amino acid sequence of SEQ ID NO: 49. The cysteine-substituted / hydrophobic amino acid-substituted TCRs of the invention may comprise 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 is Each of the TCR α and β chains of the present invention may be any of the α, β and β chains. In this regard, the α chain of the TCR of the present invention may independently comprise the amino acid sequences of SEQ ID NOs: 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, The α chain may comprise any of the β chains of the TCR. In this regard, the β chain of the TCR of the present invention has the amino acid sequence of SEQ ID NO: 51, 53, 55 or 57. Thus, the TCR of the present invention may comprise 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 NOs:50 and 51, the sequence The TCR of the present invention may comprise the amino acid sequences of both SEQ ID NOs: 52 and 53, both SEQ ID NOs: 54 and 55, or both SEQ ID NOs: 56 and 57. Preferably, the TCR of the present invention comprises (1) both SEQ ID NOs: 50 to 51; (3) both of SEQ ID NOs: 54 to 55; or (4) both of the amino acid sequences of SEQ ID NOs: 56 to 57.

[0039] The present invention also provides a polypeptide comprising a functional portion of any of the TCRs described herein. As used herein, the term "polypeptide" includes oligopeptides and refers to a single chain of amino acids linked by one or more peptide bonds.

[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 portion, 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. , a functional portion comprising 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 may comprise 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 α-chain), SEQ ID NO:13 (CDR2 of the α-chain), SEQ ID NO:14 (CDR3 of the α-chain), SEQ ID NO:15 (CDR3 of the α-chain), SEQ ID NO:16 (CDR3 of the α-chain), SEQ ID NO:17 (CDR3 of the α-chain), SEQ ID NO:18 (CDR3 of the α-chain), SEQ ID NO:19 (CDR3 of the (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 In another embodiment of the invention, the polypeptide may comprise the amino acid sequence of SEQ ID NO:17 (α chain CDR1), SEQ ID NO:18 (α chain CDR2), SEQ ID NO:19 (α chain CDR3), SEQ ID NO:20 (β chain CDR1), SEQ ID NO:21 (β chain CDR2), SEQ ID NO:22 (β chain CDR3), or a combination thereof. In another embodiment of the invention, the polypeptide may comprise the amino acid sequence of SEQ ID NO:25 (α chain CDR1), SEQ ID NO:26 (α chain CDR2), SEQ ID NO:27 (α chain CDR3), SEQ ID NO:28 (α chain CDR4), SEQ ID NO:29 (α chain CDR5), SEQ ID NO:30 (α chain CDR6), SEQ ID NO:31 (α chain CDR7), SEQ ID NO:32 (α chain CDR8), SEQ ID NO:33 (α chain CDR9), SEQ ID NO:34 (α chain CDR10), SEQ ID NO:35 (α chain CDR11), SEQ ID NO:36 (α chain CDR22), SEQ ID NO:37 (α chain CDR3), SEQ ID NO:38 (α chain CDR12), SEQ ID NO:39 (α chain CDR13), SEQ ID NO:40 (α chain CDR14), SEQ ID NO:41 (α chain CDR15), SEQ ID NO:42 (α chain CDR16), SEQ ID NO:43 (α chain CDR17), SEQ ID NO:44 (α chain CDR18), SEQ ID NO:45 (α chain CDR19), SEQ ID NO:50 (α chain CDR110), SEQ ID NO:51 (α chain CDR111), SEQ ID NO:52 (α chain CDR122), SEQ ID NO:53 (α chain CDR133), SEQ ID NO:54 (α chain CDR111), SEQ ID NO: 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 In another embodiment of the invention, the polypeptide may comprise a combination of amino acid sequences of SEQ ID NO: 33 (CDR1 of the alpha chain), SEQ ID NO: 34 (CDR2 of the alpha chain), SEQ ID NO: 35 (CDR3 of the alpha chain), SEQ ID NO: 36 (CDR4 of the alpha chain), SEQ ID NO: 37 (CDR5 of the alpha chain), SEQ ID NO: 38 (CDR6 of the alpha chain), SEQ ID NO: 39 (CDR7 of the alpha chain), SEQ ID NO: 40 (CDR8 of the alpha chain), SEQ ID NO: 41 (CDR9 of the alpha chain), SEQ ID NO: 42 (CDR10 of the alpha chain), SEQ ID NO: 43 (CDR11 of the alpha chain), SEQ ID NO: 44 (CDR22 of the alpha chain), SEQ ID NO: 45 (CDR33 of the alpha chain SEQ ID 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 The polypeptide may comprise any combination of the amino acid sequences of SEQ ID NO:9. (b) both of SEQ ID NOs: 17 to 22; (c) both of SEQ ID NOs: 25 to 30; or (d) both of the amino acid sequences of SEQ ID NOs: 33 to 38.

[0043] In one embodiment of the present invention, the polypeptide of the present invention comprises, for example, In this regard, the polypeptide may comprise a variable region of a TCR of the present invention comprising a combination of the sequence The polypeptide may comprise the amino acid sequence of SEQ ID NO:15 (alpha chain variable region), SEQ ID NO:16 (beta chain variable region), both SEQ ID NOs:15 and 16, SEQ ID NO:23 (alpha chain variable region), SEQ ID NO:24 (beta chain variable region), both SEQ ID NOs:23 and 24, SEQ ID NO:31 (alpha chain variable region), SEQ ID NO:32 (beta chain variable region), both SEQ ID NOs:31 and 32, SEQ ID NO:39 (alpha chain variable region), SEQ ID NO:40 (beta chain variable region), or both SEQ ID NOs:39 and 40. Preferably, the polypeptide comprises the amino acid sequence 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 is a constant domain of the TCR of the present invention. In this regard, the polypeptide may further comprise a region as set forth in SEQ ID NO:41 (human constant region for the α chain), SEQ ID NO:42 (human constant region for the β chain), SEQ ID NO:43 (human constant region for the β chain), SEQ ID NO:44 (WT mouse constant region for the α chain), SEQ ID NO:45 (WT mouse constant region for the β chain), SEQ ID NO:46 (human constant region for the α chain), SEQ ID NO:47 (human constant region for the α chain), SEQ ID NO:48 (human constant region for the β chain), SEQ ID NO:49 (human constant region for the α chain), SEQ ID NO:50 (human constant region for the β chain), SEQ ID NO:51 (human constant region for the α chain), SEQ ID NO:52 (human constant region for the β chain), SEQ ID NO:53 (human constant region for the α chain), SEQ ID NO:54 (human constant region for the α chain), SEQ ID NO:55 (human constant region for the β chain), SEQ ID NO:56 (human constant region for the α chain), SEQ ID NO:57 (human constant region for the β chain), SEQ ID NO:58 (human constant region for the α chain), SEQ ID NO:59 (human constant region for the β chain), SEQ ID NO:60 (human constant region for the α chain), SEQ ID NO:61 (human constant region for the β chain), SEQ ID NO:62 (human constant region for the α chain), SEQ ID NO:63 (human constant region for the β chain), SEQ ID NO:64 (human constant region for the α chain), SEQ ID NO:65 (human constant region for the β chain), SEQ ID NO:66 (human constant region for the α chain), SEQ ID NO:67 (human constant region for the β chain), SEQ ID NO:68 (human constant region for the α chain), SEQ ID NO:69 (human constant region for the β chain), S SEQ ID NO:47 (substituted mouse constant region of the β chain), SEQ ID NO:48 (cysteine-substituted and hydrophobic amino acid-substituted mouse constant region of the α chain), SEQ ID NO:50 (cysteine-substituted and hydrophobic amino acid-substituted mouse constant region of the α chain), No. 49 (mouse constant region with cysteine ​​substitutions in the α chain and hydrophobic amino acid substitutions), SEQ ID NO: The polypeptide may further comprise the amino acid sequence of 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, as with other aspects of the invention, the polypeptide comprises the amino acid sequence of any of the CDR regions or variable regions described herein. (i) both of SEQ ID NOs: 44 and 45; (ii) both of SEQ ID NOs: 46 and 47, in combination with any of the regions (iii) both SEQ ID NOs: 48 and 49; (iv) both SEQ ID NOs: 41 and 42; or (v) SEQ ID NOs: It further comprises both amino acid sequences of Nos. 41 and 43.

