KRAS_G12V mutant antigen-specific TCR and redirected CD4 T cells co-expressing such TCR and CD8

Abstract

The present invention relates to a T cell receptor (TCR) that specifically binds to a KRAS_G12V mutant antigen, a fusion protein or complex containing a TCR, a nucleic acid encoding a TCR, genetically modified cells containing the same, and a method for producing genetically modified cells. The present invention also relates to enhancing T cell function by coexpressing an exogenous CD8 molecule and a TCR gene in T cells. The present invention provides uses of the TCR and genetically modified cells in the detection, prevention, and / or treatment of cancers associated with the KRAS_G12V mutant antigen.

Description

[Technical Field]

[0001] The present invention generally relates to the field of genetics. Specifically, the present invention relates to a T cell receptor (hereinafter referred to as "TCR") that specifically binds to a KRAS_G12V mutant antigen, a genetically modified cell expressing the TCR, and a method for preparing the genetically modified cell. The present invention further relates to coexpression of an exogenous CD8 molecule and the TCR in a T cell to enhance T cell function. The present invention also provides uses of the TCR and the genetically modified cell in the detection, prevention, and / or treatment of cancers associated with the KRAS_G12V mutant antigen. [Background technology]

[0002] The RAS gene was the first human oncogene discovered and encodes the RAS protein, which plays a central role in many important cell signaling networks. The RAS gene is the most frequently mutated oncogene in human cancers. Activation of the RAS protein due to mutations in the RAS gene has been identified in approximately one-fifth of all human tumors.

[0003] The KRAS gene (Kirsten rat sarcoma viral oncogene homolog) encodes the KRAS protein, a small GTPase that belongs to the RAS superprotein family.

[0004] Within cells, the KRAS protein alternates between an inactive and an active state. When bound to guanosine diphosphate (GDP), the KRAS protein is inactivated, whereas when bound to guanosine triphosphate (GTP), the KRAS protein is activated, allowing it to activate downstream signaling pathways. In most cells, the KRAS protein is inactivated.

[0005] Of the mutations in the KRAS gene, 97% involve mutations in the 12th or 13th amino acid residue. One of the most common mutations is G12V. Structural studies have revealed that the G12V mutation in KRAS disrupts GAP activity, maintaining the KRAS protein bound to GTP, locking the KRAS protein in a tyrosine kinase active state and causing continuous activation of downstream signaling pathways (e.g., PI3K signaling pathway, MAPK signaling pathway, PI3K and Ral-GEFs signaling pathway). Activation of these downstream signaling pathways stimulates cell proliferation and migration, ultimately leading to tumor formation.

[0006] In human cancers, the KRAS gene is one of the most well-known oncogenes in oncology and was once considered an "undruggable" target. KRAS mutations are found in nearly 90% of pancreatic cancers, 30-40% of colon cancers, 17% of endometrial cancers, 15-20% of lung cancers (including lobular lung cancer), as well as bile duct cancer, cervical cancer, and bladder cancer.

[0007] In recent years, covalent inhibitors of mutant KRAS have been developed. These target mutant KRAS via an allosteric site, reducing its affinity for GTP and "locking" its activity. For example, Amgen's sotorasib (AMG510), which is administered to patients with non-small cell lung cancer (NSCLC) harboring the KRAS_G12C mutation, is a KRAS_G12C inhibitor. Mirati Therapeutics' MRTX1257 is also a KRAS_G12C inhibitor and is currently in preclinical development.

[0008] Currently, there are no therapeutic drugs for other KRAS mutations, such as the KRAS_G12V mutation. There is a need in this field to develop immune cells (e.g., TCR-T cells) specific to the KRAS_G12V mutation antigen and to effectively detect, prevent, and treat cancers associated with the KRAS_G12V mutation antigen. Summary of the Invention

[0009] As a result of extensive research, the present inventors have isolated a T cell receptor (TCR) that specifically binds to the KRAS_G12V mutant antigen and prepared lymphocytes recombinantly expressing the TCR. By using such lymphocytes, the specific binding of the TCR to the KRAS_G12V mutant antigen makes it possible to detect the presence of cancer associated with the KRAS_G12V mutant antigen in mammals, and / or to kill cancer cells expressing the KRAS_G12V mutant antigen through an in vivo immune response against target cells expressing the KRAS_G12V mutant antigen, thereby fulfilling the above-mentioned needs.

[0010] Thus, according to a first aspect, the present invention provides an isolated or purified T cell receptor (TCR) that specifically binds to a KRAS_G12V mutant antigen, and preferably the TCR comprises an α chain and a β chain, each of the α chain and β chain comprising three complementarity determining regions (CDRs), the amino acid sequence of CDR3 of the α chain being selected from SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48 and variants thereof in which one or two amino acid residues have been changed, and the amino acid sequence of CDR3 of the β chain being selected from SEQ ID NOs: 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224 and variants thereof in which one or two amino acid residues have been changed.

[0011] According to one embodiment, in the TCR of the present invention, the amino acid sequence of the CDR3 of the α chain and the amino acid sequence of the CDR3 of the β chain are: (i) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 3, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 179, or a variant thereof in which one or two amino acid residues have been changed; (ii) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 6, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 182, or a variant thereof in which one or two amino acid residues have been changed; (iii) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 9, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 185, or a variant thereof in which one or two amino acid residues have been changed; (iv) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 12, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 188, or a variant thereof in which one or two amino acid residues have been changed; (v) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 15, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 191, or a variant thereof in which one or two amino acid residues have been changed; (vi) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 18, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 194, or a variant thereof in which one or two amino acid residues have been changed; (vii) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 21, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 197, or a variant thereof in which one or two amino acid residues have been changed; (viii) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 24, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 200, or a variant thereof in which one or two amino acid residues have been changed; (ix) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 27, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 203, or a variant thereof in which one or two amino acid residues have been changed; (x) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 30, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 206, or a variant thereof in which one or two amino acid residues have been changed; (xi) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 33, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 209, or a variant thereof in which one or two amino acid residues have been changed; (xii) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 36, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 212, or a variant thereof in which one or two amino acid residues have been changed; (xiii) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 39, or a variant thereof in which one or two amino acid residues have been changed from said sequence; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 215, or a variant thereof in which one or two amino acid residues have been changed from said sequence; (xiv) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 42, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 218, or a variant thereof in which one or two amino acid residues have been changed; (xv) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 45, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 221, or a variant thereof in which one or two amino acid residues have been changed; or (xvi) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 48, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 224, or a variant thereof in which one or two amino acid residues have been changed. is.

[0012] According to one embodiment, the amino acid sequences of the three complementarity determining regions (CDRs) contained in the α chain and the three CDRs contained in the β chain of the TCR of the present invention are: (i) α chain CDR1, CDR2, CDR3 amino acid sequences shown in SEQ ID NOs: 1, 2, 3, or variants thereof in which one or two amino acid residues have been changed from each of the above sequences; and β chain CDR1, CDR2, CDR3 amino acid sequences shown in SEQ ID NOs: 177, 178, 179, or variants thereof in which one or two amino acid residues have been changed from each of the above sequences; (ii) α chain CDR1, CDR2, CDR3 amino acid sequences shown in SEQ ID NOs: 4, 5, 6, or variants thereof in which one or two amino acid residues have been changed from each of the above sequences; and β chain CDR1, CDR2, CDR3 amino acid sequences shown in SEQ ID NOs: 180, 181, 182, or variants thereof in which one or two amino acid residues have been changed from each of the above sequences; (iii) α chain CDR1, CDR2, CDR3 amino acid sequences shown in SEQ ID NOs: 7, 8, and 9, or variants in which one or two amino acid residues have been changed from each of the above sequences; and β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 183, 184, and 185, or variants in which one or two amino acid residues have been changed from each of the above sequences; (iv) α chain CDR1, CDR2, CDR3 amino acid sequences shown in SEQ ID NOs: 10, 11, and 12, or variants thereof in which one or two amino acid residues have been changed from each of the above sequences; and β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 186, 187, and 188, or variants thereof in which one or two amino acid residues have been changed from each of the above sequences; (v) α chain CDR1, CDR2, CDR3 amino acid sequences shown in SEQ ID NOs: 13, 14, and 15, or variants thereof in which one or two amino acid residues have been changed from each of the above sequences; and β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 189, 190, and 191, or variants thereof in which one or two amino acid residues have been changed from each of the above sequences; (vi) α chain CDR1, CDR2, CDR3 amino acid sequences shown in SEQ ID NOs: 16, 17, and 18, or variants thereof in which one or two amino acid residues have been changed from each of the above sequences; and β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 192, 193, and 194, or variants thereof in which one or two amino acid residues have been changed from each of the above sequences; (vii) α chain CDR1, CDR2, CDR3 amino acid sequences shown in SEQ ID NOs: 19, 20, and 21, or variants thereof in which one or two amino acid residues have been changed from each of the above sequences; and β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 195, 196, and 197, or variants thereof in which one or two amino acid residues have been changed from each of the above sequences; (viii) α chain CDR1, CDR2, CDR3 amino acid sequences shown in SEQ ID NOs: 22, 23, and 24, or variants thereof in which one or two amino acid residues have been changed from each of the above sequences; and β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 198, 199, and 200, or variants thereof in which one or two amino acid residues have been changed from each of the above sequences; (ix) α chain CDR1, CDR2, CDR3 amino acid sequences shown in SEQ ID NOs: 25, 26, and 27, or variants thereof in which one or two amino acid residues have been changed from each of the above sequences; and β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 201, 202, and 203, or variants thereof in which one or two amino acid residues have been changed from each of the above sequences; (x) α chain CDR1, CDR2, CDR3 amino acid sequences shown in SEQ ID NOs: 28, 29, 30, or variants thereof in which one or two amino acid residues have been changed from each of the above sequences; and β chain CDR1, CDR2, CDR3 amino acid sequences shown in SEQ ID NOs: 204, 205, 206, or variants thereof in which one or two amino acid residues have been changed from each of the above sequences; (xi) α chain CDR1, CDR2, CDR3 amino acid sequences shown in SEQ ID NOs: 31, 32, 33, or variants thereof in which one or two amino acid residues have been changed from each of the above sequences; and β chain CDR1, CDR2, CDR3 amino acid sequences shown in SEQ ID NOs: 207, 208, 209, or variants thereof in which one or two amino acid residues have been changed from each of the above sequences; (xii) α chain CDR1, CDR2, CDR3 amino acid sequences shown in SEQ ID NOs: 34, 35, and 36, or variants thereof in which one or two amino acid residues have been changed from each of the above sequences; and β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 210, 211, and 212, or variants thereof in which one or two amino acid residues have been changed from each of the above sequences; (xiii) α chain CDR1, CDR2, CDR3 amino acid sequences shown in SEQ ID NOs: 37, 38, and 39, or variants thereof in which one or two amino acid residues have been changed from each of the above sequences; and β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 213, 214, and 215, or variants thereof in which one or two amino acid residues have been changed from each of the above sequences; (xiv) α chain CDR1, CDR2, CDR3 amino acid sequences shown in SEQ ID NOs: 40, 41, and 42, or variants thereof in which one or two amino acid residues have been changed from each of the above sequences; and β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 216, 217, and 218, or variants thereof in which one or two amino acid residues have been changed from each of the above sequences; (xv) α chain CDR1, CDR2, CDR3 amino acid sequences shown in SEQ ID NOs: 43, 44, and 45, or variants thereof in which one or two amino acid residues have been changed from each of the sequences; and β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 219, 220, and 221, or variants thereof in which one or two amino acid residues have been changed from each of the sequences; or (xvi) α chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 46, 47, and 48, or variants thereof in which one or two amino acid residues have been changed from each of the above sequences; and β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 222, 223, and 224, or variants thereof in which one or two amino acid residues have been changed from each of the above sequences. is.

[0013] According to some aspects, the TCR of the invention has an alpha chain sequence as set forth in SEQ ID NO: 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173 or 175, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto. and the beta strand sequence set forth in SEQ ID NO: 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, or 379, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0014] According to some aspects, the present invention provides a T cell receptor fusion protein or T cell receptor conjugate comprising a TCR according to the first aspect of the invention and another biologically active molecule, wherein the other biologically active molecule is, for example, an antibody, cytokine, cytotoxic agent, enzyme, radioactive substance or detectable label, and the TCR and the other biologically active molecule are linked to each other with or without a linker.

