KRAS_g12d mutant antigen-specific TCR, and co-expression of TCR and CD8 to redirect CD4 t cells

Abstract

The present invention relates to a T cell receptor (TCR) specifically binding to a KRAS_G12D mutant antigen, a fusion protein or conjugate comprising the TCR, a nucleic acid encoding the TCR, an engineered cell comprising same, and a method for preparing the engineered cell. The present invention also relates to co-expressing an exogenous CD8 molecule and a TCR gene in T cells to enhance the function of the T cells. Provided are uses of the TCR and the genetically engineered cell in detection, prevention and / or treatment of cancers related to the KRAS_G12D mutant antigen.

Description

KRAS_G12D mutant antigen-specific TCR and its co-expression with CD8 redirect CD4 T cells Technical Field

[0001] The present invention generally relates to the field of immunology. Specifically, the present invention relates to a T cell receptor (hereinafter also abbreviated as TCR) that specifically binds to the KRAS_G12D mutant antigen, a genetically engineered cell expressing the TCR, and a method for preparing the genetically engineered cell. The present invention also relates to co-expressing exogenous CD8 molecules with the TCR gene in T cells to enhance the function of T cells. The present invention provides the use of the TCR and the genetically engineered cell in detecting, preventing and / or treating cancers associated with the KRAS_G12D mutant antigen. Background Art

[0002] The RAS gene was the first human oncogene discovered, and the RAS protein it encodes plays a central role in many important cellular signaling networks. RAS genes are the most commonly mutated oncogenes in human cancers. Currently, RAS protein activation caused by RAS gene mutations has been found in approximately one-fifth of all human tumors.

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

[0004] Inside cells, KRAS protein switches between inactive and active states. When bound to guanine nucleoside diphosphate (GDP), KRAS protein is inactive; when bound to guanine nucleoside triphosphate (GTP), KRAS protein is in an active state and can activate downstream signaling pathways. In most cells, KRAS protein is inactive.

[0005] When the KRAS gene mutates, the resulting mutant KRAS protein (for example, a single amino acid substitution mutation) disrupts GAP activity, causing the KRAS protein to remain bound to GTP, locking the KRAS protein in a state with tyrosine kinase activity and continuously activating downstream signaling pathways (such as the PI3K signaling pathway, the MAPK signaling pathway, the PI3K and Ral-GEFs signaling pathway, etc.). Once these downstream signaling pathways are turned on, they stimulate cell proliferation and migration, ultimately contributing to tumorigenesis.

[0006] Among all KRAS mutations, G12D (35%), G12V (29%) and G12C (21%) mutations are the most common. KRAS_G12D differs from KRAS_G12C only in the type of amino acid mutation at the same codon. KRAS_G12D is a codon mutation of the 12th amino acid residue of KRAS that introduces aspartic acid instead of cysteine, which directly manifests as epidemiological differences. KRAS_G12C is the most common KRAS mutation in NSCLC patients, while KRAS_G12D is the most common mutation in colorectal cancer and pancreatic cancer. In China, approximately 37.6% of colon cancer patients have KRAS mutations, of which nearly 32.2% have the KRAS G12D mutation subtype, which usually occurs in patients under 65 years old. Pancreatic cancer, also known as the "king of cancers," has limited treatment options. Therefore, there is a need in the art to develop specific immune cells targeting the KRAS_G12D mutant antigen, such as TCR-T cells, to effectively detect, prevent and treat cancers associated with the KRAS_G12D mutant antigen, bringing new treatment options to more patients. Summary of the Invention

[0007] The present invention provides a T cell receptor (TCR) that can specifically bind to the KRAS_G12D mutant antigen and prepares lymphocytes that recombinantly express the TCR. As a result, the presence of cancer associated with the KRAS_G12D mutant antigen in mammals can be detected through the specific binding of the TCR to the KRAS_G12D mutant antigen; and / or cancer cells expressing the KRAS_G12D mutant antigen can be killed by mediating an immune response against target cells expressing the KRAS_G12D mutant antigen in vivo, thereby meeting the above-mentioned needs.

[0008] Therefore, in one aspect, the present invention provides an isolated or purified T cell receptor (TCR) that specifically binds to the KRAS_G12D mutant antigen, preferably, the TCR comprises an α chain and a β chain, wherein the α chain and the β chain each comprise three complementarity determining regions (CDRs), and the amino acid sequence of the CDR3 of the α chain is selected from SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30 and variants having 1 or 2 amino acid residue changes with the sequence, and the amino acid sequence of the CDR3 of the β chain is selected from SEQ ID NO: 113, 116, 119, 122, 125, 128, 131, 134, 137, 140 and variants having 1 or 2 amino acid residue changes with the sequence.

[0009] In one embodiment, the amino acid sequence of CDR3 of the TCR α chain and the amino acid sequence of CDR3 of the β chain of the invention are:

[0010] (i) the α-chain CDR3 amino acid sequence set forth in SEQ ID NO: 3, or a variant thereof having one or two amino acid residue changes therefrom; and the β-chain CDR3 amino acid sequence set forth in SEQ ID NO: 113, or a variant thereof having one or two amino acid residue changes therefrom;

[0011] (ii) the α chain CDR3 amino acid sequence set forth in SEQ ID NO: 6, or a variant thereof having one or two amino acid residue changes therefrom; and the β chain CDR3 amino acid sequence set forth in SEQ ID NO: 116, or a variant thereof having one or two amino acid residue changes therefrom;

[0012] (iii) the α chain CDR3 amino acid sequence set forth in SEQ ID NO: 9, or a variant thereof having one or two amino acid residue changes therefrom; and the β chain CDR3 amino acid sequence set forth in SEQ ID NO: 119, or a variant thereof having one or two amino acid residue changes therefrom;

[0013] (iv) the α chain CDR3 amino acid sequence set forth in SEQ ID NO: 12, or a variant thereof having one or two amino acid residue changes therefrom; and the β chain CDR3 amino acid sequence set forth in SEQ ID NO: 122, or a variant thereof having one or two amino acid residue changes therefrom;

[0014] (v) the α chain CDR3 amino acid sequence set forth in SEQ ID NO: 15, or a variant thereof having one or two amino acid residue changes therefrom; and the β chain CDR3 amino acid sequence set forth in SEQ ID NO: 125, or a variant thereof having one or two amino acid residue changes therefrom;

[0015] (vi) the α chain CDR3 amino acid sequence set forth in SEQ ID NO: 18, or a variant thereof having one or two amino acid residue changes therefrom; and the β chain CDR3 amino acid sequence set forth in SEQ ID NO: 128, or a variant thereof having one or two amino acid residue changes therefrom;

[0016] (vii) the α chain CDR3 amino acid sequence set forth in SEQ ID NO: 21, or a variant thereof having one or two amino acid residue changes therefrom; and the β chain CDR3 amino acid sequence set forth in SEQ ID NO: 131, or a variant thereof having one or two amino acid residue changes therefrom;

[0017] (viii) the α chain CDR3 amino acid sequence set forth in SEQ ID NO: 24, or a variant thereof having one or two amino acid residue changes therefrom; and the β chain CDR3 amino acid sequence set forth in SEQ ID NO: 134, or a variant thereof having one or two amino acid residue changes therefrom;

[0018] (ix) the α chain CDR3 amino acid sequence set forth in SEQ ID NO: 27, or a variant thereof having one or two amino acid residue changes therefrom; and the β chain CDR3 amino acid sequence set forth in SEQ ID NO: 137, or a variant thereof having one or two amino acid residue changes therefrom;

[0019] (x) the α chain CDR3 amino acid sequence set forth in SEQ ID NO: 30, or a variant thereof having one or two amino acid residue changes therefrom; and the β chain CDR3 amino acid sequence set forth in SEQ ID NO: 140, or a variant thereof having one or two amino acid residue changes therefrom.