[0045] In one embodiment of the invention, the polypeptide of the invention comprises a TCR polypeptide as described herein. In this regard, a polypeptide of the invention may comprise 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, a polypeptide of the invention may comprise both chains of the TCR described herein. For example, a polypeptide of the invention may comprise the amino acid sequence of SEQ ID NO:50 and SEQ ID NO:51. The polypeptide may comprise both of the amino acid sequences of SEQ ID NOs: 50 to 51, both of SEQ ID NOs: 52 and 53, both of SEQ ID NOs: 54 and 55, or both of SEQ ID NOs: 56 and 57. Preferably, the polypeptide comprises the amino acid sequences of (1) both of SEQ ID NOs: 50 to 51; (2) both of SEQ ID NOs: 52 to 53; (3) both of SEQ ID NOs: 54 to 55; or (4) both of SEQ ID NOs: 56 to 57.

[0046] The present invention further relates to a protein comprising at least one polypeptide as described herein. By "protein" is meant a molecule containing one or more polypeptide chains.

[0047] In one embodiment, the protein of the present invention comprises: (a) an amino acid sequence of SEQ ID NO: 9 to 11; 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.

[0048] In another embodiment of the 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 polypeptide chain may comprise:

[0049] The protein of the invention may further comprise any of the constant regions described herein with respect to other aspects of the invention. In this regard, in an embodiment of the invention, the first polypeptide chain comprises: (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 Thr or Cys; (iii) 114 of SEQ ID NO: 46, wherein X is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; X at position 115 of SEQ ID NO:46 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) and (B) the second polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 47, where X at position 57 of SEQ ID NO: 47 is Ser or Cys. The first polypeptide chain is represented by SEQ ID NO: 41 (constant region of human α chain), SEQ ID NO: 44 (constant region of α chain) Mouse WT constant region) or SEQ ID NO: 48 (α chain cysteine ​​substitutions and hydrophobic amino acid substitutions The second polypeptide chain may further 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). WT constant region) or SEQ ID NO: 49 (β-chain cysteine ​​substitutions and hydrophobic amino acid substitutions The antibody may further comprise an amino acid sequence of a mouse constant region.

[0050] Alternatively or additionally, the protein of the invention may comprise: (1) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:50 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:51; (2) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:52 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:53; (3) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:54 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:55; or (4) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:56 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:57. In this example, the protein of the invention may be a TCR. Alternatively, This includes, for example, when the protein comprises a single polypeptide chain that comprises 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 comprises another amino acid sequence, e.g., an amino acid sequence encoding an immunoglobulin or portion thereof. In this regard, the present invention also relates to a fusion protein comprising the polypeptide of the present invention described herein, together with at least one other polypeptide. The present invention provides a fusion protein comprising at least one of the peptides. It may exist as a separate polypeptide of the protein or as a polypeptide expressed in frame (in tandem) with one of the polypeptides of the invention described herein. Other polypeptides may be present in the immune system, such as immunoglobulins, CD3, CD4, CD8, MHC molecules, CD1 molecules, and the like. The polypeptide may encode any peptidic or proteinaceous molecule, or portion thereof, including but not limited to, e.g., CD1a, CD1b, CD1c, CD1d, etc.

[0051] Fusion proteins include one or more copies of a polypeptide of the invention and / or another polypeptide. For example, a fusion protein may contain a polypeptide of the invention and one or more copies of the polypeptide. and / or 1, 2, 3, 4, 5 or more copies of another polypeptide. Suitable methods for producing the same are known in the art and include, for example, recombinant methods.

[0052] In some embodiments of the invention, the TCRs, polypeptides and proteins of the invention are In this regard, the TCRs, polypeptides and proteins of the present invention may be expressed as a single protein including a linker peptide connecting the α and β chains. The linker peptide may be included to facilitate expression of the recombinant TCR, polypeptide and / or tag in a host cell. The linker peptide may advantageously facilitate expression of the protein. The linker peptide may comprise any suitable amino acid sequence. For example, the linker peptide may comprise SEQ ID NO: 58. Upon expression of a construct comprising the linker peptide by a host cell, the linker peptide may be cleaved, resulting in separate α and β chains. In one embodiment of the invention, the TCR, polypeptide or protein is The protein can include an amino acid sequence including 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 invention comprises at least one of the polypeptides of the invention described herein. As used herein, a "recombinant antibody" refers to a recombinant (e.g., genetically engineered) protein that contains at least one of the polypeptides, antibody polypeptide chains, or antigen-binding portions thereof of the invention. The polypeptide or antigen-binding portion thereof may be an antibody heavy chain, light chain, heavy or light chain variable or constant region, single chain variable fragment (scFv), or Fc, Fab or F(ab)2' fragment, etc. The antibody polypeptide chain or antigen-binding portion thereof may be present as a separate polypeptide of a recombinant antibody. Alternatively, the antibody polypeptide chain or antigen-binding portion thereof may be present as a polypeptide expressed in frame (in tandem) with a polypeptide of the invention. The antibody polypeptide or antigen-binding portion thereof may be the polypeptide of any antibody or any antibody fragment, including any of the antibodies and antibody fragments described herein.

[0054] Within the scope of the present invention are the TCRs, polypeptides or proteins of the invention described herein. As used herein, the term "functional variant" refers to a variant that retains a biological activity of the TCR, polypeptide or protein that it is a variant of. retains substantial or significant sequence identity to the parent TCR, polypeptide or protein A functional variant refers to a TCR, polypeptide or protein that has a similar or similar function to the TCR, polypeptide or protein. For example, the parent TCR may have antigen specificity or the parent polypeptide or protein may have a specific to a similar or similar extent to that of the parent TCR, polypeptide or protein or a TCR as described herein that retains the ability to specifically bind to a greater extent. This includes variants of the polypeptide or protein (parent TCR, polypeptide or protein). With reference to a parent TCR, polypeptide or protein, for example, a functional variant has at least about 30% amino acid sequence identity with the parent TCR, polypeptide or protein, respectively. , 50%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identical.

[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 are defined as those where one amino acid with certain physical and / or chemical properties is replaced by another amino acid with the same 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, No. 57, (1) both of SEQ ID NOs: 50 to 51; (2) both of SEQ ID NOs: 52 to 53; (3) both of SEQ ID NOs: 54 to 55; or (4) both of SEQ ID NOs: 56 to 57. For example, the TCR, polypeptide or protein of the present invention may essentially consist of the amino acid sequences of SEQ ID NOs: 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 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: The TCR of the present invention may essentially consist of both amino acid sequences (several may be selected from the group consisting of amino acid sequences 39 to 40). The polypeptide or protein is: (a) all of SEQ ID NOs: 9 to 14; (b) all of SEQ ID NOs: 17 to 22 (c) all of SEQ ID NOs: 25 to 30; or (d) essentially all of the amino acid sequences of SEQ ID NOs: 33 to 38.

[0058] The TCRs, polypeptides and proteins of the invention may be of any length, i.e., contain any number of amino acids, provided that the TCRs, polypeptides or proteins retain their biological activity, e.g., the ability to specifically bind to mutant KRAS; detect cancer in a mammal; or treat or prevent cancer in a mammal. For example, polypeptides may range from about 50 to about 5000 amino acids in length, such as 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino acids in length, etc. In this regard, polypeptides of the invention also include oligopeptides.

[0059] The TCRs, polypeptides and proteins of the invention contain a nucleotide sequence which replaces one or more naturally occurring amino acids. Such synthetic amino acids may 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, cysteine, 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-aminocyclopentadiene α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminobutyric acid, Aminopropionic acid, homophenylalanine and alpha-tert-butylglycine.

[0060] The TCRs, polypeptides and proteins of the present invention may be modified by glycosylation, amidation, carboxylation, etc. The carboxyl groups may be substituted, phosphorylated, esterified, N-acylated, cyclized, e.g. via disulfide bridges, or converted into acid addition salts, and / or optionally dimerized or polymerized, or conjugated.

[0061] The TCRs, polypeptides and / or proteins of the invention may be prepared, for example, by synthesis of the present invention, e.g., de novo synthesis. Polypeptides and proteins can be produced recombinantly using the nucleic acids described herein by standard recombinant techniques, see, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual(4th ed.) Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Alternatively, The TCRs, polypeptides and / or proteins described herein may be commercially synthesized by companies such as, for example, Synpep (Dublin, Calif.), Peptide Technologies Corp. (Gaithersburg, Md.) and Multiple Peptide Systems (Sandiego, Calif.). The polypeptides and proteins may be synthetic, recombinant, isolated and / or purified.

[0062] Within the scope of the present invention are the TCRs, polypeptides or proteins of the invention (functional parts thereof). or variants thereof), a nucleic acid, a recombinant expression vector, a host cell, a host cell population, or a conjugate, e.g., a bioconjugate, comprising either an antibody or an antigen-binding portion thereof. Conjugates and methods of synthesizing conjugates are generally known in the art.