[0015] The present invention also provides nucleic acids encoding the α chain and / or β chain of the TCR of the present invention.

[0016] Additionally, the present invention provides a vector, preferably a plasmid, shuttle plasmid, phagemid, cosmid, expression vector, retroviral vector, adenoviral vector and / or homology directed repair (HDR) vector, comprising one or more of the nucleic acids described above.

[0017] According to a second aspect, the present invention provides a modified cell transformed with the vector described above and expressing a TCR as described in the first aspect.

[0018] According to some aspects, the present invention provides methods for preparing TCR-T cells expressing an exogenous TCR of the present invention through a targeting strategy that does not use a viral vector.

[0019] According to some aspects, the present invention provides a method for editing the genome of a human cell, comprising: From N-terminus to C-terminus: (i) a sequence encoding a first cleavable linker polypeptide; (ii) a sequence encoding the β chain of a TCR described in the first aspect; (iii) a sequence encoding a second cleavable linker polypeptide; and (iv) a sequence encoding the α chain variable region of a TCR according to the first aspect into a target region of exon 1 of an endogenous T cell receptor (TCR) alpha chain constant region gene in a human cell; and The method further comprises the step of: providing said first cleavable linker polypeptide and said second cleavable linker polypeptide are the same or different viral 2A peptides;

[0020] The exogenous TCR-expressing cells prepared by the above method have high binding affinity to the VVVGAVGVGK-HLA-A*11:01 complex and / or VVGAVGVGK-HLA-A*11:01, and have potent killing activity against SW620 cells (overexpressing HLA-A*11:01 and KRAS G12V+) in vitro.

[0021] According to some aspects, the method of preparing a cell expressing an exogenous TCR is carried out by knocking out the endogenous TCR and knocking in the exogenous TCR using CRISPR / Cas9 technology and homologous recombination technology.

[0022] In a third aspect, the present invention provides methods and modified cells with improved cell therapy efficacy.

[0023] In some aspects, the present invention provides for the co-expression of exogenous TCR and CD8aa molecules in T cells. In some aspects, the present invention provides for the co-expression of exogenous TCR and CD8ab molecules in T cells.

[0024] Coexpression of CD8aa and / or CD8ab molecules with TCRs in CD8+ and CD4+ T cells has beneficial effects on CD8+ and CD4+ T cell function. In particular, coexpression of MHC class I TCRs with CD8 molecules in CD4+ T cells reprograms CD4+ T cells into multifunctional hybrid T cells with both cytotoxic and natural helper functions.

[0025] According to a fourth aspect, the present invention provides the use of a TCR as described in the first aspect, or a modified cell obtainable by the second aspect and the third aspect, for the detection, prevention and / or treatment of cancer related to the KRAS_G12V mutant antigen.

[0026] The preferred embodiments of the invention, as described in detail below, can be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments that are presently preferred. It is to be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Brief explanation of the drawings]

[0027] [Figure 1] Figure 1A shows the targeting strategy for knocking in an exogenous TCR into the TRAC locus using gRNA002, and Figure 1B shows the targeting strategy for knocking out the TRBC1 and TRBC2 loci using gRNA004. [Figure 2]Figure 2 is a schematic diagram showing the results of TCR gene editing efficiency detected by flow cytometry. Flow cytometry data were analyzed as a cell distribution map of four quadrants (Q1, Q2, Q3, and Q4). Q2 represents the cell population that has undergone endogenous TCR knockout (KO) and exogenous TCR knockin (KI) and expresses nwTCR. Q3 represents wild-type T cells that have not undergone gene editing. Q4 represents KO cells that have undergone endogenous TCR knockout. [Figure 3A-C] 3A to 3P show examples of flow cytometry results of CD4+ T cells and CD8+ T cells transfected with different nwTCRs by electroporation and stained with pMHC tetramer. For CD4+ T cells and CD8+ T cells transfected by electroporation with nwTCR-0125, nwTCR-0126, nwTCR-0127, nwTCR-1708, nwTCR-1862, nwTCR-2162, nwTCR-2241, nwTCR-2308, or nwTCR-2563, the exemplified staining results are obtained by staining with labeled VVVGAVGVGK-HLA-A*11:01 tetramer; and for CD4+ T cells and CD8+ T cells transfected by electroporation with nwTCR-2310, nwTCR-2390, nwTCR-2392, nwTCR-2424, nwTCR-2561, nwTCR-2595, or nwTCR-2629, the exemplified staining results are obtained by staining with labeled VVVGAVGVGK-HLA-A*11:01 tetramer. For T cells, the staining results shown are from staining with labeled VVGAVGVGK-HLA-A*11:01 tetramer. [Figure 3D-F] Same as above. [Figure 3G-I] Same as above. [Figure 3J-L] Same as above. [Figure 3M-O] Same as above. [Figure 3P] Same as above. [Figure 4A-B]4A to 4H show experimental results and EC50 values ​​of binding affinity assays of T cells expressing each nwTCR for the short peptide represented by VVVGAVGVGK (SEQ ID NO: 381) or VVGAVGVGK (SEQ ID NO: 382) and presented by HLA-A*11:01. Here, FIGS. 4A, 4C, 4D, and 4G show the binding affinity for the short peptide represented by VVVGAVGVGK (SEQ ID NO: 381), and FIGS. 4B, 4E, 4F, and 4H show the binding affinity for the short peptide represented by VVGAVGVGK (SEQ ID NO: 382). [Figure 4C-E] Same as above. [Figure 4F-H] Same as above. [Figure 5A-C] Figures 5A to 5E show the in vitro killing effect of T cells expressing each nwTCR on the colorectal cancer (CRC) cell line SW620 (overexpressing HLA-A*11:01 and KRASG12V+) target cells. In the figures, "Blank" indicates target cells to which no nwTCR-expressing T cells were added. [Figure 5D-E] Same as above. [Figure 6] Figure 6 shows the fluorescence imaging results of killing of SW620 cells (overexpressing HLA-A*11:01 and KRASG12V+) by nwTCR-2404-expressing T cells. In the figure, "Blank" indicates target cells to which nwTCR-2404-expressing T cells had not been added. [Figure 7] Figure 7 shows real-time analysis data on the killing of SW620 cells (overexpressing HLA-A*11:01 and KRASG12V+) by T cells expressing nwTCR-2404. The results demonstrate that the gene-edited T cells have a specific killing effect on SW620 cells (overexpressing HLA-A*11:01 and KRASG12V+). In the figure, "Blank" indicates target cells to which nwTCR-expressing T cells were not added. [Figure 8] Figure 8A shows the targeting strategy of nwTCR-CD8a. Figure 8B shows the targeting strategy of nwTCR-CD8ab. [Figure 9] Figure 9A shows flow cytometry results of CD4+ T cells and CD8+ T cells transfected with nwTCR-1708 by electroporation and stained with pMHC tetramer. Figure 9B shows flow cytometry results of CD4+ T cells and CD8+ T cells transfected with nwTCR-1708-CD8a by electroporation and stained with pMHC tetramer. Figure 9C shows flow cytometry results of CD4+ T cells and CD8+ T cells transfected with nwTCR-1708-CD8ab by electroporation and stained with pMHC tetramer. DETAILED DESCRIPTION OF THE INVENTION

[0028] Before describing the present invention in detail, it should be noted that the present invention is not limited to the specific methods and experimental conditions described herein, as such methods and conditions may vary. Furthermore, the terms used in this disclosure are for the purpose of describing particular embodiments and are not intended to be limiting.

[0029] I. Definition Unless otherwise defined, all technical and scientific terms used in this disclosure have the meanings commonly understood by one of ordinary skill in the art. For purposes of the present invention, these terms are defined below.

[0030] The term "about" when used in connection with a numerical value is intended to include numerical values ​​within a range of 10% below the stated numerical value and 10% above the stated numerical value.

[0031] The word "and / or" when used in connection with two or more alternatives should be understood to mean any one of those alternatives, or any two or more of those alternatives.

[0032] In the present disclosure, the word "comprise" or "include" is intended to include the stated elements, integers, or steps, but is not intended to exclude other elements, integers, or steps. In the present disclosure, the word "comprise" or "include" also encompasses situations consisting of the stated elements, integers, or steps, unless otherwise specified. For example, when referring to an antibody variable region "comprising" a particular sequence, it is intended to encompass an antibody variable region consisting only of that particular sequence.

[0033] The "RAS protein family" belongs to a large family of small GTPases. RAS proteins can be constitutively activated by a single amino acid mutation. Mutant RAS protein products are involved in signal transduction during the early stages of tumorigenesis in many human cancers. Various human cancers, including lung cancer (e.g., lung adenocarcinoma), ovarian cancer (e.g., epithelial ovarian cancer), pancreatic cancer, prostate cancer, endometrial cancer, and colorectal cancer, express mutant RAS proteins.

[0034] Kirsten rat sarcoma viral oncogene homolog (KRAS) is an important member of the RAS protein family. KRAS is regulated by the upstream epidermal growth factor receptor (EGFR) family, and EGFR signaling activates SOS proteins, which then regulates KRAS activation. The transition between inactive and active states of the KRAS protein in cells is determined by the molecules to which KRAS binds. The guanosine nucleotide exchange factor (GEF) catalyzes the binding of KRAS to GTP, thereby activating KRAS, while the GTPase-activating protein (GAP) promotes the hydrolysis of KRAS-bound GTP to GDP, thereby inactivating KRAS. Activated KRAS regulates downstream signaling pathways, such as MAPK and PI3K, which are involved in cell proliferation and cell migration. Mutations in KRAS maintain their active state by continuously binding GTP, leading to continuous activation of downstream signaling pathways and promoting tumorigenesis.

[0035] The term "antigen" refers to a molecule that is specifically detected by an organism's immune system.

[0036] "KRAS_G12V mutant antigen" refers to a KRAS protein that has a G12V mutation. The G12V mutation is specifically detected by an organism's immune system. "G12V" and "G12V mutation" are used interchangeably to refer to a substitution of glycine at position 12 of the KRAS protein with valine.

[0037] The T cell receptor (TCR) is a protein on the surface of T cells that specifically recognizes antigenic peptides bound to the major histocompatibility complex (MHC). When the TCR binds to the antigenic peptide and MHC, the T lymphocyte is activated by signaling and initiates the subsequent immune response. There are four TCR genes in the human genome. Two of these genes encode the light chain of the TCR: the TRA gene encodes TCRα, and the TRG gene encodes TCRγ. Two of the TCR genes encode the heavy chain: the TRB gene encodes TCRβ, and the TRD gene encodes TCRδ. The heavy and light chains of the TCR form a heterodimer to form the complete TCR. There are two types of TCRs in humans: TCRα / β and TCRγ / δ. 95% of T cells are αβ T cells expressing TCRα / β, and 5% are γ / δ T cells expressing TCRγ / δ. This ratio varies depending on the individual's developmental process and disease state (e.g., leukemia), and also differs between species.

[0038] The mature heavy chain TCR gene is composed of four gene segments (VDJC): the variable region (V), diversity region (D), joining region (J), and constant region (C), whereas the light chain TCR gene lacks the D region (VJC). The heavy and light chains of TCR each have three complementarity-determining regions (CDRs). CDR1 and CDR2 are relatively conserved and are involved in MHC recognition. CDR3 is the primary CDR involved in antigen recognition.

[0039] TCR genes are the most complex and diverse genes in the human genome. There are approximately 2 × 10 16 ~10 18Each T cell contains 100 T cells, each expressing a different TCR. This complexity is due to three main factors: (i) structural diversity: The VDJC / VJC structure of mature TCRs is generated through complex rearrangements. The genome contains 65–100 V gene segments, 2 D gene segments, and 13 J gene segments. During TCR rearrangement, one of each of these three types of segments is selected. This results in high TCR diversity. (ii) junction flexibility: During rearrangement, random insertion or deletion of non-templated nucleotides frequently occurs in the VD and DJ junction regions, further increasing the diversity of the CDR3 region. (iii) somatic mutation: The mutation frequency of the D region of T cells is approximately 1,000 times higher than normal.

[0040] As known in the art, the terms "polynucleotide" and "nucleic acid," used interchangeably in this disclosure, refer to a chain of nucleotides of any length, including DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides, or their bases and / or analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase.

[0041] The sequence identity between multiple sequences is calculated as follows.