[0020] In one embodiment, the amino acid sequences of the three complementarity determining regions (CDRs) comprised by the α chain and the amino acid sequences of the three CDRs comprised by the β chain of the TCR of the present invention are:

[0021] (i) the α-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, or variants thereof having one or two amino acid residues altered therefrom; and the β-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 111, 112, and 113, or variants thereof having one or two amino acid residues altered therefrom;

[0022] (ii) the α-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, or variants thereof having one or two amino acid residue changes therefrom; and the β-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 114, 115, and 116, or variants thereof having one or two amino acid residue changes therefrom;

[0023] (iii) the α chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, or variants thereof having one or two amino acid residue changes therefrom; and the β chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 117, 118, and 119, or variants thereof having one or two amino acid residue changes therefrom;

[0024] (iv) the α-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, or variants thereof having one or two amino acid residue changes therefrom; and the β-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 120, 121, and 122, or variants thereof having one or two amino acid residue changes therefrom;

[0025] (v) the α-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, or variants thereof having one or two amino acid residue changes therefrom; and the β-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 123, 124, and 125, or variants thereof having one or two amino acid residue changes therefrom;

[0026] (vi) the α-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, or variants thereof having one or two amino acid residue changes therefrom; and the β-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 126, 127, and 128, or variants thereof having one or two amino acid residue changes therefrom;

[0027] (vii) the α-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, or variants thereof having one or two amino acid residues altered therefrom; and the β-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 129, 130, and 131, or variants thereof having one or two amino acid residues altered therefrom;

[0028] (viii) the α chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, or variants thereof having one or two amino acid residue changes therefrom; and the β chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 132, 133, and 134, or variants thereof having one or two amino acid residue changes therefrom;

[0029] (ix) the α chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 25, 26, and 27, or variants thereof having one or two amino acid residues altered therefrom; and the β chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 135, 136, and 137, or variants thereof having one or two amino acid residues altered therefrom;

[0030] (x) the α chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, or variants thereof having one or two amino acid residues altered compared to those sequences; and the β chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 138, 139, and 140, or variants thereof having one or two amino acid residues altered compared to those sequences.

[0031] In some embodiments, the TCR of the invention comprises an alpha chain sequence as set forth in SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, or 109, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; and a beta chain sequence as set forth in SEQ ID NO: 219, 221, 223, 225, 227, 229, 231, 233, 235, or 237, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0032] In some embodiments, the present invention provides a T cell receptor fusion protein or a T cell receptor conjugate comprising the TCR described in the first aspect of the invention and other biologically active molecules, wherein the other biologically active molecules are, for example, antibodies, cytokines, cytotoxic agents, enzymes, radioactive substances, detectable labels, and there may or may not be a linker between the TCR and the other biologically active molecules.

[0033] The present invention also provides nucleic acids encoding the TCR α chain and / or β chain of the present invention.

[0034] Furthermore, the present invention provides a vector, preferably a plasmid, a shuttle plasmid, a phagemid, a cosmid, an expression vector, a retroviral vector, an adenoviral vector and / or a homologous recombination repair (HDR) vector, comprising one or more nucleic acids as described above.

[0035] In a second aspect, the present invention provides an engineered cell transformed with the above vector and expressing the TCR described in the first aspect of the present invention.

[0036] In some embodiments, the present invention provides a method for preparing TCR-T cells using a targeting strategy that does not use a viral vector to express the exogenous TCR of the present invention.

[0037] In some embodiments, the present invention provides a method for editing the genome of a human cell, the method comprising inserting the following nucleic acid sequence into a target region of exon 1 of an endogenous T cell receptor (TCR) α chain constant region gene in a human cell, the nucleic acid sequence comprising from N-terminus to C-terminus:

[0038] (i) a sequence encoding a first cleavable linker polypeptide;

[0039] (ii) a sequence encoding the β chain of the TCR according to the first aspect of the present invention;

[0040] (iii) a sequence encoding a second cleavable linker polypeptide;

[0041] (iv) a sequence encoding the α chain variable region of the TCR according to the first aspect of the present invention;

[0042] The first cleavable linker polypeptide and the second cleavable linker polypeptide are the same or different viral 2A peptides.

[0043] The cells expressing exogenous TCR prepared by the method have high binding affinity to the VVVGADGVGK-HLA-A*11:01 complex and / or VVGADGVGK-HLA-A*11:01, and

[0044] Both the CL-40 cell line (HLA-A*11:01+, KRAS G12D+) and the SNU-601 cell line (HLA-A*11:01+, KRAS G12D+) had strong in vitro killing effects.

[0045] In some embodiments, the method for preparing cells expressing exogenous TCRs is performed by knocking out endogenous TCRs and knocking in exogenous TCRs using CRISPR / Cas9 technology and homologous recombination technology.

[0046] In a third aspect, the present invention provides methods and engineered cells for improving cell therapy.

[0047] In some embodiments, the present invention co-expresses exogenous TCR and CD8ab molecules in T cells.

[0048] By co-expressing CD8ab molecules with TCR genes in CD8+ and CD4+ T cells, the functions of CD8+ and CD4+ T cells were beneficially affected. In particular, by co-expressing MHC class I TCR and CD8 molecules in CD4+ T cells, CD4+ T cells were reprogrammed into multifunctional hybrid T cells that possess both cytotoxic effector functions and natural helper functions.

[0049] In a fourth aspect, the present invention provides uses of the TCR described in the first aspect and the engineered cells obtained in the second and third aspects in detecting, preventing and / or treating cancers associated with the KRAS_G12D mutant antigen. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The preferred embodiments of the present invention described in detail below will be better understood when read in conjunction with the following drawings. For the purpose of illustrating the present invention, the drawings show presently preferred embodiments. However, it should be understood that the present invention is not limited to the precise arrangements and means of the embodiments shown in the drawings.

[0051] Figure 1A shows a schematic diagram of the targeting strategy for knocking exogenous TCR into the TRAC site using gRNA002.

[0052] Figure 1B shows the targeting strategy for knocking out TRBC1 and TRBC2 sites using gRNA004.

[0053] Figure 2 shows a schematic diagram of the results of flow cytometry detection of TCR gene editing efficiency (Day 7). Flow cytometry data analysis is a cell distribution diagram divided into four quadrants (Q1, Q2, Q3, and Q4), where Q2 is a cell population that has completed endogenous TCR knockout (KO) and exogenous TCR knockin (KI) and expresses nwTCR; Q3 is wild-type T cells that have not undergone gene editing; and Q4 is KO cells that have completed endogenous TCR knockout.

[0054] Figures 3A-3J illustrate flow cytometry results of CD4+ T cells and CD8+ T cells electroporated with different nwTCRs and stained with pMHC tetramers (VVVGADGVGK-HLA-A11:01 tetramer) (Day 7).

[0055] FIG4A shows the binding affinity test results and EC50 values ​​of T cells expressing each nwTCR to the short peptide represented by SEQ ID NO: 239 presented by HLA-A*11:01.

[0056] FIG4B shows the binding affinity test results and EC50 values ​​of T cells expressing each nwTCR to the short peptide represented by SEQ ID NO: 240 presented by HLA-A*11:01.

[0057] Figures 5A-5C show the real-time fluorescence imaging results of T cells expressing various nwTCRs specifically killing the CL-40 (KRAS G12D+) cell line (0 hours at the beginning of cell co-incubation, and red fluorescence signals appearing after 18 hours of the killed target cells). "Blank" in the figure indicates that only target cells were present and no T cells expressing any nwTCR were added.

[0058] Figures 5D-5F show the real-time fluorescence imaging results of T cells expressing various nwTCRs specifically killing the SNU-601 (KRAS G12D+) cell line (0 hours at the start of cell co-incubation, and red fluorescence signals appearing in the killed target cells 18 hours later). "Blank" in the figure indicates that only target cells were present and no T cells expressing any nwTCR were added.

[0059] Figures 6A-6C show real-time analysis of CL-40 cell killing by T cells expressing various nwTCRs. The results demonstrate that gene-edited T cells have a specific killing effect on CL-40 cells. "Blank" in Figures 6A-6C indicates that only CL-40 cells were present, without the addition of T cells expressing any nwTCR.

[0060] Figures 6D-6F show real-time analysis of T cells expressing various nwTCRs killing SNU-601 cells. The results demonstrate that gene-edited T cells have a specific killing effect on SNU-601 cells. "Blank" in Figures 6D-6F indicates that only CL-40 cells were present, without the addition of T cells expressing any nwTCR.

[0061] FIG7 shows a diagram of the targeting strategy of nwTCR-CD8ab.

[0062] Figures 8A-8F show the flow cytometry results of CD4+T cells, CD8+T cells, and CD8+CD4+T cells electroporated with nwTCR-2985, nwTCR-2985-CD8ab, nwTCR-4536, nwTCR-4536-CD8ab, nwTCR-6670-2, and nwTCR-6670-2-CD8ab, respectively, stained with pMHC tetramers (Day 7). DETAILED DESCRIPTION

[0063] Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific methods and experimental conditions in this specification, because the methods and conditions can be varied. In addition, the terminology used herein is only for describing specific embodiments and is not intended to be limiting.

[0064] I. Definition

[0065] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present invention, the following terms are defined below.

[0066] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 10% less than the specified numerical value and an upper limit that is 10% greater than the specified numerical value.

[0067] The term "and / or" when used to link two or more alternatives should be understood to mean any one of the alternatives or any two or more of the alternatives.