[0063] One embodiment of the present invention is a method for the treatment of a TCR, polypeptide or protein comprising administering to a subject the The present invention provides a nucleic acid comprising a nucleotide sequence encoding any one of the following: As used herein, "nucleic acid" includes "polynucleotide", "oligonucleotide" and "nucleic acid molecule" and generally refers to a polymer of DNA or RNA, which may be single-stranded or double-stranded, which may contain natural, non-natural or modified nucleotides, and which may contain natural, non-natural or modified internucleotide linkages, such as phosphoramidate or phosphorothioate linkages instead of the phosphodiesters found between the nucleotides of unmodified oligonucleotides. In one embodiment, a nucleic acid comprises a complementary DNA (cDNA). A nucleic acid may contain insertions, deletions, inversions and / or substitutions. It is generally preferred that the nucleic acid does not contain any insertions, deletions, inversions, and / or substitutions, however, as discussed herein, in some instances it may be preferred that the nucleic acid contains one or more insertions, deletions, inversions, and / or substitutions.

[0064] Preferably, the nucleic acids of the invention are recombinant. As used herein, the term "recombinant" refers to (i) a natural or synthetic nucleic acid segment that is capable of replicating in a living cell. or (ii) a molecule constructed outside a living cell by adding to a nucleic acid molecule comprising the nucleic acid sequence described in (i) above. "In vitro replication" refers to a molecule obtained by replication of something that has been replicated. For the purposes of this specification, replication can be in vitro replication or in vivo replication.

[0065] Nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, e.g., Green and Sambrook et al., supra. For example, nucleic acids can be synthesized using naturally occurring nucleotides or various modified nucleotides (e.g., phosphorothioate derivatives and acridine substituted nucleotides) designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed upon hybridization. Examples of modified nucleotides that can be used to generate nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, and 5-iodouracil. ), hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosyl cucurbitate, -osine (β-D-galactosylqueosine), inosine, N 6 -Isopentenyl ade Nin 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) acid (v)), wybutoxosine, pseudouracil , queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil (4-thiouracil), 5-methyluracil, uracil-5-oxyacetate methyl uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxy Examples of suitable amines include, but are not limited to, 3-(3-amino-3-N-2-carboxypropyl)uracil and 2,6-diaminopurine. One or more of the nucleic acids can be purchased commercially from companies such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0066] The nucleic acid may encode any of the TCRs, polypeptides or proteins described herein. In one embodiment of the present invention, the nucleic acid may comprise any nucleotide sequence of SEQ ID NO: 63 to 70 (Table 1). In one embodiment, the nucleic acid comprises the nucleotide sequence of both of SEQ ID NOs: 63 to 64, both of SEQ ID NOs: 65 to 66, both of SEQ ID NOs: 67 to 68, or both of SEQ ID NOs: 69 to 70.

[0067] [Table 1]

[0068] In one embodiment of the invention, the nucleic acid is a TCR, polypeptide or These include codon-optimized nucleotide sequences that code for any of the proteins. Without being bound by any particular theory or mechanism, it is believed that codon optimization of a nucleotide sequence increases the translation efficiency of an mRNA transcript. Codon optimization of a nucleotide sequence can include replacing a native codon with another codon that codes for the same amino acid but can be translated by a more readily available tRNA in the cell, thus increasing translation efficiency. Optimization of a nucleotide sequence can also reduce secondary structures in the mRNA that interfere with translation, thus increasing translation efficiency.

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

[0070] Nucleotide sequences that hybridize under stringent conditions preferably hybridize under high stringency conditions. By "high stringency conditions" is meant that a nucleotide sequence specifically hybridizes to a target sequence (any nucleotide sequence of the nucleic acids described herein) in an amount detectably greater than non-specific hybridization. High stringency conditions include conditions that allow for a high degree of hybridization, ranging from random sequences that happen to have a few small regions (e.g., 3-10 bases) that match the nucleotide sequence to sequences that match exactly. These conditions include conditions that distinguish between polynucleotides with complementary sequences or polynucleotides that contain only scattered minor mismatches. Such small regions of complementarity melt more easily than full-length complements of 14-17 bases or more, and they can be easily distinguished by high stringency hybridization. Relatively high stringency conditions include low salt and / or high temperature conditions, such as those provided by about 0.02-0.1 M NaCl or equivalent at a temperature of about 50-70° C. Such high stringency conditions include conditions that are suitable for use in the preparation of polynucleotides having complementary sequences or polynucleotides that contain only scattered minor mismatches. Such small regions of complementarity melt more easily than full-length complements of 14-17 bases or more, and they can be easily distinguished by high stringency hybridization. Relatively high stringency conditions include low salt and / or high temperature conditions, such as those provided by about 0.02-0.1 M NaCl or equivalent at a temperature of about 50-70° C. The conditions are particularly suitable for detecting the expression of any of the TCRs of the present invention, as they tolerate little, if any, mismatch between the nucleotide sequence and the template or target strand. It is generally understood that conditions can be made more stringent by increasing the

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

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

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

[0074] The recombinant expression vector of the present invention may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host cell. Suitable vectors include vectors that are suitable for propagation and expansion, such as plasmids and viruses, or vectors that are suitable for the propagation and expansion of host cells. Vectors include those designed for transcription, transcription, or expression of ribosomal DNA. The recombinant expression vector may be selected from the group consisting of pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4 and λNM1149 may also be used. Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-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 a MSGV1 vector.

[0075] The recombinant expression vectors of the invention can be prepared using standard recombinant DNA techniques, for example as described in Green and Sambrook, supra. Constructs can be prepared to contain replication systems that are functional in prokaryotic or eukaryotic host cells. Replication systems can be derived, for example, from ColE1, 2μ plasmid, lambda, SV40, bovine papilloma virus, and the like.

[0076] Desirably, the recombinant expression vector is a vector that can be either DNA-based or RNA-based, as appropriate. In particular, the vector includes control sequences, e.g., transcriptional and translational initiation and termination codons, that are specific to the type of host cell (e.g., bacterial, fungal, plant, or animal) into which the vector will be introduced, taking into consideration the type of host cell into which the vector will be introduced.

[0077] The recombinant expression vector may contain one or more marker genes to allow for the selection of transformed or transfected host cells. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in auxotrophic host cells to provide prototrophy, etc. Suitable marker genes for the expression vectors of the present invention include, for example, the neomycin / G418 resistance gene, the hygromycin resistance gene, the histidinol resistance gene, the tetracycline resistance gene and the ampicillin resistance gene.

[0078] The recombinant expression vector comprises a nucleotide sequence encoding a TCR, a polypeptide or a protein. a nucleotide sequence encoding a TCR, polypeptide or protein, The vector may comprise a native or non-native promoter operably linked to a complementary or hybridizing nucleotide sequence. The selection of a promoter, e.g., strong, weak, inducible, tissue-specific, and developmental stage-specific, is within the ordinary skill of one of ordinary skill in the art. Similarly, it is within the ordinary skill of one of ordinary skill in the art to combine a nucleotide sequence with a promoter. The promoter may be a non-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long terminal repeat of murine stem cell virus.

[0079] The recombinant expression vectors of the invention can be designed for either transient expression, stable expression, or both, and can be made for constitutive or inducible expression.

[0080] Additionally, the recombinant expression vector can be made to contain a suicide gene. As used herein, the term "suicide gene" refers to a gene that kills a cell that expresses the suicide gene. A suicide gene can be a gene that confers sensitivity to a reagent, e.g., a drug, on a cell that expresses the gene, causing the cell to die when the cell is in contact with or 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 (PND) gene, and the like. These include the cytoside phosphorylase, nitroreductase, and inducible caspase 9 gene systems. .

[0081] Another embodiment of the present invention further provides a host cell comprising any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that may contain a recombinant expression vector of the present invention. The host cell may be a eukaryotic cell, e.g., a plant, an animal, a fungus or an algae, or a prokaryotic cell, e.g., a bacterium or a protozoan. The host cell may be a cultured cell or a primary cell, i.e., directly isolated from an organism, e.g., a human. The host cell may be an adherent cell or a suspension cell, i.e., a cell that grows in a suspension. Suitable host cells are known in the art and include, e.g., DH5α E. coli cells, Chinese hamster ovarian measles (Cham. cerevisiae), and the like. Examples of such cells include ovarian cells, monkey VERO cells, COS cells, and HEK293 cells. For purposes of amplification or replication, the host cells are preferably prokaryotic cells, e.g., DH5α cells. For purposes of producing recombinant TCRs, polypeptides or proteins, host cells are preferred. Preferably, the host cell is a human cell. While the host cell can be of any cell type, derived from any type of tissue, and at any stage of development, the host cell is preferably a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). More preferably, the host cell is a human cell. Often the host cell is a T cell.