[0042] To determine the identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., for optimal alignment, gaps may be introduced in either or both of the first amino acid or nucleic acid sequence and the second amino acid or nucleic acid sequence, or non-homologous sequences may be deleted for comparison). In a preferred embodiment, for comparison purposes, the length of the aligned reference sequence is at least 30%, preferably at least 40%, more preferably at least 50% or 60%, and even more preferably at least 70%, 80%, 90%, or 100% of the total length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If the same amino acid residue or nucleotide occupies the same position in the first sequence and the corresponding position in the second sequence, the molecules are identical at this position.

[0043] A mathematical algorithm can be used to compare two sequences and calculate the percent identity between them. In a preferred embodiment, the identity between two amino acid sequences is determined using the Needleman and Wunsch algorithm ((1970) J. Mol. Biol., 48: 444-453; available at http: / / www.gcg.com) integrated into the GAP program (GCG software package), using a Blossum 62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6. In another preferred embodiment, the identity between two nucleotide sequences is determined using the GAP program of the GCG software package (available at http: / / www.gcg.com), using a NWSgapdna, CMP matrix, a gap weight of 40, 50, 60, 70, or 80, and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred parameter set (and that which should be used unless otherwise specified) is a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0044] Identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller (1989, CABIOS, 4:11-17) as incorporated into the ALIGN program (version 2.0), using a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4.

[0045] The term "antigen presenting cells" or "APCs" refers to cells of the immune system, such as helper cells (e.g., B cells, dendritic cells, etc.), that present foreign antigens on their surface complexed with major histocompatibility complexes (MHC). T cells can recognize these complexes using the T cell receptor (TCR). APCs process antigens and present them to T cells.

[0046] The term guide RNA (gRNA) refers to a target DNA-specific RNA that forms a complex with a Cas protein and guides the Cas protein to the target DNA, whereby the Cas protein introduces a double-strand break at the cleavage site of the target DNA. In the present invention, the guide RNA may be composed of two RNAs, i.e., a CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA), or a single guide RNA (sgRNA) created by fusing the necessary portions of the crRNA and tracrRNA.

[0047] Ribonucleoproteins (RNPs) are complexes that have gene editing functions and are formed by the complexation of Cas9 protein and gRNA.

[0048] The CRISPR / Cas9 gene editing system is primarily composed of two components: the Cas9 protein, which acts as a "wrench," and the CRISPR guide RNA, which acts as a "screw." The guide RNA identifies the target site and recruits and activates the Cas9 protein, while the Cas9 protein cuts the target DNA.

[0049] The term "recombinant," when used with respect to, for example, a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein, or the alteration of a naturally occurring nucleic acid or protein.

[0050] The term "target site" refers to a region within a target genome that is to be modified or repaired. The term "target DNA sequence" refers to any segment of a DNA sequence. The DNA sequence near the target site allows for integration of an exogenous sequence into the target site. Such integration includes, but is not limited to, gene knock-in (KI). In certain embodiments, the target DNA sequence is a double-stranded DNA sequence, examples of which include, but are not limited to, a DNA sequence within the chromosomal genome of a cell, a DNA sequence outside the chromosomal genome of a cell (e.g., a mitochondrial genome), a plasmid, a virus, etc.

[0051] As used herein, the term "site-directed recombination" refers to non-random integration of an exogenous sequence into a specific target site, such as integration 5' upstream, 3' downstream, or between target sites.

[0052] As used herein, the term "exogenous DNA sequence" refers to a DNA sequence that is desired to be site-specifically recombined into a target site. The exogenous DNA sequence may be a sequence that is not present in the target site, or may be a modified sequence.

[0053] The terms "donor DNA" or "donor nucleic acid sequence" refer to a polynucleotide containing a polynucleotide sequence to be expressed and inserted into a target site in a target genome. In some embodiments, the donor DNA further contains a sequence homologous to the genome sequence (hereinafter also referred to as a "homology arm"). "Homology" means that multiple DNA sequences are similar. The homology arm is sufficient for homologous recombination with the homologous genome sequence. The homology arm may, for example, contain at least 50 to 3,500 or more bases in length.

[0054] The term "homology directed repair (HDR)" refers to repair by homology-based recombination. HDR allows for highly efficient and specific insertion of a donor DNA template (encoding a sequence of interest) into a target genomic site. HDR is a repair pathway initiated by double-stranded damage in cellular DNA. HDR occurs when a DNA fragment homologous to the damaged DNA is present in the cell nucleus. HDR vectors refer to vectors for electroporation transfection using CRISPR / Cas9 and homologous recombination techniques. HDR efficiency refers to the efficiency of gene knock-in using electroporation transfection using CRISPR / Cas9 and homologous recombination techniques.

[0055] A synonymous mutation refers to a mutation in which the genetic code is degenerate, i.e., there are often two or more codons that determine a single amino acid, and the third nucleotide of the triplet codon is substituted, resulting in a neutral mutation that does not change the amino acid composition. In this case, the mutation of the third nucleotide of the triplet codon does not change the encoded amino acid, so this mutation is considered a synonymous mutation.

[0056] As used herein, the term "vector" refers to a construct capable of delivering one or more genes or sequences of interest to a host cell and preferably expressing the genes or sequences in the host cell. Examples of vectors include, but are not limited to, viral vectors, plasmids, cosmids, or phage vectors. A vector may contain a nucleic acid sequence, such as an origin of replication, that allows the gene or sequence of interest to replicate in a host cell. A vector may also contain one or more selectable marker genes and other genetic elements known to those skilled in the art. The vector is preferably an expression vector containing a nucleic acid of the invention, preferably operably linked to a sequence that allows its expression.

[0057] The term "operatively linked to" refers to a functional linkage between a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a protein of interest so that they function as a whole. Such functional linkages in recombinant vectors can be formed using recombinant techniques well known in the art, including site-specific DNA cleavage and ligation using enzymes well known in the art.

[0058] As used herein, the term "genetically engineered cells" includes cells into which exogenous nucleic acid has been introduced and the progeny of such cells. Genetically engineered cells include "transfected cells." This term includes the primary transfected cell and its progeny (regardless of the number of passages). Such progeny need not contain nucleic acids identical to those of the parent cell and may contain mutations. The present disclosure also includes mutant progeny that have the same function or biological activity as the cells screened or selected from the originally transfected cells.

[0059] As used herein, the terms "subject" and "individual" refer to an animal, preferably a mammal, more preferably a human, in need of remission and / or treatment of a cancer associated with the KRAS_G12V mutant antigen. Examples of mammals include, but are not limited to, livestock, race animals, pets, primates, horses, dogs, cats, mice, and rats.

[0060] Adoptive cell transfer therapy (ACT) is a therapy in which immune-active cells are isolated from a subject or patient in vivo, activated, expanded, modified in vitro through gene editing, and then re-administered into the patient's body to kill target cells.

[0061] II. T Cell Receptors (TCRs) of the Invention and Nucleic Acids Encoding the TCRs The wild-type human KRAS protein is 188 amino acid residues long, has a molecular weight of approximately 21.6 kD, and has a glycine residue at position 12. 83% of genetic mutations in KRAS occur at position 12, with one of the most significant being a glycine-to-valine mutation at position 12 (sometimes abbreviated as G12V).

[0062] The present invention provides an isolated or purified TCR having antigen specificity for a KRAS peptide having a G12V mutation that is presented by a human leukocyte antigen (HLA) class I molecule. The KRAS peptide having a G12V mutation that is presented by a human leukocyte antigen (HLA) class I molecule has any length suitable for binding to any HLA class I molecule.

[0063] According to some embodiments, the KRAS peptide having a G12V mutation is a mutant KRAS peptide having a length of about 9 to about 10 amino acid residues, comprising about 9 to about 10 consecutive amino acid residues in a KRAS protein having a G12V mutation. According to some embodiments, the TCR of the present invention has antigen specificity for a KRAS peptide having a G12V mutation, and is a mutant KRAS peptide having a length of about 9 or about 10 amino acid residues. Examples of KRAS peptides having a G12V mutation recognized by the TCR of the present invention include a short peptide consisting of amino acids 7 to 16 of KRAS represented by VVVGAVGVGK (SEQ ID NO: 381) (sometimes abbreviated as "KRAS_G12V_7-16 peptide" in this disclosure) and a short peptide consisting of amino acids 8 to 16 of KRAS represented by VVGAVGVGK (SEQ ID NO: 382) (sometimes abbreviated as "KRAS_G12V_8-16 peptide" in this disclosure).

[0064] The T cell receptor (TCR) is a molecule present on the surface of T cells that is responsible for recognizing antigenic peptide-MHC complexes (i.e., pMHC). Specific binding of the TCR to an antigenic peptide-MHC complex leads to a series of biochemical reactions mediated by associated enzymes, co-receptors, and accessory molecules, resulting in T cell activation. The TCR heterodimer is composed of an α-chain and a β-chain in 95% of T cells, and a γ-chain and a δ-chain in 5% of T cells.

[0065] Each chain of the TCR is a member of the immunoglobulin superfamily and consists of an N-terminal immunoglobulin (Ig) variable (V) domain, an Ig constant (C) domain, a transmembrane region (i.e., transmembrane domain), and a short C-terminal cytoplasmic tail. The variable regions of the TCR α and β chains each contain three highly variable regions, or complementarity-determining regions (CDRs), with CDR3 of each variable region being the primary CDR responsible for recognizing processed antigens. CDR2 is thought to recognize MHC molecules.

[0066] The constant domain of the TCR consists of a short linking sequence in which cysteine ​​residues form disulfide bonds linking the TCR α and β chains.

[0067] During T cell maturation, the TCR and CD3 form a TCR / CD3 complex. The formation of the TCR / CD3 complex typically follows the following sequence: First, the three peptide chains CD3γ, δ, and ε form two types of heterodimers, γ-ε and δ-ε, to form a stable core complex, to which TCRαβ (or TCRγδ) binds. Subsequently, the ζ-ζ or ζ-η dimer binds to the TCRαβ (or TCRγδ) / CD3γεδε complex, which is ultimately transported to the T cell surface. Signals are transmitted from the TCR into the cell via the TCR / CD3 complex.

[0068] Signaling from the TCR / CD3 complex is enhanced by simultaneous binding of MHC to specific co-receptors: in helper T cells, the co-receptor is the CD4 molecule, which is specific for MHC class II, whereas in cytotoxic T cells, the co-receptor is the CD8 molecule, which is specific for MHC class I.

[0069] As used herein, the term "T cell receptor" has its conventional meaning in the art and is used to refer to a molecule that recognizes peptides presented by MHC molecules. TCR molecules are heterodimers having two chains, α and β (or sometimes γ and δ).

[0070] The TCR of the present invention recognizes the KRAS_G12V mutant antigen with specific affinity. The KRAS_G12V mutant antigen is degraded intracellularly by proteasomes into short peptides of 8 to 10 amino acids in length (e.g., the KRAS_G12V_7-16 peptide shown in SEQ ID NO: 381 and / or the KRAS_G12V_8-16 peptide shown in SEQ ID NO: 382). These short peptides are presented on the cell surface as peptide / MHC complexes (pMHC) by MHC class I. Some pMHCs have been confirmed to be associated with various cancers and may be potential targets for TCR therapy.

[0071] The present invention provides isolated or purified T cell receptor (TCR) α and / or β chains. The TCRs of the present invention may be hybrid TCRs comprising sequences from two or more species. For example, because mouse TCRs can be expressed more efficiently in human T cells than human TCRs, the TCRs of the present invention may comprise a human variable region and a mouse constant region.

[0072] According to one aspect, the TCR of the present invention comprises an α chain and a β chain, each of which comprises three complementarity determining regions (CDRs), the amino acid sequence of the CDR3 of the α chain being selected from SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48 and variants in which one or two amino acid residues have been changed from said sequences, and the amino acid sequence of the CDR3 of the β chain being selected from SEQ ID NOs: 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 209, 212, 215, 218, 221, 224 and variants in which one or two amino acid residues have been changed from said sequences.