[0068] As used herein, the terms "comprising" or "including" are intended to include the recited elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, the context of consisting of the recited elements, integers, or steps is also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0069] The "RAS protein family" belongs to a large family of small GTPases. RAS proteins can become constitutively activated due to single amino acid mutations. Mutated RAS protein products are involved in signal transduction in the early stages of tumorigenesis in many human cancers. Mutated RAS proteins are expressed in a variety of human cancers (e.g., lung cancer (e.g., lung adenocarcinoma), ovarian cancer (e.g., epithelial ovarian cancer), pancreatic cancer, prostate cancer, endometrial cancer, and colorectal cancer).

[0070] Kirsten rat sarcoma viral oncogene homolog (KRAS protein) is a key member of the RAS protein family. KRAS is regulated upstream by the epidermal growth factor receptor (EGFR) family. EGFR signaling can activate SOS protein, thereby regulating KRAS activation. The inactivation and activation state transitions of KRAS protein within the cell are determined by the molecules it binds to. Guanine nucleotide exchange factors (GEFs) catalyze KARS binding to GTP, activating KRAS. The GTPase-activating protein (GAP) promotes the hydrolysis of KRAS-bound GTP to GDP, leading to KRAS inactivation. Activated KRAS regulates downstream signaling pathways such as MAPK and PI3K, which are associated with cell proliferation and migration. KRAS mutations cause it to continuously bind to GTP, maintaining its activation state and leading to persistent activation of downstream signaling pathways, thereby promoting tumorigenesis.

[0071] The term "antigen" is any molecule that can be specifically detected by an organism's immune system.

[0072] "KRAS_G12D mutant antigen" refers to a KRAS protein with the G12D mutation, which can be specifically detected by the immune system of an organism. "G12D" or "G12D mutation" are used interchangeably and refer to the replacement of glycine at position 12 of the KRAS protein with aspartic acid.

[0073] 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 through signal transduction, leading to the subsequent immune response. The human genome contains four TCR genes: two encoding light chain TCRs: the TRA gene encoding TCR α and the TRG gene encoding TCR γ; and two encoding heavy chain TCRs: the TRB gene encoding TCR β and the TRD gene encoding TCR δ. The heavy chain TCR and light chain TCR form a heterodimer to form a complete TCR. In humans, there are two types of TCRs: TCR α / β and TCR γ / δ. 95% of T cells express TCR α / β, known as αβ T cells; 5% of T cells express TCR γ / δ, known as γ / δ T cells. This ratio varies during individual development and in disease states (such as leukemia), and also varies between species.

[0074] A mature heavy-chain TCR gene consists of four gene segments: the variable region (V), the diversity region (D), the joining region (J), and the constant region (C) (VDJC). Light-chain TCRs lack the D region (VJC). Both heavy and light-chain TCRs have three complementarity-determining regions (CDRs), which play a major role in antigen recognition. CDR1 and CDR2 are relatively conserved and are responsible for MHC recognition; CDR3 is the primary CDR responsible for antigen recognition.

[0075] TCR gene is the most complex gene in the human genome and also the gene with the highest degree of variation. 16 -10 18 There are three main factors that contribute to the diversity of TCRs: (i) compositional diversity: the VDJC / VJC structure of mature TCRs is generated through complex rearrangements. There are 65-100 V gene segments, 2 D gene segments, and 13 J gene segments in the genome. During TCR recombination, one segment must be selected from each of the three segments, which gives TCRs a high degree of diversity; (ii) junctional mobility: during the rearrangement process, non-templated nucleotides are often randomly inserted or deleted in the VD and DJ junction regions, further increasing the diversity of the CDR3 region; (iii) somatic mutations: the mutation frequency of the T cell D region is about 1000 times that of normal.

[0076] As known in the art, "polynucleotide" or "nucleic acid" used interchangeably herein refer to a chain of nucleotides of any length and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate capable of being incorporated into a chain by DNA or RNA polymerase.

[0077] Calculation of sequence identity between sequences is performed as follows.

[0078] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment or non-homologous sequences can be discarded for comparison purposes). In a preferred embodiment, the length of the reference sequence compared for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.

[0079] The comparison of sequences and calculation of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm, which has been integrated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and 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 percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http: / / www.gcg.com), using the NWSgapdua.CMP matrix and 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 the one that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0080] The percent 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), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4.

[0081] The term "antigen presenting cell" or "APC" refers to a cell of the immune system, such as a helper cell (e.g., B-cell, dendritic cell, etc.), that presents foreign antigens complexed with the major histocompatibility complex (MHC) on its surface. T cells can recognize these complexes using their T cell receptors (TCRs). APCs process and present the antigens to T cells.

[0082] The term "guide RNA (gRNA)" refers to an RNA specific for a target DNA that can form a complex with a Cas protein and bring the Cas protein to the target DNA, thereby introducing a double-strand break at the site of the target DNA. In the present invention, the guide RNA can be composed of two RNAs, namely, CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA), or the guide RNA can be a single-stranded guide RNA (sgRNA) produced by fusing the essential parts of crRNA and tracrRNA.

[0083] Ribonucleoprotein (RNP) is a complex formed by Cas9 protein and gRNA with gene editing function.

[0084] The CRISPR / Cas9 gene editing system consists of two main components: the Cas9 protein, which acts like a "wrench," and the CRISPR guide RNA, which acts like a "thread." The guide RNA locates the target site and recruits and activates the Cas9 protein; the Cas9 protein then cuts the target DNA.

[0085] The term "recombinant" when applied 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 by the alteration of a native nucleic acid or protein.

[0086] The term "target site" refers to any DNA sequence in the target genome that is to be modified or repaired. The DNA sequence near the target site allows the integration of exogenous sequences at the target site, including but not limited to gene knock-in (KI). In a specific embodiment, the target DNA sequence is a double-stranded DNA sequence, including but not limited to, a DNA sequence in the chromosomal genome of a cell, a DNA sequence outside the chromosomal genome of a cell (e.g., the mitochondrial genome), a DNA sequence of a plasmid, a virus, etc.

[0087] In the present invention, the term "site-directed recombination" refers to the integration of an exogenous sequence into a specific target site in a non-random manner, including integration into the 5' upstream, 3' downstream, or between target sites of a specific target site.

[0088] In the present invention, the term "exogenous DNA sequence" refers to a DNA sequence that is desired to be site-directedly recombined into a target site. The exogenous DNA sequence may be a sequence that does not exist or is altered at the target site.

[0089] The term "donor DNA" or "donor nucleic acid sequence" refers to a polynucleotide comprising a polynucleotide sequence of interest to be expressed that is inserted into a target site in the target genome. In certain embodiments, the donor DNA further comprises a sequence homologous to the genomic sequence (also referred to as a "homology arm"). "Homologous" means a similar DNA sequence. Homologous arms are sufficient for homologous recombination with homologous genomic sequences. For example, homology arms can comprise at least 50-3500 or more bases in length.

[0090] The term "homology-directed DNA repair (HDR)" refers to a repair method based on homologous recombination that can be used to specifically and efficiently insert a donor DNA template (encoding the target sequence) into the target genomic site. It is a repair pathway initiated after double-strand damage to cellular DNA. HDR can only occur when a DNA fragment homologous to the damaged DNA exists in the cell nucleus. An HDR vector can refer to a vector used for electroporation transfection using CRISPR / Cas9 and homologous recombination technology. HDR efficiency can refer to the gene knock-in efficiency of electroporation transfection using CRISPR / Cas9 and homologous recombination technology.

[0091] A synonymous mutation is a neutral mutation. The genetic code is degenerate, meaning there is usually more than one codon that determines an amino acid. Substitution of the third nucleotide in a triplet codon often does not change the amino acid composition. Although the third nucleotide in the triplet codon mutates, the encoded amino acid remains unchanged. This mutation is considered a synonymous mutation.

[0092] As used herein, "vector" refers to a construct that is capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the gene or sequence in the host cell. Examples of vectors include, but are not limited to, viral vectors, plasmids, cosmids, or phage vectors. The vector may comprise a nucleic acid sequence that allows the gene or sequence of interest to replicate in the host cell, such as an origin of replication. The vector may also comprise one or more selectable marker genes and other genetic elements well known to those skilled in the art. The vector is preferably an expression vector comprising a nucleic acid according to the present invention, and the nucleic acid is effectively connected to a sequence that allows the expression of the nucleic acid.

[0093] The term "operably linked" refers to a functional connection between a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein so as to perform the overall function. Operable connections with recombinant vectors can be prepared using gene recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes well known in the art.