[0082] For purposes herein, a T cell can be, for example, a cultured T cell, e.g., a primary T cell, or a cultured T cell. The T cells may be any T cell, such as a T cell from a line, e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal. If obtained from a mammal, the T cells may be derived from blood, bone marrow, lymph nodes, thymus, or other tissue. T cells can be obtained from a number of sources, including, but not limited to, human tissues or body fluids. T cells can also be enriched or purified. Preferably, the T cells are human T cells. T cells are CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells, e.g., Th1 and Th2 cells, CD4 + T cells, CD8 + These include, but are not limited to, T cells (e.g., cytotoxic T cells), tumor infiltrating lymphocytes (TILs), memory T cells (e.g., central memory T cells and effector memory T cells), naive T cells, etc. The T cells may be of any type and 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 is free of at least one other cell, e.g., a recombinant expression vector. Host cells (e.g., T cells) or cells other than T cells, e.g., B cells, macrophages, neutrophils The cell population may be a heterogeneous population comprising host cells that comprise any of the described recombinant expression vectors, as well as erythrocytes, hepatocytes, endothelial cells, epithelial cells, muscle cells, brain cells, etc. Alternatively, the cell population may be a substantially homogenous population, where the population comprises primarily (e.g., consists essentially of) host cells that comprise the recombinant expression vector. The population may also be a clonal population of cells, where all cells of the population are clones of a single host cell that comprises the recombinant expression vector, such that all cells of the population comprise the recombinant expression vector. In one embodiment of the present invention, the cell population is a clonal population that comprises host cells that comprise the recombinant expression vectors 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 ), 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. For example, the pharmaceutical composition may comprise a polypeptide and a nucleic acid, or two or more different TCRs. Alternatively, the pharmaceutical composition may comprise, for example, a chemotherapeutic agent, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, ), gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, bilirubin Other medicinal actives, such as vinblastine, vincristine, etc. The composition may comprise the TCR material of the present invention in combination with an agent(s) or drug(s).

[0087] Preferably, the carrier is a pharma- ceutically acceptable carrier. For pharmaceutical compositions, the carrier may be any of those contemplated for routine use with the particular TCR material of the invention. Methods for preparing compositions that can be administered are known or apparent to those skilled in the art and are described, for example, in Remington :The Science and Practice of Pharmacy, 22 nd Ed., Pharmaceutical Press (2012) Preferably, the pharma- ceutically acceptable carrier is one that has no detrimental side effects or toxicity under the conditions of use.

[0088] The choice of carrier will be determined in part by the particular TCR material of the invention and by the particular method used to administer the TCR material of the invention. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the invention. Suitable formulations may include any formulation 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 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 invention is administered by injection (e.g., intravenously). When the TCR material of the invention is a host cell expressing a TCR of the invention, a pharma- ceutical acceptable carrier for the injectable cell is used. The carrier used may be, for example, normal saline (about 0.90% w / v NaCl in water, Any isotonic carrier may be included, such as about 300 mOsm / L NaCl, or about 9.0 g NaCl per L of water), NORMOSOL R electrolyte solution (Abbott, Chicago, Ill.), PLASMA-LYTE A (Baxter, Deerfield, Ill.), about 5% dextrose in water or Ringer's lactate. In one embodiment, the pharma- ceutically acceptable carrier is supplemented with human serum albumin.

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

[0091] Many assays are known in the art for determining the amount to be administered. For purposes of the present invention, the extent to which target cells are lysed or different doses of T cells are given, respectively, is used. A given dose of such T cells is administered to one mammal of a set of mammals. Assays involving comparing the degree of IFN-γ secreted by T cells expressing a TCR, polypeptide or protein of the invention upon administration of a given dose can be used to determine the initial dose to administer to a mammal. The degree to which target cells are lysed or IFN-γ is secreted upon administration of a dose can be assayed by methods known in the art.

[0092] The dosage of the TCR material of the invention will also be determined by the existence, nature, and extent of any adverse side effects that may accompany administration of a particular TCR material of the invention. Typically, the attending physician will take into consideration factors such as age, weight, general health, diet, sex, the TCR material of the invention to be administered, the route of administration, and the severity of the condition being treated. The dosage of the TCR material of the invention with which to treat an individual patient is determined taking into account various factors, such as the severity of the cancer being treated, etc. In embodiments in which the TCR material of the invention is a cell population, the number of cells administered per injection is, for example, about 1×10 6 ~Approx. 1×10 12 Changes in cells or beyond In one embodiment, 1×10 6 Less than 100 cells may be administered.

[0093] Those skilled in the art will appreciate that modifications can be made to enhance the therapeutic or prophylactic effects of the TCR materials of the present invention. The inventive TCR materials of the invention can be prepared by any number of methods. For example, the TCR material of the present invention can be modified to inhibit the activation of chemotherapeutic agents. The conjugation may be either direct or indirect, via cross-linking. The practice of conjugating compounds to chemotherapeutic agents is known in the art. One of skill in the art will be able to easily conjugate the compounds to the TCR materials of the present invention. Sometimes, cross-linking and / or chemotherapeutic agents can enhance the function of the TCR material of the invention, i.e., binding to mutant KRAS. It is recognized that sites on the TCR material of the invention that are not necessary for the function of the TCR material of the invention are ideal sites for binding cross-linking and / or chemotherapeutic agents, provided that they do not interfere with the ability to bind to, or detect, treat or prevent cancer.

[0094] The pharmaceutical composition, TCR, polypeptide, protein, nucleic acid, recombinant expression vector, It is expected that the host cells and cell populations may be used in methods of treating or preventing cancer. Without being bound to a particular theory, it is believed that the TCRs of the invention specifically bind to mutant KRAS such that the TCR (or associated polypeptide or protein of the invention), when expressed by a cell, can mediate an immune response against target cells expressing mutant KRAS. In this regard, the invention provides a method of treating or preventing cancer in a mammal, comprising administering to the mammal an effective amount of a pharmaceutical composition, TCR, polypeptide or protein, any of the pharmaceutical compositions, TCRs, polypeptides or proteins described herein, the present invention, or a combination thereof, in an amount effective to treat or prevent cancer in the mammal. Nucleotides encoding any of the TCRs, polypeptides, or proteins described herein. Any nucleic acid or recombinant expression vector comprising the sequence of any of the TCRs described herein, This includes administering to a mammal any host cell or population of cells that contains a recombinant vector encoding either the polypeptide or protein.

[0095] One embodiment of the present invention provides any of the pharmaceutical compositions, TCRs, polypeptides or proteins described herein for use in the treatment or prevention of cancer in a mammal. Nucleotides encoding any of the TCRs, polypeptides, or proteins described herein. Any nucleic acid or recombinant expression vector comprising the TCR sequence described herein. The present invention provides any host cell or population of cells that contains 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, it is intended to provide a means by which those of skill in the art can potentially There are various degrees of treatment or prevention that recognize that the therapeutic effect of the present invention has potential benefit or therapeutic effect. In this regard, the present method may provide any amount of any level of cancer treatment or prevention in a mammal. Moreover, the therapeutic effect provided by the present method may be any amount of any level of cancer treatment or prevention in a mammal. The treatment or prevention performed may include treatment or prevention of one or more conditions or symptoms of the cancer being treated or prevented. For example, treatment or prevention may include promoting tumor regression. Also, for purposes herein, "prevention" may include delaying the onset of cancer, or a symptom or condition thereof. Alternatively or additionally, "prevention" may encompass preventing or delaying the recurrence of cancer, or a symptom or condition thereof.

[0097] Also provided is a method for detecting the presence of cancer in a mammal, the method comprising: (i) detecting a cancer cell in a mammal; The TCRs, polypeptides, proteins, nucleic acids and recombinant expression vectors of the present invention as described in the specification. The method includes contacting either the host cell, cell population or pharmaceutical composition with a sample comprising 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 methods of the present invention for detecting cancer in a mammal, the cellular sample may be a sample comprising whole cells, a lysate thereof or a fraction of a whole cell lysate, e.g., a nuclear or cytoplasmic fraction, a total protein fraction or a nucleic acid fraction.

[0099] For purposes of the detection methods of the present invention, the contacting can be performed in vitro or in a mammal. Preferably, the contacting is in vitro.

[0100] Detection of the complex may be by any number of methods known in the art, including, for example, the TCRs, polypeptides, proteins, nucleic acids, recombinant proteins, and the like of the invention described herein. The present vectors, host cells, or cell populations can be labeled with a detectable label, such as, for example, radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), enzymes (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

[0101] For the purposes of the methods of the present invention, a host cell or cell population may be administered, which may be allogeneic or autologous to the mammal. Preferably, the cells are autologous to the mammal.