[0073] According to one aspect, the TCR of the present invention comprises an α chain and a β chain, and the amino acid sequences of the three complementarity determining regions (CDRs) contained in the α chain of the TCR of the present invention and the amino acid sequences of the three CDRs contained in the β chain are: (i) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 3, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 179, or a variant thereof in which one or two amino acid residues have been changed; (ii) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 6, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 182, or a variant thereof in which one or two amino acid residues have been changed; (iii) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 9, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 185, or a variant thereof in which one or two amino acid residues have been changed; (iv) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 12, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 188, or a variant thereof in which one or two amino acid residues have been changed; (v) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 15, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 191, or a variant thereof in which one or two amino acid residues have been changed; (vi) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 18, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 194, or a variant thereof in which one or two amino acid residues have been changed; (vii) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 21, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 197, or a variant thereof in which one or two amino acid residues have been changed; (viii) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 24, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 200, or a variant thereof in which one or two amino acid residues have been changed; (ix) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 27, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 203, or a variant thereof in which one or two amino acid residues have been changed; (x) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 30, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 206, or a variant thereof in which one or two amino acid residues have been changed; (xi) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 33, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 209, or a variant thereof in which one or two amino acid residues have been changed; (xii) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 36, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 212, or a variant thereof in which one or two amino acid residues have been changed; (xiii) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 39, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 215, or a variant thereof in which one or two amino acid residues have been changed; (xiv) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 42, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 218, or a variant thereof in which one or two amino acid residues have been changed; (xv) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 45, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 221, or a variant thereof in which one or two amino acid residues have been changed; or (xvi) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 48, or a variant thereof in which one or two amino acid residues have been changed; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 224, or a variant thereof in which one or two amino acid residues have been changed. is.

[0074] According to one aspect, the TCR of the invention comprises an alpha chain sequence as set forth in SEQ ID NO: 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173 or 175, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto. and the β chain sequence set forth in SEQ ID NO: 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377 or 379, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. Preferably, the constant region of the TCR of the present invention is a murine constant region.

[0075] According to some aspects, the amino acid residue changes in the TCR mutants of the present invention are substitutions, additions, or deletions of amino acid residues within the α chain sequence set forth in any one of SEQ ID NOs: 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, or 175, and within the β chain sequence set forth in any one of SEQ ID NOs: 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, or 379, provided that such TCR mutants retain or improve their ability to bind to KRAS_G12V mutant antigen epitope peptide-MHC complexes. In one aspect, the substitutions are conservative substitutions. Examples of conservative substitutions are shown in Table A below.

[0076] [Table A]

[0077] Amino acids can be classified according to common properties of their side chains. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu; Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn; Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0078] Non-conservative substitutions are those that convert a residue from one of these classes into a residue from another class.

[0079] According to some embodiments, the TCR of the present invention can recognize and bind to an epitope peptide of a mutant KRAS protein presented by an HLA class I molecule, and induce an immune response.

[0080] According to some embodiments, the HLA class I molecule may be any HLA-A molecule. According to one example, the HLA class I molecule is an HLA-A11 molecule. The HLA-A11 molecule may be any HLA-A11 molecule. Examples of HLA-A11 molecules include, but are not limited to, HLA-A*11:01, HLA-A*11:02, HLA-A*11:03, or HLA-A*11:04. Of these, the HLA class I molecule is preferably an HLA-A*11:01 molecule. The HLA-A*11:01 molecule is the most common HLA-A molecule in Asians.

[0081] The present invention also relates to nucleic acids encoding the TCRs of the present invention or portions thereof. Examples of TCR portions include one or more CDRs; one or more variable regions; an α chain; or a β chain. The nucleic acid may be double-stranded or single-stranded, and may be RNA or DNA. The nucleic acid sequence may be codon-optimized to achieve high expression in mammalian production cells. Codon usage in mammalian cells and various other organisms is well known in the art. Codon optimization may include removal of mRNA instability motifs and cryptic splice sites.

[0082] The TCRs of the present invention may be linked to other biologically active molecules through modification by various techniques (e.g., gene fusion, chemical conjugation, etc.). TCRs that can be linked to other biologically active molecules include TCR heterodimers or soluble forms thereof, more preferably soluble single-chain TCRs. Other biologically active molecules include various biologically active effectors, specific examples of which include antibodies, cytokines, cytotoxic substances, enzymes, radioactive substances, detectable markers, etc. A linker may or may not be present between the TCR and other biologically active molecule.

[0083] In some embodiments, the TCR fusion protein is a fusion of a TCR and an antibody, including, for example, an intact antibody (e.g., IgG, IgM, or IgA class), a fragment thereof (e.g., Fv, Fab, Fab', Fab'-SH, F(ab')2, diabody, single-chain antibody (e.g., scFv), single-domain antibody, or multispecific antibody (e.g., bispecific antibody).

[0084] In some embodiments, the TCR fusion protein is a fusion of a TCR with a cytokine, such as an interleukin (e.g., IL-2), a chemokine (e.g., MIP-1β), a growth factor (e.g., GCSF), or the like.

[0085] According to some embodiments, a TCR conjugate is a molecule in which a TCR is covalently linked to a cytotoxic agent (e.g., adriamycin).

[0086] According to some embodiments, the TCR conjugate is a TCR conjugate in which the TCR is fused to a radioactive material (e.g., I 125 ) is a molecule covalently bonded to

[0087] According to some embodiments, a TCR conjugate is a molecule in which a TCR is covalently linked to a detectable marker (e.g., a fluorescent marker).

[0088] The T cell receptor fusion proteins or T cell receptor complexes of the invention can be used in a variety of applications, including in vivo detection of cells and / or imaging of cells or tissues, and therapeutic applications such as in vivo or in vitro killing of target cells or tissues that express the KRAS_G12V mutant antigen and specifically bind to the TCR.

[0089] III. Vectors Comprising Nucleic Acids Encoding the TCRs of the Present Invention The present invention also relates to a vector comprising a nucleic acid encoding a TCR of the present invention. In one embodiment, the vector is a pUC57-Simple vector (purchased from GenScript Biotech Corporation). In yet another embodiment, a pUC57-HA vector is used. This is an optimized vector based on the pUC57-Simple vector, formed by retaining only the Ori and Amp sequences of the pUC57-Simple vector, replacing the Amp sequence with a Kana sequence, and adding left and right homology arm (HA) sequences (approximately 800 bp) corresponding to the TRAC locus.

[0090] Such vectors enable the introduction of nucleic acids encoding the TCRs of the present invention into cells such as T cells, NK cells, and stem cells (e.g., pluripotent stem cells, induced pluripotent stem cells (iPSCs), etc.), thereby enabling the production of modified cells that express KRAS_G12V mutant antigen-specific TCRs.

[0091] The KRAS_G12V mutant antigen-specific TCR refers to a TCR that specifically binds to and immunologically recognizes the G12V mutant KRAS with high affinity. For example, approximately 1 × 10 4 ~Approx. 1×10 5 After co-culture of T cells with antigen-presenting cells such as T2 cells or K562 cells pulsed with G12V mutant KRAS, IFN-γ secretion was induced, and TCR EC 50 is about 1×10 -7 M or less (e.g., 1×10 -8 M or less, 1×10 -9 M or less, 1×10 -10 M or less), the TCR is considered to be antigen-specific for G12V mutant KRAS. The HLA class I molecule may be any of the HLA class I molecules described in the present disclosure (e.g., an HLA-A*11:01 molecule).

[0092] Preferably, the vector is configured to confer sustained, high-level expression of the introduced exogenous TCR within the engineered cell (e.g., engineered T cell), allowing the introduced exogenous TCR to successfully compete with the endogenous TCR for a finite pool of CD3 molecules. Alternatively, increasing the supply of CD3 molecules may also increase expression of the exogenous TCR in the genetically engineered cell. That is, the vector optionally includes a CD3-γ, CD3-δ, CD3-ε, and / or CD3-ζ gene. In one embodiment, the vector includes a CD3-ζ gene. Additionally, one or more separate vectors encoding the CD3 gene(s) may be provided along with a vector encoding the exogenous TCR and co-transfected into the cell.

[0093] The form of such a vector is not limited to a homology-directed repair (HDR) vector, but may also be a viral vector, such as a lentiviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes viral vector, a retroviral vector, or a baculoviral vector for intracellular genome editing.

[0094] Genome editing technology involves inserting, deleting, or replacing nucleic acids within cellular genomic DNA. Genetically modified T cells using genome editing technology in human primary T cells have shown excellent efficacy in clinical trials of various adoptive immunotherapy drugs. Among these, chimeric antigen receptors (CARs) or T cell receptors (TCRs) are often used to modify human primary T cells to recognize specific target epitopes. These modified T cells can specifically kill specific target cells.

[0095] Common TCR gene editing methods can be divided into two types based on the gene transfer method. One type involves random gene transfer, such as lentivirus (LV) systems, adeno-associated virus (AAV) systems, and transposon systems. The other type involves zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) technology. CRISPR technology, among others, recognizes and edits DNA via gRNA guidance and then inserts large gene fragments site-specifically through homologous recombination, offering the advantage of easy modification and scalability.

[0096] IV. Preparation of Modified Cells A viral vector can be used to introduce a target TCR into cells. However, the viral vector method, which introduces exogenous TCR α / β genes into cells without knocking out the cell's endogenous TCR, may result in mismatching with the exogenous TCR α or β chain. While the problem of mismatching with the exogenous TCR α or β chain can be alleviated by means such as disulfide bond modification or conversion to a mouse constant region, the viral vector is randomly inserted into the cell genome, so there remains a risk of disrupting other genes.

[0097] Alternatively, a desired TCR can be introduced into a cell using a non-viral vector, allowing the exogenous TCR α / β gene to be precisely integrated into a specific genomic gene of the cell. According to some embodiments, a gene editing method that does not use a viral vector can be used to knock out the endogenous T cell receptor α and β chains of a human T cell, while simultaneously knocking in nucleotides encoding the desired exogenous T cell receptor α and β chains into the exons of the TRAC gene using CRISPR / Cas9 technology and homologous recombination technology. As a result, expression of the endogenous TCR is inhibited, and the endogenous TCR promoter is used to express the desired exogenous TCR α and β chains.

[0098] According to one embodiment, nucleotides encoding the desired heterologous T cell receptor α and β chains are knocked into exon 1 of the endogenous TRAC gene. This eliminates the need to insert the TRAC gene into the heterologous knock-in fragment, shortening the length of the fragment for gene knock-in and making gene knock-in easier. Compared to techniques that use viral vectors to express TCRs, techniques for expressing TCRs using non-viral vector approaches serve as a rapid, simple, and cost-effective method for introducing exogenous TCR α / β genes into cells.

[0099] IV.1 Knockout site selection TCR is a dimer consisting of a TCRα chain and a TCRβ chain. The TCRα chain gene is formed by rearrangement of the TRAV, TRAJ, and TRAC genes. The TRAV and TRAJ genes each contain multiple distinct sequences. During rearrangement, only one of these multiple sequences is randomly selected and expressed. If the TRAV and TRAJ genes are selected as knockout sites, it is difficult to avoid the generation of random TCRα chain genes. Since there is only one TRAC gene, knocking out the TRAC gene results in the knockout of any random TCRα chain gene, making TRAC a suitable knockout site. The TCRβ chain gene is formed by rearrangement of the TRBV, TRBJ, TRBD, and TRBC genes. Because the TRBV and TRBJ genes each contain multiple distinct sequences, the TRBV and TRBJ genes are inappropriate knockout sites. The TRBC gene includes both TRBC1 and TRBC2, which contain partially identical sequences, and a common sequence can be selected as the knockout site. By knocking out the common sequence, any random TCRβ gene is knocked out.

[0100] In some embodiments, one or more of the endogenous TRAC gene, the endogenous TRBC1 gene, and / or the TRBC2 gene are knocked out. In some embodiments, the endogenous TRAC gene and the endogenous TRBC1 and TRBC2 genes are knocked out simultaneously. This increases the efficiency of endogenous TCR knockout and reduces the risk of mismatch between the exogenous TCR and the endogenous TCR derived from endogenous TCR expression.

[0101] Nuclease-based genome editing tools, including meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), megaTAL nucleases, and CRISPR / CRISPR-associated protein 9 (CRISPR / Cas9), can be used to target and disrupt endogenous TRAC and TRBC genes through double-strand breaks and DNA repair via non-homologous end joining (NHEJ).

[0102] IV.2 Knock-in site selection Because the endogenous TRAC gene is unique and required for all TCRα expression, the endogenous TRAC site is selected as the knock-in site for the exogenous TCRα / β gene. This allows the endogenous TCR to be removed, and at the same time, the exogenous TCRα / β gene of the present invention (hereinafter sometimes referred to as the "nwTCR gene") can be expressed using the endogenous TCR promoter of human-derived T cells without the need for additional TRAC gene addition. This allows the size of the knock-in fragment to be reduced, thereby improving the efficiency of gene editing.