[0094] As used herein, the term "engineered cell" refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such cells. Engineered cells include "transfected cells," which include the primary transfected cell and its progeny, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny having the same function or biological activity as the cell screened or selected in the initial transfected cell are included herein.

[0095] As used herein, "subject" or "individual" refers to an animal, preferably a mammal, and more preferably a human, that is in need of alleviation and / or treatment of a KRAS_G12D mutant antigen-associated cancer. Mammals also include, but are not limited to, farm animals, racing animals, pets, primates, horses, dogs, cats, mice, and rats.

[0096] Adoptive Cell Transfer Therapy (ACT) refers to the isolation of immune-active cells from the subject or patient, activation and amplification, gene editing and other treatments in vitro, and then back-infusion into the patient to kill the target cells.

[0097] II. T Cell Receptors (TCRs) and TCR-Encoding Nucleic Acids of the Invention

[0098] The wild-type human KRAS protein is 188 amino acid residues long and has a molecular weight of approximately 21.6 kDa. It contains a glycine residue at position 12. Of the KRAS gene mutations, 83% involve mutations at position 12, one of which is a glycine-to-aspartic acid mutation (herein referred to as G12D).

[0099] The present invention provides isolated or purified TCRs that have antigenic specificity for a KRAS peptide with a G12D mutation presented by a human leukocyte antigen (HLA) class I molecule. The KRAS peptide with a G12D mutation presented by a human leukocyte antigen (HLA) class I molecule has any length suitable for binding to any HLA class I molecule.

[0100] In some embodiments, the KRAS peptide with the G12D mutation has a length of about 9 to about 10 amino acid residues, including any consecutive about 9 to about 10 amino acid residues in the KRAS protein with the G12D mutation. In some embodiments, the TCR of the present invention has antigenic specificity for a KRAS peptide with the G12D mutation, wherein the mutant KRAS peptide has a length of about 9 amino acid residues or about 10 amino acid residues. Examples of KRAS peptides with the G12D mutation that can be recognized by the TCR of the present invention are a short peptide VVVGADGVGK (SEQ ID NO: 239) of the amino acid sequence of KRAS from position 7 to position 16 (also referred to herein as "KRAS_G12D_7-16 peptide"); and a short peptide VVGADGVGK (SEQ ID NO: 240) of the amino acid sequence of KRAS from position 8 to position 16 (also referred to herein as "KRAS_G12D_8-16 peptide").

[0101] 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 the antigenic peptide-MHC complex triggers T cell activation through a series of biochemical events mediated by associated enzymes, co-receptors, and accessory molecules. In 95% of T cells, the TCR heterodimer is composed of α and β chains, while in 5% of T cells, the TCR heterodimer is composed of γ and δ chains.

[0102] Each chain of the TCR is a member of the immunoglobulin superfamily and has an N-terminal immunoglobulin (Ig) variable (V) domain, an Ig constant (C) domain, a cell membrane-spanning region (i.e., a transmembrane region), and a short cytoplasmic tail at the C-terminus. In the variable domains of the TCR α and β chains, each variable domain has three hypervariable regions or complementary determining regions (CDRs), of which CDR3 in each variable domain is the primary CDR responsible for recognizing processed antigens. CDR2 is believed to recognize MHC molecules.

[0103] The constant domain of TCRs consists of a short linker sequence in which cysteine ​​residues form disulfide bonds, creating a connection between the TCR α and β chains.

[0104] During T cell maturation, the TCR and CD3 form the TCR / CD3 complex. The TCR / CD3 complex formation process typically proceeds in the following order: First, the three peptide chains CD3γ, δ, and ε form two heterodimers, γ-ε and δ-ε, forming a stable complex core. TCRαβ (or TCRγδ) then binds to these heterodimers. Subsequently, the ζ-ζ or ζ-η dimer binds to the TCRαβ (or TCRγδ) / CD3γεδε complex, which is then transferred to the T cell surface. Signals are transmitted from the TCR to the cell via the TCR / CD3 complex.

[0105] The signal from the TCR / CD3 complex is enhanced by the simultaneous binding of MHC to a specific co-receptor. In helper T cells, this co-receptor is the CD4 molecule, which is specific for MHC class II, while in cytotoxic T cells, this co-receptor is CD8, which is specific for MHC class I.

[0106] In this article, the term "T cell receptor" has the conventional meaning in the art and is used to refer to a molecule that can recognize peptides presented by MHC molecules. The molecule is a heterodimer of two chains α and β (or optionally γ and δ).

[0107] The TCRs of the present invention provide specific affinity recognition for the KRAS_G12D mutant antigen. The KRAS_G12D mutant antigen is degraded intracellularly by the proteasome into short peptides of 8 to 10 amino acids, such as the KRAS_G12D_7-16 peptide set forth in SEQ ID NO:239 and / or the KRAS_G12D_8-16 peptide set forth in SEQ ID NO:240. These short peptides are presented on the cell surface by MHC class I as peptide / MHC complexes (pMHC). Some pMHCs have been shown to be associated with various cancers and, therefore, could be potential targets for TCR therapy.

[0108] The present invention provides isolated or purified T cell receptor (TCR) α chain and / or β chain. The TCR of the present invention can be a hybrid TCR comprising sequences derived from more than one species. For example, considering that murine TCR can be expressed more effectively than human TCR in human T cells, TCR can comprise human variable regions and murine constant regions.

[0109] In one embodiment, the TCR of the present invention comprises an α chain and a β chain, wherein the α chain and the β chain each comprise three complementarity determining regions (CDRs), and wherein the amino acid sequence of the TCR α chain CDR3, which is primarily responsible for antigen recognition, is selected from SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, and variants thereof having 1 or 2 amino acid residue changes, and the amino acid sequence of the β chain CDR3 is selected from SEQ ID NOs: 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, and variants thereof having 1 or 2 amino acid residue changes.

[0110] In one embodiment, the TCR of the present invention comprises an α chain and a β chain, wherein the amino acid sequence of the three complementarity determining regions (CDRs) comprised by the α chain and the amino acid sequence of the three CDRs comprised by the β chain are:

[0111] (i) the α-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, or variants thereof having one or two amino acid residues altered therefrom; and the β-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 111, 112, and 113, or variants thereof having one or two amino acid residues altered therefrom;

[0112] (ii) the α-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, or variants thereof having one or two amino acid residue changes therefrom; and the β-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 114, 115, and 116, or variants thereof having one or two amino acid residue changes therefrom;

[0113] (iii) the α chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, or variants thereof having one or two amino acid residue changes therefrom; and the β chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 117, 118, and 119, or variants thereof having one or two amino acid residue changes therefrom;

[0114] (iv) the α-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, or variants thereof having one or two amino acid residue changes therefrom; and the β-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 120, 121, and 122, or variants thereof having one or two amino acid residue changes therefrom;

[0115] (v) the α-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, or variants thereof having one or two amino acid residue changes therefrom; and the β-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 123, 124, and 125, or variants thereof having one or two amino acid residue changes therefrom;

[0116] (vi) the α-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, or variants thereof having one or two amino acid residue changes therefrom; and the β-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 126, 127, and 128, or variants thereof having one or two amino acid residue changes therefrom;

[0117] (vii) the α-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, or variants thereof having one or two amino acid residues altered therefrom; and the β-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 129, 130, and 131, or variants thereof having one or two amino acid residues altered therefrom;

[0118] (viii) the α chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, or variants thereof having one or two amino acid residue changes therefrom; and the β chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 132, 133, and 134, or variants thereof having one or two amino acid residue changes therefrom;

[0119] (ix) the α chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 25, 26, and 27, or variants thereof having one or two amino acid residues altered therefrom; and the β chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 135, 136, and 137, or variants thereof having one or two amino acid residues altered therefrom;

[0120] (x) the α chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, or variants thereof having one or two amino acid residues altered compared to those sequences; and the β chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 138, 139, and 140, or variants thereof having one or two amino acid residues altered compared to those sequences.

[0121] In one embodiment, the TCR of the invention comprises an alpha chain sequence as set forth in SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107, or 109, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; and a beta chain sequence as set forth in SEQ ID NO: 219, 221, 223, 225, 227, 229, 231, 233, 235, or 237, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. Preferably, the constant region of the TCR of the invention is a mouse constant region.

[0122] In some embodiments, the amino acid residue changes in the TCR variants of the present invention are substitutions, additions, or deletions of amino acid residues in any of the alpha chain sequences set forth in SEQ ID NOs: 91, 93, 95, 97, 99, 101, 103, 105, 107, or 109, or any of the beta chain sequences set forth in SEQ ID NOs: 219, 221, 223, 225, 227, 229, 231, 233, 235, or 237, provided that the TCR variant retains or improves its ability to bind to an epitope peptide-MHC complex of a KRAS-G12D mutant antigen. In one embodiment, the substitutions are conservative substitutions. Examples of conservative substitutions are provided in Table A below.