[0102] In the context of the methods of the present invention, cancer is any of the following: acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal or anorectum, eye cancer, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder or pleura, cancer of the nose, nasal cavity or middle ear, cancer of the oral cavity, cancer of the vagina, cancer of the vulva, chronic lymphocytic leukemia, chronic bone marrow cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumors, neurological cancer, and the like. The cancer may include any of glioma, Hodgkin's lymphoma, hypopharyngeal cancer, renal cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, oropharyngeal cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, ureteral cancer and bladder cancer. The preferred cancer is pancreatic, colorectal, lung, endometrial, ovarian or prostate cancer. Preferably In another preferred embodiment, the cancer is a cancer expressing a mutant KRAS amino acid sequence having a G12D mutation.

[0103] The mammal referred to in the methods of the present invention may be any mammal. As used herein, the term "mammal" refers to any mammal, including but not limited to rodent mammals, such as mice and hamsters, and lagomorph mammals, 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 Bovidae, including Bovidae (cattle) and Suidae (pigs), or the order Equidae, including Equidae (horses). Most preferably, the mammal is from the order Primates, Ceboids, or Simoides (monkeys) or Anthropoids (humans and apes). A particularly preferred mammal is a human.

[0104] The following examples further illustrate the present invention but, of course, should not be construed as in any way limiting its scope. EXAMPLES

[0105] Next-generation sequencing The QIAGEN AllPrep DNA / RNA kit (Qiagen, Venlo, Netherlands) was used as a manufacturer's Following the suggestion of One sample (Tu-Pri) was formalin fixed and paraffin embedded. FFPE samples were then extracted using the Covaris truXTRAC™ FFPE DNA kit according to the manufacturer's instructions (Covaris, Woburn, MA). Whole-exome libraries were generated using the Agilent Technologies SureSelectXT Target Enrichment System for paired-end libraries in combination with Human All Exon V6 RNA bait (Agilent Technologies, Santa Clara, CA, USA). A genomic construct and exon capture of approximately 20,000 coding genes were prepared. Whole-exome sequencing was then performed on a NEXTSEQ 500 benchtop sequencer (Illumina, San Diego, CA, USA). We sequenced the WES libraries using 3 μg of gDNA from fresh tumor tissue samples and 200 ng of gDNA from FFPE tumor samples according to the manufacturer's protocol. Libraries were prepared using the Illumina High Output Flow Cell Kit (300 cycles). Paired-end sequencing was performed initially using v1 of the reagent / flow cell kit, followed by subsequent runs of the same library preparation in v2 of the reagent / flow cell kit. The Illumina TruSeq RNA strand library prep kit was used according to the manufacturer's protocol. 2 μg of total RNA was used to prepare an RNA-seq library. The RNA-seq library was subjected to paired-end sequencing on a NextSeq 500 benchtop sequencer (Illumina, San Diego, CA, USA). I sang.

[0106] Alignment, processing and variant calling For WES, alignment was performed against human genome build hg19 using the novoalignMPI program from Novocraft (Selangor, Malaysia) (novocraft.com / ). Duplicates were marked using the Picard MARKDUPLICATES tool. Insertion / deletion realignment and base recalibration were performed following the GATK best practices workflow (broadinstitute.org / gatk / ). After data cleanup, pileup files were generated using the samtools utility (samtools.sourceforge.net) and Varscan2 platform independent variant caller (varscan.sourceforge.net) was used to call somatic variants using the following criteria: ≥10 tumor and normal reads, ≥10% variant allele frequency, and ≥4 tumor variant reads. Then, the data were analyzed using the Annovar software. We used Atool (annovar.openbioinformatics.org) to characterize these variants. Annotated.

[0107] For RNA-seq, alignments were performed against human genome build hg19 using the two-pass method in STAR (github.com / alexdobin / STAR). Duplicates were marked using the Picard MARKDUPLICATES tool. Reads were split and trimmed using the GATK SplitNTrim tool. Then, insertion / deletion realignment and base recalibration were performed using the GATK toolbox. Finally, pileup files were generated using the realigned bam files and the mpileup tool of samtools. Finally, the VARSCAN2 platform was used to generate the pileup files. Variants were called using a group-independent variant caller.

[0108] WES and RNA-seq were performed on three metastatic fresh tumor samples (Tu-1, Tu-2A, and Tu-2B). Variants with a minimum exome frequency of 7% and a minimum of three alternative reads were then manually curated using the Integrated Genomics Viewer (IGV) tool (Broad Institute, Cambridge, MA) to remove false positive calls likely due to sequencing or mapping errors. To focus on mutations that may represent distinct clonal populations, two or more tumor samples were used. Based on detection in the samples, 61 mutations were selected for further analysis. One of these 61 mutations is KRAS (Table 2). These included a minimum of one WES and one RNA-seq library. These included 29 mutations identified in and 32 mutations identified in WES libraries derived from two or more metastatic lesions.

[0109] [Table 2]

[0110] Generation of tumor-infiltrating lymphocytes (TILs), infused TILs and antigen-presenting dendritic cells (DCs) TILs, infused TILs and dendritic cells were generated as described in Tran et al., Science, 350:1387-90 (2015). Briefly, to generate TILs, surgically excised tumors were cut into 24 pieces of approximately 1-2 mm in size, and each piece was placed separately in a well of a 24-well plate containing 2 ml of complete medium (CM) with high dose IL-2 (6000 IU / ml, Chiron, Emeryville, CA). CM was supplemented with 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. The DCs were incubated in 10% CO2 medium. 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). Fresh DC medium was added to the cultures on days 2-3. DCs were cryopreserved on days 4 or 5 after the initiation of culture. DCs were used in experiments between days 4 and 6 after the initiation of culture.

[0112] Identification of mutation-reactive T cells and co-culture experiments Detailed methods are 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 minigene encoding the mutation was generated on the other side, with 12 adjacent amino acids, and synthesized in tandem to generate a tandem minigene (TMG) construct. Five TMGs (TMG1-5) encoding 61 mutations were generated and transcribed in vitro into RNA, allowing processing and presentation of all mutations in the context of the patient's own HLA-I and HLA-II molecules (Table 4). , and electroporated into autoantigen-presenting DCs. TMG for wild-type and mutant KRAS The HLA data shown in Table 4 is taken from Bai et al., BMC Genomics, 15:325 (2014). The genomic DNA sequence was determined from next-generation sequencing data using the algorithm PHLAT as described below. Twenty-four individual patient-derived TIL cultures were cocultured with these TMG-expressing DCs, and T cell reactivity was determined by IFN-γ enzyme-linked immunospot (ELISPOT) assays (Figure 1A) and flow cytometric analysis of the T cell activation markers 4-1BB and OX40. Several TIL cultures were identified that were reactive to TMG-1. To identify which mutant antigen in TMG-1 was recognized by TIL culture #6, peptides encoded in TMG-1 were synthesized (ThermoFisher Scientific (Waltham, MA) and GenScript Inc. (Piscataway Township, NJ)) and then DCs were then pulsed individually overnight and continued to be co-cultured with TIL culture #6 (Figure 1B).

[0113] [Table 3]

[0114] [Table 4]

[0115] The following HPLC purified peptides (GenScript Inc.): wild type (WT)-9-mer: GAGGVGKSA (sequence no. No. 7); mutated (G12D)-9-mer: GADGVGKSA (SEQ ID NO: 8); WT-10-mer: GAGGVGKSAL (SEQ ID NO: No. 5); G12D-10-mer:GADGVGKSAL (SEQ ID NO: 6) was used in peptide titration 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. In addition, 5'RACE was performed using constant primers for the TCR-alpha and beta chains (SMARTER RACE cDNA amplification kit, Clontech). The sequences of the inhibitors are 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 modified (2 min instead of 3 min) and the kit's program was The PCR products of the TCR were then subjected to standard agarose gel electrophoresis and gel electrophoresis. The products were isolated by extraction (zymogen) and then sequenced (Macrogen, Seoul, Korea).

[0118] The second and third ranked TCRs in the infusion bag also expressed KRAS G12D It is reactive, Long KRAS G12D DCs were isolated by first stimulating apheresis samples 40 days after cell transfer with DCs pulsed overnight with peptide. After overnight stimulation, T cell activation markers were then isolated. Vβ5.2 positive and negative CD8+ T cells with upregulated 4-1BB were separately selected by FACS. Vβ5.2+ cells were further amplified prior to 5'RACE as described above, followed by TOPO-TA cloning of the TCR PCR products and sequencing of individual colonies to identify the TCR-alpha and beta chains. Multiplex PCR of single-cell TCR was performed on Vβ5.2-negative cells to identify the TCR-alpha and beta chains as described in Pasetto et al., Cancer Immunol. Res., (2016).