[0103] In some embodiments, the expression construct of the nwTCR is cloned into a targeting vector (e.g., pUC57-S vector), and the nwTCR is knocked into the constant region of the TCR α chain site-specifically via the designed homology arms. Expression of the nwTCR is regulated by the transcriptional regulatory sequence of the gene locus. The knock-in site has a higher level of regulation by the endogenous promoter than other sites, ensuring continuous and stable expression of the nwTCR gene.

[0104] IV.3 Modified Cells The present invention provides modified cells that express an exogenous TCR.

[0105] In some embodiments, the modified cells expressing a TCR are prepared from cells derived from blood, bone marrow, lymph, or lymphoid organs, such as lymphocytes or stem cells, including, but not limited to, T cells, NK cells, etc., and stem cells, such as, for example, pluripotent stem cells and induced pluripotent stem cells (iPSCs).

[0106] The cells are usually primary cells, e.g., cells isolated directly from a subject, and / or cells isolated from a subject and frozen. The cells may be allogeneic and / or autologous.

[0107] In some embodiments, the RNPs and plasmids are used to transduce CD3 / CD28-activated primary cells (e.g., sorted CD4+ and CD8+ T cells) by electroporation using CRISPR / Cas9 and homologous recombination techniques, thereby preparing modified TCR cells.

[0108] In some embodiments, sgRNAs are designed to target the endogenous TRAC gene, and sgRNAs are designed to target the endogenous TRBC1 and TRBC2 genes.

[0109] The Cas9 protein, guided by the sgRNA, binds to and cleaves a specific site in the target genome. The double-stranded break in the endogenous TRAC gene created by the RNP allows homologous recombination in the presence of donor DNA bearing homologous arms, resulting in site-specific insertion of the target nwTCR gene.

[0110] According to a specific embodiment, the specific site in the target genome to which the Cas9 protein guided by the sgRNA binds is located within exon 1 of the TRAC gene, and the Cas9 protein cleaves that specific site. The sgRNA recognition sequence and PAM sequence efficiently targeted by the designed and validated sequence comprise the nucleotide sequence shown as TCAGGGTTCTGGATATCTGT-GGG (i.e., the sgRNA recognition sequence shown in SEQ ID NO: 383 - the PAM sequence shown in SEQ ID NO: 385, where "-" separates the CRISPR / Cas9 recognition site and the PAM sequence).

[0111] According to a specific embodiment, the specific site in the target genome to which the Cas9 protein guided by the sgRNA binds is located within exon 1 of the TRBC1 and TRBC2 genes, and the Cas9 protein cleaves the specific site. The sgRNA recognition sequence and PAM sequence efficiently targeted by the designed and validated sequence comprise the nucleotide sequence shown as CTGCCTGAGCAGCCGCCTGA-GGG (i.e., the sgRNA recognition sequence shown in SEQ ID NO: 384 - the PAM sequence shown in SEQ ID NO: 385, where "-" separates the CRISPR / Cas9 recognition site and the PAM sequence).

[0112] CRISPR / Cas systems can include Cas components in the form of Cas proteins or nucleic acids encoding the Cas proteins.

[0113] In the present invention, the Cas protein may be any Cas protein as long as it has endonuclease activity or nickase activity when complexed with a guide RNA.

[0114] Preferably, the Cas protein is a Cas9 protein or a variant thereof, or a functional fragment thereof.

[0115] Cas proteins include, but are not limited to, proteins isolated from organisms such as Streptococcus sp., preferably Streptococcus pyogenes, or recombinant proteins.

[0116] In one embodiment, the Cas protein comprises a Cas9 from Streptococcus pyogenes, e.g., a Cas9 having the amino acid sequence set forth in SEQ ID NO: 405.

[0117] In other embodiments, the Cas protein comprises, but is not limited to, an amino acid sequence having at least 50% identity, preferably at least 60, 70, 80, 90, 95, 97, 98, or 99% identity to the amino acid sequence set forth in SEQ ID NO:405.

[0118] In the present invention, a nucleic acid encoding a Cas protein may be present in a vector, e.g., a plasmid containing a Cas coding sequence, under the control of a promoter such as a CMV promoter or a CAG promoter. When the Cas protein is Cas9, the sequence encoding Cas9 may be derived from, for example, a Streptococcus sp., preferably Streptococcus pyogenes. For example, the Cas9-encoding nucleic acid may comprise a nucleotide sequence encoding SEQ ID NO: 405. Furthermore, the Cas9-encoding nucleic acid may comprise a nucleotide sequence having at least 50% identity, preferably 60, 70, 80, 90, 95, 97, 98, or 99% identity, to the nucleotide sequence encoding SEQ ID NO: 405, but is not limited thereto.

[0119] In one embodiment, the donor DNA comprises a 5' homology arm, a sequence encoding a cleavable linker polypeptide, an exogenous TCR α / β gene or a functional fragment thereof, and a 3' homology arm in its sequence. After the sequence encoding the cleavable linker polypeptide is expressed, the cleavable linker polypeptide is cleaved. In some embodiments, the cleavable linker polypeptide sequence comprises a 2A ribosomal skip element, such as T2A, E2A, P2A, and F2A.

[0120] In one embodiment, the donor DNA is contained in a targeting vector. The basic targeting vector used as a backbone is not particularly limited, as long as it has a prokaryotic replication origin and a selectable marker for propagation of the vector in bacteria.

[0121] In a preferred embodiment, in order to increase expression of the exogenous TCR α / β gene or a fragment thereof, a sequence encoding a cleavable linker polypeptide and a signal peptide sequence are linked to the N-terminus of the exogenous TCR α chain gene and the N-terminus of the exogenous TCR β chain gene, respectively, in the targeting vector.

[0122] According to a particular embodiment, the targeting vector for knocking in the nwTCR gene sequence comprises the following operatively linked constructs: 5'-2A ribosomal skipping element-SP-TCRβ-2A ribosomal skipping element-SP-TRAV-TRAJ-3', where SP refers to the signal peptide coding sequence.

[0123] The targeting vector for knocking in the nwTCR gene sequence, the RNP complex, and cells are mixed, and a step of delivering the nwTCR gene sequence into the cells is carried out. In some embodiments, the delivery step is selected from electroporation, transfection, deformation of the cell membrane by physical means, lipid nanoparticles (LNPs), virus-like particles (VLPs), and sonication. In some embodiments, the delivery step includes electroporation.

[0124] According to some aspects, the modified cells are primary cells.

[0125] According to some aspects, the modified cells are isolated cells, wherein the isolated cells are cells isolated from a subject.

[0126] In some embodiments, the modified cells are ex vivo cultured cells. In some embodiments, the ex vivo cultured cells comprise stimulated cells. In some embodiments, the stimulated cells comprise cytokine-stimulated T cells. Optionally, the cytokine-stimulated T cells comprise CD3-stimulated T cells, CD28-stimulated T cells, or CD3 and CD28-co-stimulated T cells. In some embodiments, the cytokine-stimulated T cells are cultured in the presence of IL7, IL15, or a combination thereof. In some embodiments, the cytokine-stimulated T cells are cultured in the presence of IL2.

[0127] In some embodiments, the modified cells are stem cells, such as hematopoietic stem cells (HSCs). Because stem cells do not express CD3 molecules, introducing the nwTCR gene into HSCs does not result in cell surface expression of the TCR. However, when the stem cells differentiate into lymphoid progenitor cells that migrate to the thymus, CD3 expression begins and the introduced nwTCR is expressed on the surface of thymocytes. The advantages of this approach include the following: Because expression of the introduced nwTCR chains inhibits expression of endogenous TCR chains and inhibits rearrangement of endogenous TCR genes to form functional TCRα and β genes, once generated, mature T cells express only the introduced nwTCR and little or no expression of endogenous TCR chains. Another advantage of this approach is that TCR-modified stem cells provide a continuous source of mature T cells with the desired antigen specificity. Upon differentiation, nwTCR-modified stem cells generate T cells expressing the TCR of the present invention.

[0128] V. Methods for detecting, preventing, or treating cancer associated with KRAS_G12V mutant antigen The present invention relates to a method for preventing or treating cancer associated with a KRAS_G12V mutant antigen, comprising administering to a subject in need thereof a modified cell, TCR nucleic acid, vector, or pharmaceutical composition of the present invention. In some embodiments, the method comprises administering a polynucleotide encoding a TCR. In some embodiments, the method comprises administering a vector comprising a polynucleotide encoding a TCR. In some embodiments, the method comprises administering an effective amount of a modified cell of the present invention.

[0129] According to some aspects, the modified cells, TCR nucleic acids, vectors, or pharmaceutical compositions of the present invention are used to prevent or treat cancers associated with the KRAS_G12V mutant antigen. Without being limited by theory, it is believed that the TCRs of the present invention can specifically bind to the KRAS_G12V mutant antigen, thereby mediating an immune response against target cells expressing the KRAS_G12V mutant antigen.

[0130] Treatment or prevention includes treatment or prevention of one or more symptoms of the cancer being treated or prevented, such as promoting tumor regression, delaying the onset of cancer or its symptoms, and preventing or delaying the recurrence of cancer or its symptoms.

[0131] The present invention also provides a method for detecting the presence of cancer in a mammal. Such a method comprises: (i) contacting a sample comprising one or more cells from a mammal with any of the TCRs of the present invention, a population of cells expressing a TCR of the present invention, or a pharmaceutical composition comprising a population of cells expressing a TCR of the present invention, as described in this disclosure, to form a complex; and (ii) detecting the complex, wherein the detected complex indicates the presence of cancer in the mammal. The contacting may be performed in vitro or in vivo in a living mammal. In one embodiment, the contacting is performed in vitro. The complex can be detected by various methods known in the art. In one embodiment, the TCRs of the present invention or cell populations expressing the TCRs of the present invention can be labeled with a detectable marker, such as a radioisotope, a fluorescent substance (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE), etc.), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase, etc.), and an elemental particle (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE), etc.), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase, etc.), and an elemental particle (e.g., a gold particle), etc.

[0132] The present invention further provides a method for inducing anti-tumor immunity, which comprises administering an effective amount of modified cells according to the present invention to a subject, wherein the tumor is a KRAS_G12V mutant antigen-associated tumor.

[0133] The present invention provides methods for inducing an immune response in a subject, the method comprising administering an effective amount of modified cells of the present invention. In some embodiments, the immune response is a T cell-mediated immune response. In some embodiments, the T cell-mediated immune response is directed against one or more target cells. In some embodiments, the modified immune cells comprise a TCR of the present invention. In some embodiments, the target cells are KRAS_G12V mutant antigen-associated cancer cells.

[0134] According to some aspects, donor T cells for T cell therapy are obtained from the patient (e.g., in the case of autologous T cell therapy). According to other embodiments, donor stem cells to be differentiated into T cells for T cell therapy are obtained from a subject other than the patient.

[0135] The T cells can be administered in a therapeutically effective amount. For example, a therapeutically effective amount of T cells can be at least about 10 per kg of body weight. 4 cells, at least about 10 5 cells, at least about 10 6 cells, at least about 10 7 cells, at least about 10 8 cells, at least about 10 9 cells, or at least about 10 10 It is a cell.

[0136] The cancer referred to in the various methods of the present invention may be any cancer. Examples of cancer include, but are not limited to, acute lymphoblastic cancer, acute myeloid leukemia, chronic lymphoblastic leukemia, chronic myeloid cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, brain tumor, glioma, nasopharyngeal cancer, eye cancer, oral cancer, cervical cancer, esophageal cancer, liver cancer, intrahepatic bile duct cancer, gallbladder cancer, lung cancer, bone cancer, breast cancer, gastrointestinal tumor, colon cancer, small intestine cancer, large intestine cancer, rectal cancer, stomach cancer, skin cancer, melanoma, multiple myeloma, cervical cancer, endometrial cancer, uterine cancer, ovarian cancer, ureter cancer, bladder cancer, penile cancer, testicular cancer, pancreatic cancer, prostate cancer, kidney cancer, soft tissue cancer, thyroid cancer, etc. Preferably, the cancer is lung cancer, pancreatic cancer, large intestine cancer, endometrial cancer, ovarian cancer, or prostate cancer.