[0123] Table A

[0124] Amino acids can be grouped according to common side chain properties:

[0125] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu; Ile;

[0126] (2) Neutral hydrophilic: Cys, Ser, Thr, Asn; Gln;

[0127] (3) Acidic: Asp, Glu;

[0128] (4) Basic: His, Lys, Arg;

[0129] (5) Residues that affect chain direction: Gly, Pro;

[0130] (6) Aromatic: Trp, Tyr, Phe.

[0131] Non-conservative substitutions will exchange a member of one of these classes for a member of another class.

[0132] In some embodiments, the TCR of the present invention is capable of recognizing and binding to an epitope peptide of a mutant KRAS protein presented by an HLA class I molecule, thereby eliciting an immune response.

[0133] In some embodiments, the HLA class I molecule is any HLA-A molecule, for example, the HLA class I molecule is an HLA-A11 molecule. The HLA-A11 molecule can be any HLA-A11 molecule. Examples of HLA-A11 molecules include, but are not limited to, HLA-A*11:01, HLA-A*11:02, HLA-A*11:03, or HLA-A*11:04. Preferably, the HLA class I molecule is an HLA-A*11:01 molecule. The HLA-A*11:01 molecule is the most common HLA-A molecule in Asians.

[0134] The present invention also relates to nucleic acids encoding TCRs of the present invention or portions thereof, such as one or more CDRs; one or more variable regions; an alpha chain; or a beta chain. The nucleic acid can be double-stranded or single-stranded and can be RNA or DNA. The nucleic acid sequence can 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 can also include removal of mRNA instability motifs and hidden splice sites.

[0135] The TCR of the present invention can be modified by various methods (e.g., gene fusion, chemical conjugation, etc.) to enable the TCR to be linked to other bioactive molecules. The TCR that can be linked to other bioactive molecules can be a TCR heterodimer or a soluble form thereof, more preferably a soluble, single-chain TCR. The other bioactive molecules can be various bioactive effectors, such as antibodies, cytokines, cytotoxic agents, enzymes, radioactive substances, detectable labels, etc. There may or may not be a linker between the TCR and the other bioactive molecule.

[0136] In some embodiments, the TCR fusion protein is a fusion of a TCR with an antibody, including a complete antibody (e.g., IgG, IgM, or IgA class) or a fragment thereof (e.g., Fv, Fab, Fab', Fab'-SH, F(ab')2; diabody; single-chain antibody (e.g., scFv); single-domain antibody); and multispecific antibodies (e.g., bispecific antibodies).

[0137] In some embodiments, the TCR fusion protein is a TCR fused to a cytokine, such as an interleukin (eg, IL-2), a chemokine (eg, MIP-1β), or a growth factor (eg, GCSF).

[0138] In some embodiments, the TCR conjugate is a covalent linking of the TCR to a cytotoxic agent, such as doxorubicin.

[0139] In some embodiments, a TCR conjugate is a TCR covalently linked to a radioactive substance, such as I 125 .

[0140] In some embodiments, the TCR conjugate is a TCR covalently linked to a detectable label, such as a fluorescent label.

[0141] The T cell receptor fusion proteins or T cell receptor conjugates of the present invention can be used in various applications, including in vivo detection of cells and / or imaging of cells or tissues, as well as therapeutic uses, such as killing target cells or target tissues expressing KRAS_G12D mutant antigens with specific binding TCR in vivo or in vitro.

[0142] III. Vectors containing nucleic acids encoding the TCRs of the present invention

[0143] The present invention also relates to vectors comprising nucleic acids encoding the TCRs of the present invention. In one embodiment, the vector is a pUC57-Simple vector (available from GenScript Biotech). In another embodiment, a pUC57-HA vector is used, which is an optimized vector based on the pUC57-Simple vector. It retains only the Ori and Amp sequences of the pUC57-Simple vector, then replaces the Amp sequence with a Kana sequence, and adds the left and right homology arms (HA) sequences of the TRAC site (about 800bp).

[0144] The vector transfers the nucleic acid encoding the TCR of the present invention into cells, such as T cells, NK cells, stem cells, e.g., pluripotent stem cells, induced pluripotent stem cells (iPSCs), such that the engineered cells express a TCR specific for the KRAS_G12D mutant antigen.

[0145] The KRAS_G12D mutant antigen-specific TCR refers to a TCR that can specifically bind to and immunologically recognize the G12D mutant KRAS with high affinity. For example, about 1×10 4 to about 1×10 5 After T cells expressing TCR were co-cultured with antigen-presenting cells such as T2 cells or K562 cells pulsed with G12D mutant KRAS and overexpressing HLA class I molecules, the T cells were expressed with an EC50 of about 1×10 -4 M or less (e.g., 1×10 -6 M or less, 1×10 -7 M or less, 1×10 -8 If the TCR induces IFN-γ secretion when the TCR is expressed in a KRAS mutant with a G12D mutation (M or less), the TCR is considered to have antigenic specificity for the KRAS mutant with a G12D mutation. The HLA class I molecule can be any HLA class I molecule described herein (eg, an HLA-A*11:01 molecule).

[0146] Preferably, the vector allows for sustained high-level expression of the introduced exogenous TCR in engineered cells (e.g., engineered T cells), and the introduced exogenous TCR can successfully compete with the endogenous TCR for a limited pool of CD3 molecules. Alternatively, increasing the supply of CD3 molecules can also increase exogenous TCR expression in gene-modified cells. Therefore, the vector optionally comprises genes for CD3-γ, CD3-δ, CD3-ε, and / or CD3-ζ. In one embodiment, the vector comprises a gene for CD3-ζ. In addition, one or more separate vectors encoding CD3 genes can also be provided for co-transfer into cells with the exogenous TCR encoding vector.

[0147] The vector form is not limited to homologous recombination repair (HDR) vectors, and can also be a viral vector. The viral vector can be a lentiviral vector, an adenoviral vector, an adeno-associated virus (AAV) vector, a herpes virus vector, a retroviral vector, or a baculoviral vector, which is used to edit the cell genome.

[0148] Genome editing technology refers to the technology of inserting, deleting or replacing nucleic acids in the cell's genomic DNA. T cells genetically modified using gene editing technology for primary human T cells have demonstrated excellent therapeutic effects in clinical trials of various adoptive immunotherapy drugs. Among them, chimeric antigen receptors (CAR) or T cell receptors (TCR) are often used to modify primary human T cells to achieve recognition of certain specific target epitopes. These modified T cells can specifically kill specific target cells.

[0149] Common TCR gene editing methods can be broadly divided into two categories based on the method of gene integration. One is random gene integration, including lentivirus (LV) systems, adeno-associated virus (AAV) systems, and transposon systems. The other is precise gene editing, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) technology. CRISPR technology uses gRNA to identify and edit DNA, and achieves site-specific insertion of large gene fragments through homologous recombination, offering advantages such as ease of operation and greater scalability.

[0150] IV. Preparation of Engineered Cells

[0151] Viral vectors can be used to introduce the target TCR into cells. However, this method of introducing exogenous TCRα / β genes into cells using viral vectors does not knock out the endogenous TCR in the cells, which may cause mispairing of the exogenous TCRα and β chains. Even if the mispairing problem of the exogenous TCRα and β chains can be reduced by modifying the disulfide bonds or replacing the mouse constant region, the viral vector is randomly inserted into the cell genome, which still brings the potential risk of damaging other genes.

[0152] Alternatively, non-viral vectors can be used to introduce the target TCR into cells, precisely integrating the exogenous TCR α / β genes into specific genomic sites in the cells. In some embodiments, non-viral gene editing methods can use CRISPR / Cas9 technology and homologous recombination to knock out the endogenous T cell receptor α and β chains of human T cells and knock in the exons of the TRAC gene encoding the exogenous target T cell receptor α and β chains. This approach not only disrupts endogenous TCR expression, but also uses the endogenous TCR promoter to express the exogenous target TCR α and β.

[0153] In one embodiment, exogenous target T cell receptor α and β chain encoding nucleotides are knocked into exon 1 of the endogenous TRAC gene, and this exogenous knock-in fragment does not require the addition of the TRAC gene, thereby reducing the length of the gene knock-in fragment and simplifying the difficulty of gene knock-in. Compared with the technology of expressing TCR using viral vectors, the technology of expressing TCR using non-viral vectors can be used as a fast, simple, and low-cost method to introduce exogenous TCRα / β genes into cells.