[0119] The fourth KRAS G12DThe reactive TCR (ranked 45th in the infusion bag) was Using a single cell technology approach, a distinct TIL fragment (TIL fragment #5) was identified. Briefly, TIL culture #5 was cloned into a control group that expresses TMG-1 (KRAS G12D After 4 hours (h), TILs were harvested and subjected to a Fluidigm C1 system (Fluidigm, San Francisco, CA) to prepare single-cell RNA-seq samples according to the manufacturer's protocol. The single-cell RNA-seq samples were then sequenced using the Illumina MiSeq system. The data were analyzed using 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 KRAS in the sample G12D To determine the frequency of reactive T cells, G12D TCR sequences of reactive T cell clones were first identified and these sequences were interrogated against TCR-Vβ deep sequencing data from the indicated samples. The number of in-frame productive TCR reads in the samples ranged from 522,499 to 1,990,345.

[0121] Flow Cytometry Antibodies The following anti-human flow cytometry antibodies were used: 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, Vβ5.2-PE (BioLegend) and Vβ5.2-PE (Beckman Coulter) were used in this report. A fluorescent dye-conjugated anti-mouse TCR-beta constant region antibody (H57-597, eBioscience) was used. The transduction efficiency of TCR was evaluated using the IO Test beta Mark TCR V kit. Repertoire was evaluated (Beckman Coulter).

[0122] Identification of mutant reactive T cells and generation of infusion products Using previously described methods (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)), we tested whether TILs derived from patient 4095 could recognize somatic mutations expressed in her metastatic lung tumors. TIL culture #6 had the highest frequency of KRAS G12D Contains reactive CD8+ T cells Therefore, as described in Tran et al., Science, 344:641-5 (2014), 2 A rapid amplification process was carried out for 2 weeks.

[0123] KRAS G12D In vivo tracking of specific T cell clones Patient's infusion product, 3 separate pulmonary nodules prior to treatment, progressive lesion (lesion 3), and cell infusion T cell receptor (TCR) sequences were identified in gDNA isolated from peripheral blood at various time points before and after immunization. We performed post-sequencing (Adaptive Biotechnologies, Seattle WA) to identify KRAS G12D The frequency of reactive TCR sequences was interrogated.

[0124] KRAS G12D Assessment of specific TCR reactivity 4 KRASG12D Reactive TCRs were identified and TCR-alpha and beta chain sequences were synthesized and then cloned into the MSGV1 retroviral vector (GenScript Inc.). Retroviral supernatants encoding the TCRs were generated and used to transduce autologous peripheral blood T cells as described in Tran et al., Science, 344:641-5 (2014). The TCR-transduced T cells were then transformed into various Autologous peripheral blood mononuclear cells (PBMCs) loaded with serially diluted doses of the KRAS peptide or bound KRAS with or without stable expression of the HLA-C*08:02 allele G12D positive The cells were co-cultured with pancreatic cancer cell lines (Tran et al., Science, 350:1387-90 (2015)). IFN-γ ELISPOT assays and flow cytometry analysis of T cell activation markers 4-1BB and OX40 were performed. T cell reactivity was determined the next day by immunoblotting (Tran et al., Science, 350:1387-90 (2015)).

[0125] Example 1 In this embodiment, KRAS G12D Demonstrate the in vivo frequency of mutant-reactive CD8+ T cells. The patient was a 49-year-old woman with colorectal adenocarcinoma and multiple bilateral lung metastases. She had previously undergone 12 cycles of FOLFOX chemotherapy after sigmoidectomy and partial cystectomy. This was followed by radiation therapy of 4182 cGy to the bladder suture line. Shortly after this, she developed an increase in the number and size of bilateral pulmonary nodules that were FDG-rich. A biopsy of the right lower lobe nodule revealed nodules. The findings were consistent with metastatic colorectal adenocarcinoma.

[0126] Patients were enrolled in an institutional review board-approved phase II clinical trial (ClinicalTrials.gov number, NCT01174121) designed to test whether adoptive transfer of ex vivo expanded tumor-infiltrating lymphocytes (TILs) containing T cells targeting cancer mutations could mediate regression of metastatic solid tumors. Baseline CT scans revealed pulmonary disease as the sole source of cancer progression. Three pulmonary lesions were identified using video-assisted thoracic surgery (VATS). The tumors were then excised and 24 individual TIL cultures were generated from multiple tumor fragments. To identify mutations, we also performed whole-exome and transcriptome sequencing in three lesions. Sequencing was performed (Table 2). Each TIL culture was evaluated for reactivity to these mutations. G12D It was found that the CD8+ T cells specifically recognized the mutation. (Figure 1A and B). G12D The TIL cultures with the highest frequency of reactive CD8+ T cells were selected. The numbers of selected cells were expanded for treatment (Figure 1B and C). Prior to cell infusion, patients received cyclophosphamide at 60 mg / kg for 2 days, followed by 25 mg / m for 5 days. 2 A nonmyeloablative, lymphodepleting chemotherapy regimen containing fludarabine (Dudley et al., J. Clin. Oncol., 23 :2346-57 (2005)). 11 A single infusion of TILs was administered, followed by five doses of 720,000 IU / kg interleukin 2 (IL-2), which was discontinued due to fatigue. The treatment was well tolerated and the patient was discharged home two weeks after cell infusion. Approximately 75% of the infusion product was KRAS G12D The majority of these T cells are involved in multiple effector The seven cells produced cytokines (IFN-γ, TNF, and IL-2) and exhibited cytolytic potential. All metastatic lung lesions regressed during the first 40 days of follow-up after cell transfer, with one lesion Six of seven lesions continued to regress or showed complete remission until one (lesion 3) progressed approximately 9 months after treatment. Approximately 9 months after cell transfer, only one progressive lesion (lesion 3) was identified, as well as a reactive lesion (lesion 2) that was PET negative and completely necrotic with no viable tumor cells on pathological analysis. A VATS resection of the left lower lung was performed to remove the tumor. The patient remained clinically unaffected for 3 months after the lung resection. There remains no.

[0127] The infused TIL products expressed at least four KRAS genes at various frequencies G12D Reactive T cell clonotypes included The most frequent three types of infusion products were One TCR is KRAS G12D While it is reactive against the fourth KRAS G12D The reactive TCR is 45 The incidence of KRAS was only 0.04% of the infusion bags (Figures 2A-2D and Table 5). G12D Reactive TCR was not detected in the peripheral blood of patients 1 week prior to infusion (frequency <0.0002%, Figures 2A-2D). G12D Dramatic differences were observed in the engraftment of TCR-reactive T cells. The most dominant clonotype of infused T cells (~7.3 × 10 10 Cells) are While the remaining KRAS G12D Reactive T cell The KRAS clone was detected at this time point (Figures 2A-2D). G12D Approximately 9 months after cell transfer, reactive T cell clones accounted for 10.4%, 4.5%, and 0.005% of total peripheral blood T cells, respectively. At that time, the most dominant T cell clone in peripheral blood was the TRBV10-02 mutant KRAS G12DIn the advanced tumors compared to the peripheral blood, the KRAS G12D Enrichment of reactive T cell clones did not appear to occur (Figures 2A-2D).

[0128] [Table 5]

[0129] Example 2 In this embodiment, KRAS G12D The specificity and sensitivity of the reactive TCR is demonstrated. The nucleotide sequence encoding the TCR was G12D Each of the four clonal types of reactive T cells was cloned into the 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 encodes the variable region of the TCR alpha chain (shown in Table 6) and the constant region of the murine TCR alpha chain, plus the P2A This was followed by a linker sequence (SEQ ID NO:58) and nucleotide sequences encoding the variable region of the TCR beta chain (shown in Table 6) and the constant region of the murine TCR beta chain. The TCR has three amino acid substitutions in the TM region of the murine constant region of the alpha chain with hydrophobic amino acids (SEQ ID NO:59). In combination, they were further modified to include cysteine ​​substitutions in the murine constant regions of both the α and β chains. The full-length amino acid sequences of each of the four TCRs are shown in Table 7. Without being bound, it is believed that the murine constant chains of TCR alpha and beta may reduce mispairing with endogenous TCRs and promote expression of the introduced TCR by host cells. In addition, enhanced expression and pairing of the introduced TCR alpha and beta chains , incorporating hydrophobic amino acids into the constant chain of the TCR alpha, and the alpha and beta chains. This could conceivably be achieved by introducing a second disulfide bond between the constant regions of the chains.