[0137] VI. Methods for Improving Cell Therapy and Engineered Cells The present invention further provides methods for improving cell therapy and the modified cells thereof.

[0138] Naturally occurring CD8+ cells express the CD8 molecule, a type I transmembrane glycoprotein expressed on the cell surface as a homodimer of two CD8a chains (sometimes abbreviated as "CD8aa" in this disclosure) and / or a heterodimer of one CD8a chain and one CD8b chain (sometimes abbreviated as "CD8ab" in this disclosure).

[0139] According to some embodiments, the T cells of the present invention co-express an exogenous TCR and a CD8aa molecule. Using a non-viral gene editing method based on CRISPR / Cas9 technology, CD8+ T cells / CD4+ T cells are gene-edited with a nucleic acid encoding the nwTCR of the present invention and a nucleic acid encoding the CD8a chain. Thus, co-expression of the exogenous nwTCR and CD8aa molecule in CD8+ T cells enhances the binding of TCR-T cells to pMHC molecules. Co-expression of the exogenous nwTCR and CD8aa molecule in CD8+ T cells increases the number of CD8aa molecules on the CD8+ T cells that are available to the exogenous nwTCR, which is expected to improve the TCR-specific cytotoxicity (including its sustained killing ability) and in vivo antitumor ability of the CD8+ T cells. When exogenous nwTCR and CD8aa molecules are coexpressed on CD4+ T cells, along with the expression of endogenous CD4 molecules, the CD4+ T cells exhibit a hybrid phenotype, which is expected to retain the intrinsic helper function of CD4+ T cells while recognizing antigens with the same affinity as natural CD8+ T cells and exerting cytotoxic activity to kill target cells.

[0140] Furthermore, according to some embodiments, the present invention provides for the co-expression of exogenous TCR and CD8ab molecules in T cells. Using a non-viral gene editing method based on CRISPR / Cas9 technology, CD8+ T cells / CD4+ T cells are gene-edited with a nucleic acid encoding the nwTCR of the present invention, a nucleic acid encoding the CD8a chain, and a nucleic acid encoding the CD8b chain. This allows co-expression of exogenous nwTCR and CD8ab molecules in CD8+ T cells / CD4+ T cells to enhance the binding of TCR-T cells to pMHC molecules. When exogenous nwTCR and CD8ab molecules are co-expressed in CD8+ T cells, the number of CD8ab molecules available to the exogenous nwTCR increases within the CD8+ T cells, which is expected to improve the TCR-specific cytotoxicity (including its sustained killing ability) and in vivo anti-tumor function of CD8+ T cells. When exogenous nwTCR and CD8ab molecules are coexpressed on CD4+ T cells, along with the expression of endogenous CD4 molecules, the CD4+ T cells exhibit a hybrid phenotype, which is expected to recognize antigens with similar affinity to natural CD8+ T cells and exert cytotoxic activity to kill target cells, while retaining the natural helper function of CD4+ T cells.

[0141] Thus, coexpression of CD8aa and / or CD8ab molecules with TCR genes in CD8+ and CD4+ T cells has beneficial effects on the function of CD8+ and CD4+ T cells. CD4+ T cells can be reprogrammed into multifunctional hybrid T cells that express both cytotoxic effector and natural helper functions using MHC class I TCR and CD8 molecules.

[0142] In order to facilitate understanding of the present invention, the following examples are provided, however, these examples are not intended to, and should not be construed in any way to, limit the scope of protection of the present invention. [Example]

[0143] The invention generally described in this disclosure can be more readily understood by reference to the following examples, which are provided for illustrative purposes and are not intended to limit the scope of the invention. They are not intended to represent that the following experiments are all or the only experiments performed.

[0144] Example 1. Generation and cloning of T cells and TCRs that recognize KRAS_G12V mutant antigens The sequences near the 12th position of KRAS, in which the 12th amino acid residue of KRAS was mutated from G to V, were chemically synthesized. That is, a short peptide represented by VVVGAVGVGK (SEQ ID NO: 381) covering the 7th to 16th amino acid residues of KRAS (sometimes abbreviated as "KRAS_G12V_7-16 peptide" in the present disclosure), and a short peptide represented by VVGAVGVGK (SEQ ID NO: 382) covering the 8th to 16th amino acid residues of KRAS (sometimes abbreviated as "KRAS_G12V_8-16 peptide" in the present disclosure).

[0145] Dendritic cells (DCs) derived from cancer patients expressing KRAS_G12V and the HLA-A*11:01 genotype were pulsed in vitro with either KRAS_G12V_7-16 or KRAS_G12V_8-16 peptides suspended in DMSO and cocultured with CD8+ T cells fractionated from the patient's peripheral blood for 10 days. As a negative control, DCs were pulsed in vitro with DMSO and then cocultured with CD8+ T cells fractionated from the patient's peripheral blood for 10 days.

[0146] Next, reactive T cells that released IFN-γ and expressed CD137 were selected by detecting the release of the cytokine IFN-γ in the culture supernatant and the expression of CD137 on CD8+ T cells. Flow cytometric staining was used to assess the binding of IFN-γ-releasing CD137-expressing reactive T cells to peptide-MHC (HLA-A*11:01) tetramers (VVVGAVGVGK-HLA-A*11:01 and VVGAVGVGK-HLA-A*11:01). Tetramers containing an irrelevant peptide were used as negative controls. Flow cytometric staining of T cells with two tetramers (VVVGAVGVGK-HLA-A*11:01 tetramer or VVGAVGVGK-HLA-A*11:01 tetramer) further confirmed the specificity of the T cells.

[0147] Sixteen specific T cell clones with desirable high affinity were selected. The antigen-specific T cell receptors (TCRs) on these 16 T cell clones that specifically bind to the KRAS_G12V_7-16 epitope peptide or the KRAS_G12V_8-16 epitope peptide were designated nwTCR-0125, nwTCR-0126, nwTCR-0127, nwTCR-1708, nwTCR-1862, nwTCR-2162, nwTCR-2241, nwTCR-2308, nwTCR-2310, nwTCR-2390, nwTCR-2392, nwTCR-2424, nwTCR-2561, nwTCR-2563, nwTCR-2595, and nwTCR-2629, respectively. These were sequenced by high-throughput paired TCR sequencing. The amino acid sequences of the paired TCR α and β chains of these 16 T cell clones were determined on a single-cell basis.

[0148] Because multiple nucleotides can be translated into the same amino acid and codon usage varies among organisms, we optimized the nucleotide sequences encoding the amino acid sequences of the TCR α and β chains to increase TCR expression in eukaryotic cells. After codon optimization, we obtained the nucleotide sequences of 16 TCRs that specifically recognize the KRAS_G12V_7-16 epitope peptide or the KRAS_G12V_8-16 epitope peptide.

[0149] Tables 1A and 1B show the amino acid and nucleotide sequence information, respectively, obtained by sequencing the α and β chains of 16 TCRs expressed by clonal T cell lines.

[0150] [Table 1A] [Table 1B]

[0151] Example 2: Preparation of KRAS_G12V mutant antigen-specific TCR-T cells from T cells In this example, the TCR gene was knocked out from primary T cells using CRISPR / Cas9 technology, and the KRAS_G12V mutant antigen-specific TCR gene was knocked in using homologous recombination technology. KRAS_G12V mutant antigen-specific TCR-T cells were then prepared and their characteristics were evaluated.

[0152] 2.1 T cell teacher and activation A mixture of CD4 T cells and CD8 T cells (sometimes abbreviated as "CD4 / CD8 T cells" in this disclosure) was enriched and selected from peripheral blood mononuclear cells (PBMCs, purchased from Shanghai Saily Biotechnology Co., donor: S2001095). The enriched and selected CD4 / CD8 T cells were aliquoted and frozen (5 × 10) for future use. 6 cells / cryotube).

[0153] Frozen tubes were thawed as needed, and the selected T cells were activated by adding a 1:100 dilution of Miltenyi T cell TransACT (Miltenyi catalog number 130-111-160) T cell activator to T cell culture medium (e.g., RPMI 1640, FBS, L-glutamine, non-essential amino acids, sodium pyruvate, HEPES buffer, 2-mercaptoethanol, and IL2 as needed). After approximately 48 hours (2 days) of culture, the cells were used for transfection by electroporation.

[0154] 2.2 Targeting Strategy and Preparation of Targeting Vectors The gRNAs used were gRNA002 and gRNA004 (see Table 2). The hit site for gRNA002 was exon 1 of the endogenous TRAC gene (Figure 1A). The hit sites for gRNA004 were exon 1 of the endogenous TRBC1 gene and exon 1 of the endogenous TRBC2 gene (Figure 1B). Cas9 enzyme was purchased from GenScriptBiotech Corporation (catalog number: Z03469).

[0155] [Table 2]

[0156] The targeting vector (sometimes referred to as HDR vector) used a pUC57-HA vector backbone, which was optimized based on the pUC57-Simple vector. Only the Ori and Amp sequences of the pUC57-Simple vector were retained, while the Amp sequence was replaced with a Kana sequence. The left and right homology arm (HA) sequences (approximately 800 bp) of the TRAC locus were inserted. The knock-in (KI) gene sequence was constructed between the left and right HA. The structure of the nwTCR KI sequence construct included: 2A or 2A mutant-SP-TCRβ-2A or 2A mutant-SP-TRAV-TRAJ (where 2A represents the ribosomal skip element); SP, the signal peptide; and four synonymous mutated bases were introduced into the TRBC gene of the targeting vector. These mutations mutated the nucleotide encoding TRBCS77 from AGC to TCC and the nucleotide encoding S78 from AGC to TCC, respectively. For nwTCR-0126, the sequence of the KI sequence construct is set forth in SEQ ID NO: 386, where HA represents the homology arm (5'HA is set forth in SEQ ID NO: 387, and 3'HA is set forth in SEQ ID NO: 391); 2A or 2A mutant is set forth in SEQ ID NO: 388 or SEQ ID NO: 390, respectively; SP represents the signal peptide sequence set forth in SEQ ID NO: 389; TCRβ represents the nucleotide sequence of nwTCR-0126 complete TRB (TCR β chain) (SEQ ID NO: 352), in which the TRBC S77-encoding nucleotides are mutated from AGC to TCC and the S78-encoding nucleotides are mutated from AGC to TCC, thereby introducing four synonymous mutated bases into the TRBC gene; TRAV represents the nucleotide sequence of nwTCR-0126 TRAV gene (SEQ ID NO: 54); and TRAJ represents the nucleotide sequence of nwTCR-0126 TRAJ gene (SEQ ID NO: 56). Similarly, KI sequence constructs of nwTCR-0125, nwTCR-0127, nwTCR-1708, nwTCR-1862, nwTCR-2162, nwTCR-2241, nwTCR-2308, nwTCR-2310, nwTCR-2390, nwTCR-2392, nwTCR-2424, nwTCR-2561, nwTCR-2563, nwTCR-2595, and nwTCR-2629 were prepared.

[0157] 2.3 Transfection by electroporation (day 2) RNPs were prepared by thoroughly mixing the sgRNA and Cas9 enzyme from Example 2.2 and incubating at room temperature for 10 minutes.

[0158] The targeting vector containing the KITCR sequence prepared in Example 2.2 was thoroughly mixed with the incubated RNP and the indicated concentrations of T cells prepared in Example 2.1 (approximately 1.25E6 T cells / electroporation tube) to knock out (KO) the endogenous TCR and knock in (KI) the exogenous TCR.

[0159] The above mixture was loaded into an electroporation device (Celetrix, catalog number CTX-1500A LE) for cell electroporation, and electroporation was performed at 480-560 V for 20 ms.

[0160] After electroporation, the electroporated transfected T cells were allowed to rest for 15 minutes, then removed from the electroporation tube and resuspended in pre-warmed ImmunoCult™ medium. (登録商標) The cells were cultured for 5 days and then characterized by flow cytometry on day 7.

[0161] 2.4 Flow cytometry analysis of nwTCR expression (day 7) The cell suspension obtained in Example 2.3 was thoroughly mixed and the cell number was determined. An appropriate amount of cells was harvested and stained with two types of labeled peptide-MHC (HLA-A*11:01) tetramers, namely, VVVGAVGVGK-HLA-A*11:01 tetramer and VVGAVGVGK-HLA-A*11:01 tetramer (abbreviated as pMHC).