[0154] IV.1 Selection of knockout sites

[0155] The TCR is a dimer composed of a TCR α chain and a TCR β chain. The TCR α chain gene is formed by rearrangement of the TRAV, TRAJ, and TRAC genes. The TRAV and TRAJ genes each contain multiple sequences that differ among each other, necessitating the random selection of one sequence for expression during rearrangement. Selecting the TRAV and TRAJ genes as knockout sites makes it difficult to prevent the generation of any random TCR α chain gene. However, there is only one TRAC gene, and knocking it out can eliminate any random TCR α chain gene, making TRAC a suitable knockout site. The TCR β chain gene is formed by rearrangement of the TRBV, TRBJ, TRBD, and TRBC genes. The TRBV and TRBJ genes each contain multiple sequences that differ among each other, making them unsuitable knockout sites. The TRBC gene, consisting of TRBC1 and TRBC2, shares some identical sequences, allowing this common sequence to be selected as a knockout site, eliminating any random TCR β gene.

[0156] 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, thereby achieving a higher efficiency of endogenous TCR knockout and reducing the risk of mispairing between the exogenous TCR and the endogenous TCR chains that may be caused by endogenous TCR expression.

[0157] Nuclease-based genome editing tools can be used to target and disrupt endogenous TRAC and TRBC genes by inducing double-strand breaks and DNA repair through nonhomologous end joining (fNHEJ). These tools include meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), megaTAL nucleases, and CRISPR / CRISPR-associated protein 9 (CRISPR / Cas9).

[0158] IV.2 Selection of knock-in sites

[0159] Since the endogenous TRAC gene is unique and all TCRɑ expressions require the TRAC gene, the exogenous TCRα / β gene knock-in site is selected as the endogenous TRAC site. Thus, while eliminating the endogenous TCR, the endogenous TCR promoter of human T cells can be used to express the exogenous TCRα / β gene of the present invention (also called "nwTCR" gene) without the need to add the TRAC gene, thereby reducing the size of the knock-in fragment and improving the efficiency of gene editing.

[0160] In some embodiments, the nwTCR expression construct is cloned into a targeting vector (e.g., pUC57-HA vector), and homology arms are designed to allow the nwTCR to be site-specifically knocked into the TCRα chain constant region, where its expression is regulated by the transcriptional regulatory sequences of that locus. Because the endogenous promoter at this knock-in site is more regulated than at other sites, sustained and stable expression of the nwTCR gene is ensured.

[0161] IV.3 Engineered Cells

[0162] The present invention provides engineered cells expressing exogenous TCRs.

[0163] In some embodiments, engineered cells expressing TCRs are prepared from cells derived from blood, bone marrow, lymph or lymphoid organs, e.g., lymphocytes including but not limited to T cells, NK cells, or stem cells, e.g., pluripotent stem cells, induced pluripotent stem cells (iPSCs).

[0164] The cells are typically primary cells, e.g., cells isolated directly from a subject and / or isolated from a subject and frozen. The cells can be allogeneic and / or autologous cells.

[0165] In some embodiments, CRISPR / Cas9 and homologous recombination technology are used to electroporate and transfect CD3 / CD28 activated primary cells (e.g., sorted CD4+ T cells and CD8+ T cells) using RNP and plasmid to prepare engineered TCR cells.

[0166] In some embodiments, sgRNAs are designed against the endogenous TRAC gene, and sgRNAs are designed against the endogenous TRBC1 and TRBC2 genes.

[0167] The Cas9 protein is guided by sgRNA to bind to a specific site in the target genome, where it cuts the target site. For double-strand breaks in the endogenous TRAC gene caused by RNP, homologous recombination can occur in the presence of donor DNA with homology arms, thereby achieving site-specific insertion of the target nwTCR gene.

[0168] In a specific embodiment, the specific site of the target genome to which the sgRNA guides the Cas9 protein to bind is exon 1 of the TRAC gene, and the Cas9 protein cuts the specific site. The designed and verified sgRNA recognition sequence and PAM sequence for efficiently targeting it include the nucleotide sequence shown as TCAGGGTTCTGGATATCTGT-GGG (sgRNA recognition sequence-PAM sequence, "-" is used to distinguish the CRISPR / Cas9 recognition site and the PAM sequence).

[0169] In a specific embodiment, the specific site of the target genome to which the sgRNA guides the Cas9 protein to bind is exon 1 of the TRBC1 and TRBC2 genes, and the Cas9 protein cuts the specific site. The designed and verified sgRNA recognition sequence and PAM sequence for efficiently targeting it include the nucleotide sequence shown in CTGCCTGAGCAGCCGCCTGA-GGG (sgRNA recognition sequence-PAM sequence, "-" is used to distinguish the CRISPR / Cas9 recognition site and the PAM sequence).

[0170] The CRISPR / Cas system may comprise a Cas component in the form of a protein or in the form of a nucleic acid encoding a Cas protein.

[0171] In the present invention, the Cas protein may be any Cas protein as long as it has endonuclease or nickase activity when complexed with a guide RNA.

[0172] Preferably, the Cas protein is a Cas9 protein or a variant or a functional fragment thereof.

[0173] The Cas protein may be a protein isolated from an organism such as Streptococcus sp., preferably Streptococcus pyogenes, or a recombinant protein, but is not limited thereto.

[0174] In one embodiment, the Cas protein comprises Cas9 derived from Streptococcus pyogenes, such as Cas9 having the amino acid sequence shown in SEQ ID NO: 254.

[0175] In another embodiment, the Cas protein comprises an amino acid sequence that is at least 50% homologous to the amino acid sequence shown in SEQ ID NO: 254, preferably at least 60, 70, 80, 90, 95, 97, 98, or 99% homologous to the amino acid sequence shown in SEQ ID NO: 254, but is not limited thereto.

[0176] In the context of the present invention, the Cas protein encoding nucleic acid may be in the form of a vector, such as a plasmid comprising a Cas encoding sequence under a promoter such as CMV or CAG. When the Cas protein is Cas9, the Cas9 encoding sequence may be derived from Streptococcus, preferably from Streptococcus pyogenes. For example, the Cas9 encoding nucleic acid may comprise a nucleotide sequence encoding SEQ ID NO: 254. In addition, the Cas9 encoding nucleic acid may comprise a nucleotide sequence having at least 50% homology to the nucleotide sequence encoding SEQ ID NO: 254, preferably a nucleotide sequence having at least 60, 70, 80, 90, 95, 97, 98, or 99% homology to the nucleotide sequence encoding SEQ ID NO: 254, but is not limited thereto.

[0177] In one embodiment, the donor DNA comprises, in order, 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. Upon expression of the sequence encoding the cleavable linker polypeptide, the cleavable linker polypeptide is cleaved. In some embodiments, the cleavable linker polypeptide sequence comprises a 2A ribosomal skipping element, such as T2A, E2A, P2A, and F2A, or variants thereof.

[0178] In one embodiment, the donor DNA is located in a targeting vector. The basic targeting vector serving as the backbone is not particularly limited, as long as it has a prokaryotic replication origin and a selectable marker for vector propagation in bacteria.

[0179] In a preferred embodiment, in order to increase the expression of exogenous TCRα / β genes or fragments thereof, a sequence encoding a cleavable linker polypeptide and a signal peptide sequence are connected to the N-termini of the exogenous TCRα chain gene and the exogenous TCRβ chain gene, respectively, in the targeting vector.

[0180] In one embodiment, the targeting vector for knocking in the nwTCR gene sequence comprises the following structures operably linked: 2A ribosomal jumping element-SP-TCR β-2A ribosomal jumping element-SP-TRAV-TRAJ

[0181] Among them, SP is the signal peptide coding sequence.

[0182] The targeting vector for knocking in the nwTCR gene sequence, the RNP complex, and the cells are mixed and the nwTCR gene sequence is delivered to the cells. In some embodiments, the delivery step is selected from the group consisting of electroporation, transfection, physical deformation of the cell membrane, lipid nanoparticles (LNPs), virus-like particles (VLPs), and sonication. In some embodiments, the delivery step includes electroporation.

[0183] In some embodiments, the engineered cells are primary cells.

[0184] In some embodiments, the engineered cell is an isolated cell, wherein the isolated cell is isolated from a subject.

[0185] In some embodiments, the engineered cells are ex vivo cultured cells. In some embodiments, the ex vivo cultured cells include stimulated cells. In some embodiments, the stimulated cells include cytokine-stimulated T cells, optionally wherein the cytokine-stimulated T cells include CD3-stimulated T cells, CD28-stimulated T cells, or CD3 and CD28-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.