[0132] [Table 7]

[0133] Autologous peripheral blood T cells were genetically modified to express one of these four TCRs. The TCRs were engineered with the constant regions of mouse TCR-alpha and beta, and thus The expression of the TCR on the cell surface was analyzed using a flow cytometer with the constant region of mouse TCR-β (mTCR-β). The TCR gene was evaluated 10 days after the modification by chromatometry. The data were gated on CD8+ T cells. The percentage of cells expressing the mouse TCR-β constant region is shown in Table 8.

[0134] [Table 8]

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

[0136] [ka]

[0137] One TCR was selectively reactive against the 10 amino acid long KRAS G12D peptide

[0138] [ka]

[0139] All TCRs were specific for the mutant and did not recognize the wild-type KRAS peptide (Fig. 3A-3D). Peptide titration experiments demonstrated that the TCRs reacted only with the wild-type KRAS peptide against autologous PBMCs. When pulsed with IgG, TCRs were able to recognize peptides at concentrations between 1 and 10 nM. was demonstrated (Figures 3A to 3D).

[0140] Two KRAS genes expressing or not expressing the HLA-C*08:02 allele G12D The TCR-modified T cells were co-cultured overnight with one of the positive pancreatic cancer cell lines (MDA-Panc48 or HPAC). IFN-γ secretion was measured by ELISPOT assay and 4-1BB expression was measured by flow cytometry. The results are shown in Figures 4A-4B. The pancreatic cancer cell lines express KRAS G12D Mutations and HLA-C*08:02A The TCR specifically recognized pancreatic cancer cell lines only when they expressed both alleles (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. Autologous peripheral blood T cells were transfected to express one of four TCRs as described in Example 2. 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, followed by expression of the indicated KRAS G12D The co-culture was continued with the reactive TCR-transduced cells. The next day, flow cytometry was performed. The cells were analyzed for expression of 4-1BB by immunofluorescence assay. The results are shown in Figures 5A-5D. Data were gated on TCR-transduced (murine TCRβ+) CD8+ T cells. HLA-C*07:01 was used as the negative control HLA allele.

[0142] All references cited in this specification, including publications, patent applications, and patents, are herein incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and to the same extent as if each reference was fully set forth herein.

[0143] The terms "a" and "b" in the context of describing the present invention (especially in the context of the claims below) The use of "an," "the," and "at least one" and similar referents should be construed as including both the singular and the plural unless otherwise indicated herein or clearly contradicted by context. The use of "one" (e.g., "at least one of A and B") is intended to be limiting unless otherwise stated herein. means one item selected from the list (A or B), or any combination of two or more of the items listed (A and B), unless otherwise specified or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are used interchangeably throughout the appended claims, unless otherwise specified. These terms should be construed as open-ended terms (i.e., meaning "including, but not limited to"). Recitation of ranges is merely intended to serve as a shorthand method of referring individually to each separate value within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if each separate value was individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as") provided herein is merely to serve as a means to better illustrate the invention. The present disclosure is intended to provide a general description, and is not intended to impose limitations on the scope of the invention unless specifically claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0144] Preferred embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors anticipate that such variations will be employed by those skilled in the art, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, this invention encompasses any combination of the above-described elements in all possible variations thereof unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

1. A recombinant expression vector comprising a nucleotide sequence encoding a KRAS G12D-specific T cell receptor (TCR), wherein the recombinant expression vector is designed for constitutive, inducible, transient, or stable expression, and wherein the TCR is: (a) an α chain complementarity determining region (CDR) 1 amino acid sequence of SEQ ID NO:9, an α chain CDR2 amino acid sequence of SEQ ID NO:10, an α chain CDR3 amino acid sequence of SEQ ID NO:11, a β chain CDR1 amino acid sequence of SEQ ID NO:12, a β chain CDR2 amino acid sequence of SEQ ID NO:13, and a β chain CDR3 amino acid sequence of SEQ ID NO:14; (b) an α chain CDR1 amino acid sequence of SEQ ID NO:17, an α chain CDR2 amino acid sequence of SEQ ID NO:18, an α chain CDR3 amino acid sequence of SEQ ID NO:19, a β chain CDR1 amino acid sequence of SEQ ID NO:20, a β chain CDR2 amino acid sequence of SEQ ID NO:21, and a β chain CDR3 amino acid sequence of SEQ ID NO:22; (c) an α chain CDR1 amino acid sequence of SEQ ID NO:25, an α chain CDR2 amino acid sequence of SEQ ID NO:26, an α chain CDR3 amino acid sequence of SEQ ID NO:27, a β chain CDR1 amino acid sequence of SEQ ID NO:28, a β chain CDR2 amino acid sequence of SEQ ID NO:29, and a β chain CDR3 amino acid sequence of SEQ ID NO:30; or (d) an α chain CDR1 amino acid sequence of SEQ ID NO:33, an α chain CDR2 amino acid sequence of SEQ ID NO:34, an α chain CDR3 amino acid sequence of SEQ ID NO:35, a β chain CDR1 amino acid sequence of SEQ ID NO:36, a β chain CDR2 amino acid sequence of SEQ ID NO:37, and a β chain CDR3 amino acid sequence of SEQ ID NO:

38. A recombinant expression vector comprising:

2. The TCR comprises: (i) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:15 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:16; (ii) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:23 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:24; (iii) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:31 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:32; (iv) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:39 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:40; (v) an amino acid sequence that is at least 99% identical to amino acids 20 to 129 of SEQ ID NO:15 and an amino acid sequence that is at least 99% identical to amino acids 22 to 133 of SEQ ID NO:16; (vi) an amino acid sequence that is at least 99% identical to amino acids 20 to 129 of SEQ ID NO:23 and an amino acid sequence that is at least 99% identical to amino acids 22 to 133 of SEQ ID NO:24; (vii) an amino acid sequence that is at least 99% identical to amino acids 20 to 129 of SEQ ID NO: 31 and an amino acid sequence that is at least 99% identical to amino acids 22 to 133 of SEQ ID NO: 32; or (viii) an amino acid sequence that is at least 99% identical to amino acids 21 to 132 of SEQ ID NO: 39 and an amino acid sequence that is at least 99% identical to amino acids 22 to 130 of SEQ ID NO:

40. The recombinant expression vector of claim 1 .

3. The TCR comprises: (1) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:50 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:51; (2) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:52 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:53; (3) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:54 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:55; (4) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:56 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:57; (5) an amino acid sequence that is at least 99% identical to amino acids 20 to 266 of SEQ ID NO:50 and an amino acid sequence that is at least 99% identical to amino acids 22 to 306 of SEQ ID NO:51; (6) an amino acid sequence that is at least 99% identical to amino acids 20 to 266 of SEQ ID NO:52 and an amino acid sequence that is at least 99% identical to amino acids 22 to 306 of SEQ ID NO:53; (7) an amino acid sequence that is at least 99% identical to amino acids 20 to 266 of SEQ ID NO:54 and an amino acid sequence that is at least 99% identical to amino acids 22 to 306 of SEQ ID NO:55; or (8) an amino acid sequence that is at least 99% identical to amino acids 21 to 269 of SEQ ID NO:56 and an amino acid sequence that is at least 99% identical to amino acids 22 to 303 of SEQ ID NO:57 The recombinant expression vector according to claim 1 or 2, comprising:

4. A recombinant expression vector comprising a nucleotide sequence encoding a polypeptide comprising a functional portion of a KRAS G12D-specific TCR, wherein the recombinant expression vector is designed for constitutive, inducible, transient, or stable expression, and wherein the functional portion is: (a) an α chain CDR1 amino acid sequence of SEQ ID NO:9, an α chain CDR2 amino acid sequence of SEQ ID NO:10, an α chain CDR3 amino acid sequence of SEQ ID NO:11, a β chain CDR1 amino acid sequence of SEQ ID NO:12, a β chain CDR2 amino acid sequence of SEQ ID NO:13, and a β chain CDR3 amino acid sequence of SEQ ID NO:14; (b) an α chain CDR1 amino acid sequence of SEQ ID NO: 17, an α chain CDR2 amino acid sequence of SEQ ID NO: 18, an α chain CDR3 amino acid sequence of SEQ ID NO: 19, a β chain CDR1 amino acid sequence of SEQ ID NO: 20, a β chain CDR2 amino acid sequence of SEQ ID NO: 21, and a β chain CDR3 amino acid sequence of SEQ ID NO: 22; (c) an α chain CDR1 amino acid sequence of SEQ ID NO:25, an α chain CDR2 amino acid sequence of SEQ ID NO:26, an α chain CDR3 amino acid sequence of SEQ ID NO:27, a β chain CDR1 amino acid sequence of SEQ ID NO:28, a β chain CDR2 amino acid sequence of SEQ ID NO:29, and a β chain CDR3 amino acid sequence of SEQ ID NO:30; or (d) an α chain CDR1 amino acid sequence of SEQ ID NO: 33, an α chain CDR2 amino acid sequence of SEQ ID NO: 34, an α chain CDR3 amino acid sequence of SEQ ID NO: 35, a β chain CDR1 amino acid sequence of SEQ ID NO: 36, a β chain CDR2 amino acid sequence of SEQ ID NO: 37, and a β chain CDR3 amino acid sequence of SEQ ID NO:

38. A recombinant expression vector comprising:

5. The functional moiety is: (i) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:15 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:16; (ii) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:23 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:24; (iii) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:31 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:32; (iv) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:39 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:40; (v) an amino acid sequence that is at least 99% identical to amino acids 20 to 129 of SEQ ID NO:15 and an amino acid sequence that is at least 99% identical to amino acids 22 to 133 of SEQ ID NO:16; (vi) an amino acid sequence that is at least 99% identical to amino acids 20 to 129 of SEQ ID NO:23 and an amino acid sequence that is at least 99% identical to amino acids 22 to 133 of SEQ ID NO:24; (vii) an amino acid sequence that is at least 99% identical to amino acids 20 to 129 of SEQ ID NO: 31 and an amino acid sequence that is at least 99% identical to amino acids 22 to 133 of SEQ ID NO: 32; or (viii) an amino acid sequence that is at least 99% identical to amino acids 21 to 132 of SEQ ID NO: 39 and an amino acid sequence that is at least 99% identical to amino acids 22 to 130 of SEQ ID NO:

40. The recombinant expression vector of claim 4 .

6. A polypeptide comprising: (1) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:50 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:51; (2) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:52 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:53; (3) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:54 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:55; (4) an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:56 and an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:57; (5) an amino acid sequence that is at least 99% identical to amino acids 20 to 266 of SEQ ID NO:50 and an amino acid sequence that is at least 99% identical to amino acids 22 to 306 of SEQ ID NO:51; (6) an amino acid sequence that is at least 99% identical to amino acids 20 to 266 of SEQ ID NO:52 and an amino acid sequence that is at least 99% identical to amino acids 22 to 306 of SEQ ID NO:53; (7) an amino acid sequence that is at least 99% identical to amino acids 20 to 266 of SEQ ID NO:54 and an amino acid sequence that is at least 99% identical to amino acids 22 to 306 of SEQ ID NO:55; or (8) an amino acid sequence that is at least 99% identical to amino acids 21 to 269 of SEQ ID NO:56 and an amino acid sequence that is at least 99% identical to amino acids 22 to 303 of SEQ ID NO:57 The recombinant expression vector according to claim 4 or 5, comprising:

7. A recombinant expression vector comprising a nucleotide sequence encoding a protein comprising a functional portion of a KRAS G12D-specific TCR, wherein the recombinant expression vector is designed for constitutive, inducible, transient, or stable expression, and wherein the functional portion is: (a) a first polypeptide chain comprising an α chain CDR1 amino acid sequence of SEQ ID NO:9, an α chain CDR2 amino acid sequence of SEQ ID NO:10, an α chain CDR3 amino acid sequence of SEQ ID NO:11, and a second polypeptide chain comprising a β chain CDR1 amino acid sequence of SEQ ID NO:12, a β chain CDR2 amino acid sequence of SEQ ID NO:13, and a β chain CDR3 amino acid sequence of SEQ ID NO:14; (b) a first polypeptide chain comprising an α chain CDR1 amino acid sequence of SEQ ID NO:17, an α chain CDR2 amino acid sequence of SEQ ID NO:18, an α chain CDR3 amino acid sequence of SEQ ID NO:19, and a second polypeptide chain comprising a β chain CDR1 amino acid sequence of SEQ ID NO:20, a β chain CDR2 amino acid sequence of SEQ ID NO:21, and a β chain CDR3 amino acid sequence of SEQ ID NO:22; (c) a first polypeptide chain comprising an alpha chain CDR1 amino acid sequence of SEQ ID NO:25, an alpha chain CDR2 amino acid sequence of SEQ ID NO:26, an alpha chain CDR3 amino acid sequence of SEQ ID NO:27, and a second polypeptide chain comprising a beta chain CDR1 amino acid sequence of SEQ ID NO:28, a beta chain CDR2 amino acid sequence of SEQ ID NO:29, and a beta chain CDR3 amino acid sequence of SEQ ID NO:30; or (d) a first polypeptide chain comprising an α chain CDR1 amino acid sequence of SEQ ID NO:33, an α chain CDR2 amino acid sequence of SEQ ID NO:34, an α chain CDR3 amino acid sequence of SEQ ID NO:35, and a second polypeptide chain comprising a β chain CDR1 amino acid sequence of SEQ ID NO:36, a β chain CDR2 amino acid sequence of SEQ ID NO:37, and a β chain CDR3 amino acid sequence of SEQ ID NO:

38. A recombinant expression vector comprising: (i) a first polypeptide chain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:15, and a second polypeptide chain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:16; (ii) the first polypeptide chain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:23, and the second polypeptide chain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:24; (iii) the first polypeptide chain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:31, and the second polypeptide chain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:32; (iv) the first polypeptide chain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:39, and the second polypeptide chain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:40; (v) the first polypeptide chain comprises an amino acid sequence at least 99% identical to amino acids 20-129 of SEQ ID NO:15, and the second polypeptide chain comprises an amino acid sequence at least 99% identical to amino acids 22-133 of SEQ ID NO:16; (vi) the first polypeptide chain comprises an amino acid sequence at least 99% identical to amino acids 20-129 of SEQ ID NO:23, and the second polypeptide chain comprises an amino acid sequence at least 99% identical to amino acids 22-133 of SEQ ID NO:24; (vii) the first polypeptide chain comprises an amino acid sequence at least 99% identical to amino acids 20-129 of SEQ ID NO:31, and the second polypeptide chain comprises an amino acid sequence at least 99% identical to amino acids 22-133 of SEQ ID NO:32; or (viii) the first polypeptide chain comprises an amino acid sequence at least 99% identical to amino acids 21-132 of SEQ ID NO:39, and the second polypeptide chain comprises an amino acid sequence at least 99% identical to amino acids 22-130 of SEQ ID NO:

40. The recombinant expression vector of claim 7. (1) a first polypeptide chain comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:50, and a second polypeptide chain comprising an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:51; (2) the first polypeptide chain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:52, and the second polypeptide chain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:53; (3) the first polypeptide chain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:54, and the second polypeptide chain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:55; (4) the first polypeptide chain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:56, and the second polypeptide chain comprises an amino acid sequence at least 99% identical to the amino acid sequence of SEQ ID NO:57; (5) the first polypeptide chain comprises an amino acid sequence at least 99% identical to amino acids 20-266 of SEQ ID NO:50, and the second polypeptide chain comprises an amino acid sequence at least 99% identical to amino acids 22-306 of SEQ ID NO:51; (6) the first polypeptide chain comprises an amino acid sequence at least 99% identical to amino acids 20-266 of SEQ ID NO:52, and the second polypeptide chain comprises an amino acid sequence at least 99% identical to amino acids 22-306 of SEQ ID NO:53; (7) the first polypeptide chain comprises an amino acid sequence at least 99% identical to amino acids 20-266 of SEQ ID NO:54, and the second polypeptide chain comprises an amino acid sequence at least 99% identical to amino acids 22-306 of SEQ ID NO:55; or (8) The first polypeptide chain comprises an amino acid sequence at least 99% identical to amino acids 21-269 of SEQ ID NO:56, and the second polypeptide chain comprises an amino acid sequence at least 99% identical to amino acids 22-303 of SEQ ID NO:

57. A recombinant expression vector according to claim 7 or 8.

10. The recombinant expression vector of any one of claims 1 to 9, wherein the recombinant expression vector is designed for constitutive expression.

11. The recombinant expression vector of any one of claims 1 to 9, wherein the recombinant expression vector is designed for inducible expression.

12. The recombinant expression vector of any one of claims 1 to 9, wherein the recombinant expression vector is designed for transient expression.

13. The recombinant expression vector of any one of claims 1 to 9, wherein the recombinant expression vector is designed for stable expression.

14. The recombinant expression vector described in any one of claims 1 to 13, wherein the recombinant expression vector is a viral vector.

15. The recombinant expression vector described in any one of claims 1 to 13, wherein the recombinant expression vector is a retroviral vector.

16. A recombinant expression vector described in any one of claims 1 to 15, wherein the recombinant expression vector contains a suicide gene.

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