[0162] Each staining reagent, containing one of two labeled peptide-MHC (HLA-A*11:01) tetramers, was prepared in advance. Also prepared were LIVE / DEAD® FixableNear-IR (purchased from Invitrogen, catalog no. L10119), CD4-FITC (purchased from BioLegend, catalog no. 357406), CD8-PerCP-cy5.5 (purchased from BioLegend, catalog no. 35L10119; CD4-FITC (BioLegend, catalog no. 357406); CD8-PerCP-cy5.5 (BioLegend, catalog no. 344710); and human TCRα / β-BV510 antibody (BioLegend, catalog no. 306734).

[0163] The collected cells were stained with two labeled peptide-MHC (HLA-A*11:01) tetramer stains, washed, and then characterized by flow cytometry.

[0164] Figures 3A-3P show examples of flow cytometry results of CD4+ and CD8+ T cells electroporated with different nwTCRs and stained with pMHC tetramers, where for nwTCR-0125, nwTCR-0126, nwTCR-0127, nwTCR-1708, nwTCR-1862, nwTCR-2162, nwTCR-2241, nwTCR-2308, and nwTCR-2563, the exemplary staining results are obtained by staining with the labeled VVVGAVGVGK-HLA-A*11:01 tetramer. For nwTCR-2310, nwTCR-2390, nwTCR-2392, nwTCR-2424, nwTCR-2561, nwTCR-2595, and nwTCR-2629, the exemplary staining results are from staining with the labeled VVGAVGVGK-HLA-A*11:01 tetramer. As shown in the figure, cells at day 7 were sorted into three populations. 1) wild-type T cells that have not been gene-edited (Q3), 2) KO cells in which endogenous TCR was completely knocked out (Q4); 3) A cell population (Q2) in which KO and KI have been completed and which expresses nwTCR.

[0165] The results of TCR gene editing efficiency detected by flow cytometry are shown schematically in Figure 2.

[0166] As shown in Figures 3A-3P, CD8+ T cells into which each nwTCR had been knocked in were able to bind to peptide-MHC complex (pMHC) tetramers. On the other hand, significant differences were observed in binding to peptide-MHC complex (pMHC) tetramers between CD4+ T cells into which each nwTCR had been knocked in. This is because, as shown in Example 1, each nwTCR in the present invention was obtained by screening CD8+ T cells. Specifically, when each nwTCR was introduced into CD8+ T cells, CD8+ T cells expressing each nwTCR were able to specifically bind to peptide-MHC complex (pMHC) tetramers. On the other hand, when each nwTCR was introduced into CD4+ T cells, the following situation was observed for CD4+ T cells expressing each nwTCR. Generally, if the affinity of the nwTCR for MHC was sufficiently strong, the TCR could bind to MHC molecules without the assistance of CD8 molecules. For example, nwTCR-0127 was predicted to have stronger binding ability to MHC molecules than nwTCR-1708. Specifically, differences were observed in the staining of CD4+ T cells with pMHC tetramers. Therefore, after editing and expressing each nwTCR in CD4+ T cells, the high-affinity nwTCR continued to bind to MHC class I antigens, while the low-affinity nwTCR had weaker binding ability to MHC class I antigens.

[0167] Example 3. In vitro functional study of KRAS_G12V mutant antigen-specific TCR-T cells The KRAS_G12V mutant antigen-specific TCR-T cells of Example 2 were selectively activated with TransACT activator (Miltenyi, catalog number 130-111-160) on day 7. The TCR-T cell culture was continued until day 14. On day 14, in vitro functional tests were performed on each TCR-T cell.

[0168] 3.1 Detection of affinity of each TCR-T cell to peptides The TCR-T cell affinity assay was performed as follows. Antigen-presenting cells (T2 cells or K562 cells overexpressing HLA-A*11:01) were collected and counted. An appropriate amount of medium (e.g., RPMI-1640 medium (Gibco, catalog number 22400089), FBS (Gibco, catalog number 10099141C)) was added to adjust the cell density to 1E6 cells / mL, and the cells were suspended. 1 mL of the cell suspension was added to each well of a 24-well plate. The cells were incubated in an incubator (37°C, 5% CO 2 ) for 2 hours. After incubation, the antigen-presenting cells were collected and washed, and 100 μl of the antigen-presenting cells at 1E6 / mL were added to the corresponding wells of a 96-well plate. The nwTCR-T cells to be detected were collected, and T cell culture medium (STEMCELL, Cat. No. 10981) was added to a cell density of 1E6 / mL, and 100 μl of the cell suspension was added to the corresponding wells of a 96-well plate. Various nwTCR-T cells were co-cultured with the antigen-presenting cells (37°C, 5% CO 2 ) and cultured for 16 hours. The cell supernatant was collected, and the IFN-γ concentration was measured using an ELISA kit (Biolegend, catalog number 430104). The binding affinity of T cells expressing each nwTCR presented by HLA-A*11:01 to the short peptide shown in SEQ ID NO: 381 or 382 was evaluated by measuring the amount of IFN-γ released.

[0169] Figures 4A to 4H show the results of binding affinity assays of T cells expressing each nwTCR for the short peptide shown in VVVGAVGVGK (sequence number 381) or VVGAVGVGK (sequence number 382), and the EC50 values ​​presented by HLA-A*11:01; Figures 4A, 4C, 4D, and 4G show the binding affinity for the short peptide shown in VVVGAVGVGK (sequence number 381); and Figures 4B, 4E, 4F, and 4H show the binding affinity for the short peptide shown in VVGAVGVGK (sequence number 382).

[0170] The results in Figures 4A to 4H suggest that after co-incubation of T2 cells presenting peptide-MHC complexes with T cells expressing each nwTCR, T cells expressing each nwTCR specifically bind to the peptide-MHC complexes and release IFN-γ. Furthermore, considering that both 9-mer and 10-mer short peptides are likely to be presented by MHC when presenting polypeptides, the results in Figures 4A to 4F of experiments using 9-mer and 10-mer short peptides indicate that T cells expressing each nwTCR specifically bind to pMHC complexes presenting 9-mer or 10-mer short peptides, respectively.

[0171] 3.2 Target cell killing by each TCR-T cell Killing assays of each TCR-T cell against target cells were performed as follows: Target cells (i.e., SW620 cell line (overexpressing HLA-A*11:01 and KRASG12V+) (purchased from Nanjing Cobioer Biosciences Co., Ltd.) were harvested, counted, and resuspended to a cell density of 1E6 cells / mL using target cell medium (RPMI-1640 medium, Gibco, catalog number 22400089, and FBS, Gibco, catalog number 10099141C). 22400089) and FBS (Gibco, catalog number 10099141C) were used to resuspend the cells to a cell density of 1E6 cells / mL. 100 μL of well-mixed target cell suspension was added to the corresponding wells to prepare an E-Plate (Agilent, Catalog No. 300600890), which was then placed in an RTCA real-time cell analyzer (Agilent, Model: xCELLigence RTCA DP) for overnight detection.

[0172] The target TCR-T cells were harvested, counted, and resuspended in an appropriate volume of T cell culture medium (STEMCELL, Catalog No. 10981). The E-Plate containing the target cells was removed and the T cell suspension was added. The E-Plate was then placed back into the RTCA analyzer for detection. The cell index was obtained over 72 hours. Each independent experiment was performed in triplicate. The RTCA software automatically calculated the slope of the intervals to evaluate the rate of change in the cell index. To demonstrate the effect of treatment, the cell index was normalized to an equal value at each normalized time point.

[0173] The in vitro killing results of each TCR-T cell against SW620 cells (overexpressing HLA-A*11:01 and KRASG12V+) are shown in Figures 5A to 5E. These results demonstrate that each TCR-T cell exhibited significant killing effects against the target cell, the SW620 cell line (overexpressing HLA-A*11:01 and KRASG12V), in vitro.

[0174] Target nwTCR-T cells were harvested and an appropriate volume of T cell culture medium (STEMCELL, catalog number 10981) was added to achieve a cell density of 1E6 / mL. 100 μL of the target nwTCR-T cell suspension was added to a well of a 96-well plate and mixed with SW620 cells (overexpressing HLA-A*11:01 and KRASG12V+) in the well of the 96-well plate. After adding 1 μL of ethidium bromide (1 mg / mL) solution and mixing thoroughly, the cell culture plate was placed in a real-time fluorescence imaging system (BioTek Lionheart) to characterize cell death. Target cells specifically recognized by T cells were stained with ethidium bromide and emitted a red fluorescent signal after entering apoptotic state.

[0175] As a result, each nwTCR-expressing T cell enabled SW620 cells (overexpressing HLA-A*11:01 and KRASG12V+) to be stained with ethidium bromide. Figure 6 shows the results of nwTCR-2424-expressing T cells at the start of co-culture with SW620 cells (0 h) and after 18 hours of co-culture (18 h). The red fluorescent signal after 18 hours of co-incubation indicates specific killing of SW620 cells by nwTCR-2424-expressing T cells.

[0176] Real-time data analysis of cell killing showed that each nwTCR-expressing T cell specifically killed SW620 cells (overexpressing HLA-A*11:01 and KRASG12V+). Figure 7 shows an example of specific killing of SW620 cells by nwTCR-2424-expressing T cells.

[0177] Example 4. Co-expression of exogenous TCR and CD8 molecules This example describes an example of redirecting CD4+ T cells by co-expressing exogenous TCR and CD8 molecules on the CD4+ T cell membrane surface using CRISPR / Cas9 and homologous recombination techniques. Furthermore, gene editing was used to successfully enhance the binding of TCR-T cells to pMHC molecules.

[0178] 4.1 T cell selection and activation T cells were either commercially available (e.g., frozen human peripheral blood CD4+CD45RA+ T cells, Stem Cell Technology, catalog no. 70029) or prepared from leukapheresis samples (day 0).

[0179] For the preparation of T cells from leukapheresis samples, CD4 / CD8 T cells were enriched and selected from leukapheresis samples. The enriched and selected CD4 / CD8 T cells were aliquoted and cryopreserved (5 × 10) for future use. 6 cells / cryotubes).

[0180] 4.2 Targeting Strategy and Targeting Vector Preparation The TCR targeting strategy and targeting vector were the same as those described in Example 2.2 above. When the TCR sequence was derived from nwTCR-1708, the KI amino acid sequence of nwTCR-1708 was as follows: 2A or 2A mutant-SP-TCRβ-2A or 2A mutant-SP-TRAV-TRAJ (SEQ ID NO: 392), and the KI nucleotide sequence of nwTCR-1708 was: 2A or 2A mutant-SP-TCRβ-2A or 2A mutant-SP-TRAV-TRAJ (SEQ ID NO: 393). As shown in Figure 3D, CD8+ T cells expressing nwTCR-1708 were able to bind to pMHC molecules bearing the amino acid sequence 7 to 16 of KRAS G12V (KRAS_G12V_7-16 peptide), but CD4+ T cells expressing nwTCR-1708 were unable to bind to pMHC molecules presenting the KRAS_G12V_7-16 peptide. In this example, by selecting CD4+ T cells that express a TCR but do not bind to pMHC tetramers after gene editing, it was shown that even with such nwTCRs, introducing a CD8 molecule into CD4 T cells after gene editing of the nwTCR can enhance CD4 T cell binding to pMHC molecules. Thus, the specific nwTCR used in this example can be replaced with any of the other nwTCRs of the present invention, and enhanced CD4 T cell binding to pMHC molecules can be achieved after gene editing of the other nwTCR.

[0181] To enable CD4+ T cells to bind to pMHC molecules containing the amino acid sequence from the 7th to the 16th amino acids of KRASG12V (KRAS_G12V_7-16 peptide), the following construct was further constructed and inserted into the pUC57-HA targeting vector (sometimes referred to as the HDR vector).

[0182] The KI amino acid sequence of nwTCR-1708-CD8a was 2A or 2A mutant-CD8a-2A or 2A mutant-SP-TCRβ-2A or 2A mutant-SP-TRAV-TRAJ (SEQ ID NO: 394).

[0183] The KI nucleotide sequence of nwTCR-1708-CD8a was 2A or 2A mutant-CD8a-2A or 2A mutant-SP-TCRβ-2A or 2A mutant-SP-TRAV-TRAJ (SEQ ID NO: 395).

[0184] A diagram of the targeting strategy of nwTCR-CD8a is shown in Figure 8A.