[0186] In some embodiments, the engineered cells are stem cells, for example, hematopoietic stem cells (HSC). Transferring the nwTCR gene to HSC will not result in TCR expression on the cell surface because stem cells do not express CD3 molecules. However, when stem cells differentiate into lymphoid precursor cells (lymphoid precursor) that migrate to the thymus, the activation of CD3 expression will result in the expression of the introduced nwTCR on the surface of thymocytes. The advantage of this method is that once mature T cells are produced, they only express the introduced nwTCR, and express very little or no endogenous TCR chains, because the expression of the introduced nwTCR chains suppresses the rearrangement of endogenous TCR gene segments to form functional TCR ɑ and β genes. Another benefit of this method is that TCR gene-modified stem cells are a continuous source of mature T cells with desired antigen-specificity. Therefore, nwTCR gene-modified stem cells produce T cells expressing the TCR of the present invention after differentiation.

[0187] V. Methods for Detecting, Preventing, or Treating Cancers Associated with the KRAS_G12D Mutant Antigen

[0188] The present invention provides a method for preventing or treating cancer associated with the KRAS_G12D mutant antigen, comprising administering to a subject in need thereof an engineered cell of the present invention, a TCR nucleic acid of the present invention, a vector, or a pharmaceutical composition. 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 an engineered cell of the present invention.

[0189] In some embodiments, the engineered cells, TCR nucleic acids, vectors, or pharmaceutical compositions of the present invention are used to prevent or treat cancers associated with the KRAS_G12D mutant antigen. Without being bound by any theory, it is believed that the TCRs of the present invention are capable of specifically binding to the KRAS_G12D mutant antigen, thereby mediating an immune response against target cells expressing the KRAS_G12D mutant antigen.

[0190] The treatment or prevention may include treatment or prevention of one or more symptoms of the cancer being treated or prevented, including promoting tumor regression, delaying the onset of cancer or its symptoms, and preventing or delaying the recurrence of cancer or its symptoms.

[0191] The present invention also provides a method for detecting the presence of cancer in a mammal. The method comprises: (i) contacting a sample comprising one or more cells from a mammal with any of the TCRs of the present invention, a cell colony expressing the TCRs of the present invention, or a pharmaceutical composition comprising a cell colony expressing the TCRs of the present invention as described herein, thereby forming a complex; and (ii) detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal. The contact can be implemented in vitro or in vivo in a mammal. In one embodiment, the contact is implemented in vitro. The complex can be detected by various means known in the art. In some embodiments, the TCRs of the present invention or cell colonies expressing the TCRs of the present invention are labeled with a detectable marker, such as a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and elemental particles (e.g., gold particles).

[0192] The present invention also provides a method for inducing anti-tumor immunity, wherein the tumor is a KRAS_G12D mutant antigen-associated tumor, and the method comprises administering an effective amount of the engineered cells of the present invention to a subject.

[0193] The present invention provides a method for inducing an immune response in a subject, comprising administering an effective amount of an engineered cell 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 engineered immune cell comprises a TCR of the present invention. In some embodiments, the target cell is a cancer cell associated with a KRAS_G12D mutant antigen.

[0194] In some embodiments, donor T cells for T cell therapy are obtained from a patient (e.g., for autologous T cell therapy). In other embodiments, donor stem cells to be differentiated into T cells for T cell therapy are obtained from a non-patient subject.

[0195] 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 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 cells / kg body weight.

[0196] The cancers mentioned in the various methods of the present invention can be any cancer, including but not limited to: acute lymphocytic cancer, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, brain cancer, 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 tumors, colon cancer, small intestine cancer, colorectal cancer, rectal cancer, stomach cancer, skin cancer, melanoma, multiple myeloma, cervical cancer, endometrial cancer, uterine cancer, ovarian cancer, ureteral cancer, bladder cancer, penile cancer, testicular cancer, pancreatic cancer, prostate cancer, kidney cancer, soft tissue cancer and thyroid cancer. Preferably, the cancer is lung cancer, pancreatic cancer, colorectal cancer, endometrial cancer, ovarian cancer or prostate cancer.

[0197] VI. Methods and Engineered Cells for Improving Cell Therapy

[0198] The present invention also provides methods and engineered cells for improving cell therapy.

[0199] Natural CD8+ cells express the CD8 molecule, a type I transmembrane glycoprotein expressed on the cell surface as a homodimer composed of two CD8a chains (herein referred to as "CD8aa") and / or as a heterodimer composed of one CD8a chain and one CD8b chain (herein referred to as "CD8ab").

[0200] In some embodiments, the present invention co-expresses exogenous TCR and CD8aa molecules in T cells. Based on the non-viral gene editing method of CRISPR / Cas9 technology, the nucleic acid encoding the nwTCR of the present invention and the nucleic acid encoding the CD8a chain are used to perform gene editing on CD8+T cells / CD4+T cells, indicating that the co-expression of exogenous nwTCR and CD8aa molecules in CD8+T cells / CD4+T cells can enhance the binding of TCR-T cells to pMHC molecules. When exogenous nwTCR and CD8aa molecules are co-expressed in CD8+T cells, due to the increase in CD8aa molecules that can be used by exogenous nwTCR in CD8+T cells, it is expected to improve the TCR-specific cytotoxicity (including its continuous killing ability) and in vivo anti-tumor function of CD8+T cells. When exogenous nwTCR and CD8aa molecules are co-expressed in CD4+ T cells, accompanied by the expression of endogenous CD4 molecules, CD4+ T cells exhibit a hybrid phenotype, which is expected to recognize antigens with an affinity similar to that of natural CD8+ T cells and kill target cells, exhibiting cytotoxic effector function; while retaining the natural helper function of CD4+ T cells.

[0201] In some embodiments, the present invention co-expresses exogenous TCR and CD8ab molecules in T cells. Based on the non-viral gene editing method of CRISPR / Cas9 technology, the nucleic acid encoding the nwTCR of the present invention, the nucleic acid encoding the CD8a chain and the nucleic acid encoding the CD8b chain are used to perform gene editing on CD8+T cells / CD4+T cells, indicating that the co-expression of exogenous nwTCR and CD8ab molecules in CD8+T cells / CD4+T cells can enhance the binding of TCR-T cells to pMHC molecules. When exogenous nwTCR and CD8ab molecules are co-expressed in CD8+T cells, due to the increase in CD8ab molecules that can be used by exogenous nwTCR in CD8+T cells, it is expected to improve the TCR-specific cytotoxicity (including its continuous killing ability) and in vivo anti-tumor function of CD8+T cells. When exogenous nwTCR and CD8ab molecules are co-expressed in CD4+ T cells, accompanied by the expression of endogenous CD4 molecules, CD4+ T cells exhibit a hybrid phenotype, which is expected to recognize antigens with an affinity similar to that of natural CD8+ T cells and kill target cells, exhibiting cytotoxic effector function; while retaining the natural helper function of CD4+ T cells.

[0202] Therefore, when CD8aa and / or CD8ab molecules are co-expressed with TCR genes in CD8+ and CD4+ T cells, they have a beneficial effect on the function of CD8+ and CD4+ T cells. CD4+ T cells can be reprogrammed into multifunctional hybrid T cells through MHC class I TCR and CD8 molecules, which have both cytotoxic effector functions and natural helper functions.

[0203] The following examples are described to assist in understanding the present invention, but are not intended to, and should not be interpreted in any way as, limiting the scope of protection of the present invention.

[0204] Example

[0205] The invention generally described herein will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the scope of the invention. These examples are not intended to represent that the experiments below are all or only the experiments performed.

[0206] Example 1. Generation and cloning of T cells and TCRs that recognize KRAS_G12D mutant antigens

[0207] Chemically synthesized sequences adjacent to the KRAS amino acid sequence after the G to D mutation at position 12, i.e., a short peptide VVVGADGVGK (SEQ ID NO: 239) of the KRAS amino acid sequence from positions 7 to 16 (also referred to herein as "KRAS_G12D_7-16 peptide"); and a short peptide VVGADGVGK (SEQ ID NO: 240) of the KRAS amino acid sequence from positions 8 to 16 (also referred to herein as "KRAS_G12D_8-16 peptide").

[0208] Dendritic cells (DCs) from a cancer patient with the HLA-A*11:01 genotype and expressing the KRAS_G12D mutant antigen were pulsed in vitro with KRAS_G12D_7-16 and KRAS_G12D_8-16 peptides resuspended in DMSO, respectively, and co-cultured with CD8+ T cells isolated from the patient's peripheral blood for 10 days. As a negative control, DCs from the patient were pulsed in vitro with DMSO and co-cultured with CD8+ T cells isolated from the patient's peripheral blood for 10 days.