[0185] The KI amino acid sequence of nwTCR-1708-CD8ab was 2A or 2A mutant-CD8a-2A or 2A mutant-CD8b-2A or 2A mutant-SP-TCRβ-2A or 2A mutant-SP-TRAV-TRAJ (SEQ ID NO: 398).

[0186] The KI nucleotide sequence of nwTCR-1708-CD8ab was: 2A or 2A mutant-CD8a-2A or 2A mutant-CD8b-2A or 2A mutant-SP-TCRβ-2A or 2A mutant-SP-TRAV-TRAJ (SEQ ID NO: 399).

[0187] A diagram of the targeting strategy of nwTCR-CD8ab is shown in Figure 8B.

[0188] 4.3 Transfection by electroporation (day 2) The sgRNA designed and synthesized in Example 2.2 was thoroughly mixed with the Cas9 enzyme and incubated at room temperature for 10 minutes to prepare RNPs.

[0189] The three targeting vectors for knocking in nwTCR-1708, nwTCR-1708-CD8a, or nwTCR-1708-CD8ab prepared in Example 4 were thoroughly mixed with the incubated RNP and T cells (1.25E6 / electroporation tube) prepared in Example 4.1 to knock out (KO) the endogenous TCR and knock in (KI) the exogenous TCR, exogenous TCR and CD8a, or exogenous TCR and CD8ab.

[0190] The above mixture was loaded into an electroporation transfection device (Celetrix, catalog number CTX-1500A LE), and cell electroporation transfection was performed at 480-560 V for 20 ms.

[0191] After electroporation, the electroporated transfected cells were allowed to stand for 15 minutes, then removed from the electroporation tube and transferred to pre-warmed ImmunoCult™ medium. (登録商標) The cells were cultured for 5 days and then transferred to T cell expansion medium (-XF T cell expansion medium, Stemcell Inc., catalog no. 10981). The cells were cultured for 5 days and then characterized by flow cytometry on day 7.

[0192] 4.4 Flow cytometry analysis of nwTCR expression (day 7) The cell suspension obtained in Example 4.3 was mixed thoroughly, the cell number was determined, and an appropriate amount of cells was collected for staining with labeled peptide-MHC (HLA-A*11:01) tetramer (VVVGAVGVGK-HLA-A11:01).

[0193] Specific antigens contained in labeled peptide-MHC (HLA-A*11:01) tetramers and LIVE / DEAD purchased from Invitrogen (登録商標) Staining solution containing FixableNear-IR (Cat. No. L10119), L10119; CD4-FITC (BioLegend, Cat. No. 357406); CD8-PerCP-cy5.5 (BioLegend, Cat. No. 344710); and human TCRα / β-BV510 antibody (BioLegend, Cat. No. 306734) were prepared in advance.

[0194] The collected cells were stained with labeled peptide-MHC (HLA-A*11:01) tetramer stain, washed, and characterized by flow cytometry. The flow cytogram showed three distinct populations on day 7. 1) Wild-type T cells that have not undergone gene editing (Q3) 2) KO cells in which endogenous TCR has been completely knocked out (Q4) 3) Cell population expressing nwTCR after KO and KI (Q2)

[0195] CD8+ T cells edited with nwTCR-1708 were able to bind to specific antigen-MHC tetramers, whereas nwTCR-1708-edited CD4+ T cells were unable to bind to specific antigen-MHC tetramers due to the lack of CD8 molecule help (Figure 9A).

[0196] When CD8a or CD8ab molecules were co-transfected with nwTCR-1708 into primary T cells (primary T cells include CD4+ and CD8+ T cells), an increased proportion of CD8+CD4+ T cells was characterized by flow cytometry. These cells were CD4+ T cells that expressed exogenous CD8 molecules and were able to specifically bind to labeled peptide-MHC (HLA-A11:01) tetramers (VVVGAVGVGK-HLA-A11:01) through the action of exogenous CD8 molecules (Figures 9B and 9C).

[0197] At the same time, the ability to redirect CD4+ T cells with this approach improved the overall gene editing efficiency of T cells (Table 3), where gene editing efficiency (GE%) was calculated as follows: Percentage of cells expressing only CD8+ in the live T cell population × percent of cells expressing only CD8+ in tetramer staining + percent of cells expressing only CD4+ in the live T cell population × percent of cells expressing only CD4+ in tetramer staining + percent of cells expressing both CD8+ and CD4+ in the live T cell population × percent of cells expressing both CD8+ and CD4+ in tetramer staining.

[0198] [Table 3]

[0199] While exemplary embodiments of the present invention have been described above, those skilled in the art will recognize that these disclosures are merely exemplary and that various other substitutions, adaptations, and modifications are possible within the scope of the present invention, and therefore, the present invention is not limited to the specific embodiments illustrated in this disclosure. [Sequence List Free Text]

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Claims

1. An isolated or purified T cell receptor (suitably abbreviated as "TCR"), wherein the TCR specifically binds to a KRAS_G12V mutant antigen and comprises an α chain and a β chain, each of the α chain and the β chain having three complementarity-determining regions (suitably abbreviated as "CDR"); The amino acid sequences of the three CDRs contained in the α chain and the three CDRs contained in the β chain are: (i) α chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 4, 5, and 6; and β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 180, 181, and 182; (ii) α chain CDR1, CDR2, CDR3 amino acid sequences shown in SEQ ID NOs: 7, 8, and 9; and β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 183, 184, and 185; (iii) α chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, respectively; and β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 177, 178, and 179, respectively; (iv) α chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 10, 11, and 12, respectively; and β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 186, 187, and 188; (v) α chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 13, 14, and 15, respectively; and β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 189, 190, and 191, respectively; (vi) α chain CDR1, CDR2, CDR3 amino acid sequences shown in SEQ ID NOs: 16, 17, and 18, respectively; and β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 192, 193, and 194, respectively; (vii) α chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 19, 20, and 21, respectively; and β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 195, 196, and 197, respectively; (viii) α chain CDR1, CDR2, CDR3 amino acid sequences shown in SEQ ID NOs: 22, 23, and 24, respectively; and β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 198, 199, and 200, respectively; (ix) α chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 25, 26, and 27, respectively; and β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 201, 202, and 203, respectively; (x) α chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 28, 29, and 30, respectively; and β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 204, 205, and 206, respectively; (xi) α chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 31, 32, and 33, respectively; and β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 207, 208, and 209, respectively; (xii) α chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 34, 35, and 36, respectively; and β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 210, 211, and 212, respectively; (xiii) α chain CDR1, CDR2, CDR3 amino acid sequences shown in SEQ ID NOs: 37, 38, and 39, respectively; and β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 213, 214, and 215, respectively; (xiv) α chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 40, 41, and 42, respectively; and β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 216, 217, and 218, respectively; (xv) α chain CDR1, CDR2, CDR3 amino acid sequences shown in SEQ ID NOs: 43, 44, and 45, respectively; and β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 219, 220, and 221, respectively; or (xvi) α chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 46, 47, and 48, respectively; and β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 222, 223, and 224, respectively; That is, TCR.

2. The TCR of claim 1, wherein the TCR further comprises a constant region, optionally a mouse constant region. Claim 3: The TCR comprises as an alpha chain sequence the sequence set forth in SEQ ID NO: 145 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and as a beta chain sequence the sequence set forth in SEQ ID NO: 349 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; the TCR comprises the sequence set forth in SEQ ID NO: 147, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, as an alpha chain sequence, and the sequence set forth in SEQ ID NO: 351, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, as a beta chain sequence; the TCR comprises the sequence set forth in SEQ ID NO: 149, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, as an alpha chain sequence, and the sequence set forth in SEQ ID NO: 353, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, as a beta chain sequence; the TCR comprises the sequence set forth in SEQ ID NO: 151, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, as an alpha chain sequence, and the sequence set forth in SEQ ID NO: 355, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, as a beta chain sequence; the TCR comprises the sequence set forth in SEQ ID NO: 153, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, as an alpha chain sequence, and the sequence set forth in SEQ ID NO: 357, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, as a beta chain sequence; the TCR comprises the sequence set forth in SEQ ID NO: 155, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, as an alpha chain sequence, and the sequence set forth in SEQ ID NO: 359, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, as a beta chain sequence; the TCR comprises the sequence set forth in SEQ ID NO: 157, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, as an alpha chain sequence, and the sequence set forth in SEQ ID NO: 361, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, as a beta chain sequence; the TCR comprises the sequence set forth in SEQ ID NO: 159, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, as an alpha chain sequence, and the sequence set forth in SEQ ID NO: 363, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, as a beta chain sequence; the TCR comprises the sequence set forth in SEQ ID NO: 161, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, as an alpha chain sequence, and the sequence set forth in SEQ ID NO: 365, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, as a beta chain sequence; the TCR comprises the sequence set forth in SEQ ID NO: 163, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, as an alpha chain sequence, and the sequence set forth in SEQ ID NO: 367, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, as a beta chain sequence; the TCR comprises the sequence set forth in SEQ ID NO: 165, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, as an alpha chain sequence, and the sequence set forth in SEQ ID NO: 369, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, as a beta chain sequence; the TCR comprises the sequence set forth in SEQ ID NO: 167, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, as an alpha chain sequence, and the sequence set forth in SEQ ID NO: 371, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, as a beta chain sequence; the TCR comprises the sequence set forth in SEQ ID NO: 169, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, as an alpha chain sequence, and the sequence set forth in SEQ ID NO: 373, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, as a beta chain sequence; the TCR comprises the sequence set forth in SEQ ID NO: 171, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, as an alpha chain sequence, and the sequence set forth in SEQ ID NO: 375, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, as a beta chain sequence; the TCR comprises an alpha chain sequence set forth in SEQ ID NO: 173, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and a beta chain sequence set forth in SEQ ID NO: 377, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; or the TCR comprises an α chain sequence that is the sequence set forth in SEQ ID NO: 175 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and a β chain sequence that is the sequence set forth in SEQ ID NO: 379 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; The TCR of claim 1.

4. 4. A nucleic acid molecule encoding a TCR according to any one of claims 1 to 3, optionally wherein the nucleic acid molecule is a codon-optimized nucleotide sequence encoding a TCR according to any one of claims 1 to 3.

5. A vector comprising the nucleic acid molecule of claim 4, wherein the vector is a plasmid, a shuttle plasmid, a phagemid, a cosmid, or an expression vector.

6. A vector comprising the nucleic acid molecule of claim 4, wherein the vector is a vector for homology directed repair (HDR), or a viral vector, optionally a lentiviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes viral vector, a retroviral vector, or a baculoviral vector.

7. 4. A T cell receptor fusion protein or T cell receptor conjugate comprising the TCR of any one of claims 1 to 3 and another biologically active molecule, optionally wherein the other biologically active molecule is an antibody, cytokine, cytotoxic agent, enzyme, radioactive substance, or detectable label, and wherein the TCR and the other biologically active molecule are linked to each other with or without a linker.

8. 4. A modified cell expressing the TCR of any one of claims 1 to 3, wherein the modified cell is optionally a modified T cell or a modified NK cell; or the modified cell is a modified stem cell, and optionally the modified cell is a modified human CD4+ T cell or a modified human CD8+ T cell, or a mixed cell population of modified human CD4+ T cell and modified human CD8+ T cell; or the modified cell is a modified hematopoietic stem cell.

9. A modified human CD4+ T cell and / or modified human CD8+ T cell expressing the TCR of any one of claims 1 to 3 and exogenous CD8a or CD8ab.

10. 10. A method for producing T cells according to claim 9, comprising transfecting CD4+ T cells and / or CD8+ T cells with exogenous CD8a or CD8ab and the TCR of any one of claims 1 to 3.

11. The method of claim 10, wherein the exogenous CD8a or CD8ab and the TCR of any one of claims 1 to 3 are expressed in the CD4+ T cells and / or CD8+ T cells from the same vector, and optionally the construct expressing the exogenous CD8a or CD8ab and the construct expressing the TCR of any one of claims 1 to 3 are separated within the same vector by a 2A element or an IRES element.

12. A pharmaceutical composition comprising the modified cells of claim 8 and / or the modified human CD4+ T cells and / or modified human CD8+ T cells of claim 9.

13. 13. Use of the pharmaceutical composition of claim 12 for the manufacture of a medicament for treating a tumor harboring the KRAS_G12V mutation.

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