[0209] The culture supernatant was then assayed for IFN-γ release and CD137 expression on CD8+ T cells, and reactive T cells that released IFN-γ and expressed CD137 were sorted. Flow cytometry-based staining was used to assess the binding of these sorted reactive T cells, which released IFN-γ and expressed CD137, to peptide-MHC (HLA-A*11:01) tetramers (VVVGADGVGK-HLA-A*11:01 and VVGADGVGK-HLA-A*11:01); tetramers containing an irrelevant peptide served as negative controls. Flow cytometric staining of T cells with both tetramers (VVVGADGVGK-HLA-A*11:01 and VVGADGVGK-HLA-A*11:01) increased confidence in T cell specificity.

[0210] Ten specific T cell clones with the desired high affinity were screened. The antigen-specific T cell receptors (TCRs) on these 10 T cell clones that specifically bind to the KRAS_G12D_7-16 epitope peptide and the KRAS_G12D_8-16 epitope peptide were named nwTCR-0884-2, nwTCR-2462, nwTCR-2918, nwTCR-2985, nwTCR-3178, nwTCR-4412, nwTCR-4536, nwTCR-5963, nwTCR-6670-2, and nwTCR-6673, respectively. The amino acid sequences of the paired TCR α and β chains on these 10 T cell clones were determined on a single-cell basis using high-throughput paired TCR sequencing.

[0211] Because multiple nucleotides can translate into the same amino acid, and codon usage frequencies vary across organisms, we optimized the codons encoding the amino acid sequences of the TCR α and β chains to increase TCR expression in eukaryotic cells. We obtained the codon-optimized nucleotide sequences of 10 TCRs that specifically recognize the KRAS_G12D_7-16 and KRAS_G12D_8-16 epitope peptides.

[0212] Table 1A and Table 1B list the amino acid sequence information and nucleotide sequence information of the α chain and β chain of the 10 TCRs expressed by the clonal T cell lines generated by sequencing, respectively.

[0213] Table 1A. Amino acid and nucleotide sequences of KRAS_G12D mutant antigen-specific TCRα chain

[0214] Table 1B. Amino acid and nucleotide sequences of KRAS_G12D mutant antigen-specific TCR β chain

[0215] Example 2. Preparation of KRAS_G12D mutant antigen-specific TCR-T cells from T cells

[0216] This example describes the preparation and characterization of KRAS_G12D mutant antigen-specific TCR-T cells by knocking out the TCR gene in primary T cells using CRISPR / Cas9 technology and knocking in the KRAS_G12D mutant antigen-specific TCR gene using homologous recombination technology.

[0217] 2.1 T cell sorting and activation

[0218] A mixture of CD4 T cells and CD8 T cells (also referred to as "CD4 / CD8 T cells" in this article) was enriched and sorted from peripheral blood mononuclear cells (PBMC, purchased from Shanghai Saili Biotechnology Co., Ltd., Donor: S2001095). The enriched and sorted CD4 / CD8 T cells were aliquoted and frozen (5x10 6 cells / cryotube) for future use.

[0219] Thaw the cryovials as needed and activate the sorted T cells by adding a 1:100 dilution of the T cell activator Miltenyi T cell TransACT (Miltenyi catalog number: 130-111-160) to T cell culture medium (e.g., RPMI 1640, FBS, L-glutamine, non-essential amino acids, sodium pyruvate, HEPES buffer, 2-mercaptoethanol, and optionally IL2). Culture the cells for approximately 48 hours (2 days) before using for electroporation transfection.

[0220] 2.2 Targeting strategy and preparation of targeting vectors

[0221] The gRNAs used were gRNA002 and gRNA004 (see Table 2). The target site of gRNA002 was located in exon 1 of the endogenous TRAC gene (Figure 1A); the target site of gRNA004 was located in exon 1 of the endogenous TRBC1 and TRBC2 genes (Figure 1B). Cas9 enzyme was purchased from GenScript Biotech, catalog number: Z03469.

[0222] Table 2. sgRNAs corresponding to TRAC and TRBC genes

[0223] The backbone of the targeting vector (also known as the HDR vector) is the pUC57-HA vector, which is an optimized vector based on the pUC57-Simple vector. It only retains the Ori and Amp sequences of the pUC57-Simple vector, then replaces the Amp sequence with the Kana sequence, and adds the left and right homologous arm (HA) sequences of the TRAC site (about 800bp). The sequence to be knocked in (KI) can be constructed between the left and right HAs. The KI sequence construct structure of nwTCR includes: 2A or its variant-SP-TCR β-2A or its variant-SP-TRAV-TRAJ, where 2A or its variant is a ribosomal skipping element; SP is a signal peptide; 4 synonymous mutant bases are introduced into the TRBC gene in the targeting vector, which respectively mutate the coding nucleotide of TRBCS77 from AGC to TCC and the coding nucleotide of S78 from AGC to TCC. For nwTCR-4536, the nucleotide sequence of its KI construct is shown in SEQ ID NO: 260, wherein: 2A or its variant encodes any one of the amino acid sequences shown in SEQ ID NO: 246, 248, 250 and SEQ ID NO: 252; SP encodes the signal peptide sequence shown in SEQ ID NO: 244; TCR β is the nucleotide sequence of the complete TRB (TCR β chain) of nwTCR-4536 (SEQ ID NO: 232), wherein the encoding nucleotide of TRBC S77 is mutated from AGC to TCC and the encoding nucleotide of S78 is mutated from AGC to TCC, thereby introducing four synonymous mutant bases into the TRBC gene; TRAV is the nucleotide sequence of the nwTCR-4536 TRAV gene (SEQ ID NO: 56); and TRAJ is the nucleotide sequence of the nwTCR-4536 TRAJ gene (SEQ ID NO: 58). Similarly, KI sequence constructs of nwTCR-0884-2, nwTCR-2462, nwTCR-2918, nwTCR-2985, nwTCR-3178, nwTCR-4412, nwTCR-5963, nwTCR-6670-2, and nwTCR-6673 were prepared.

[0224] 2.3. Electroporation transfection (Day 2)

[0225] The sgRNA of Example 2.2 was thoroughly mixed with the Cas9 enzyme and incubated at room temperature for 10 min to prepare RNP.

[0226] The targeting vector containing the KI TCR sequence prepared in Example 2.2 was thoroughly mixed with the incubated RNP and the specified concentration of T cells prepared in Example 2.1 (about 1.25E6 T cells / electroporation tube) to knock out (KO) endogenous TCR and knock in (KI) exogenous TCR.

[0227] The mixed solution was loaded into an electroporation transfection instrument (Celetrix; catalog number: CTX-1500A LE) for cell electroporation transfection. The electroporation transfection conditions were 480-560 V, 20 ms.

[0228] After electroporation, the cells were allowed to rest for 15 minutes before the electroporated T cells were removed and transferred to pre-warmed culture medium (ImmunoCult TM -XF T Cell Expansion Medium, Stemcell catalog number: 10981). After culturing the cells for 5 days, flow cytometry analysis was performed on Day 7.

[0229] 2.4. Flow Cytometric Analysis of nwTCR Expression (Day 7)

[0230] The cell suspension obtained in Example 2.3 was thoroughly mixed, the cells were counted, and an appropriate amount of cells were collected for staining with two labeled peptide-MHC (HLA-A*11:01) tetramers (VVVGADGVGK-HLA-A*11:01 tetramer and VVGADGVGK-HLA-A*11:01 tetramer, respectively, which may also be referred to as pMHC).

[0231] Prepare the peptide-MHC (HLA-A*11:01) tetramer staining solution and LIVE / DEAD containing the two markers in advance. TM Fixable Near-IR was purchased from Invitrogen, catalog number: L10119; CD4-FITC was purchased from BioLegend, catalog number: 357406; CD8-PerCP-cy5.5 was purchased from BioLegend, catalog number: 344710; anti-human TCRα / β-BV510 antibody was purchased from BioLegend, catalog number: 306734.

[0232] The collected cells were stained with the two labeled peptide-MHC (HLA-A*11:01) tetramer stains, washed, and characterized by flow cytometry.

[0233] Figures 3A-3J illustrate the staining results of different nwTCRs expressed on CD4+ and CD8+ T cells after electroporation transfection of CD4+ and CD8+ T cells with labeled pMHC tetramers (labeled VVVGADGVGK-HLA-A*11:01 tetramers). It can be seen that Day 7 cells are divided into three groups:

[0234] 1) Wild-type T cells that have not undergone gene editing (Q3);

[0235] 2) Completed KO cells with endogenous TCR knockout (Q4);

[0236] 3) KO and KI were completed, and the cell population expressing nwTCR was identified (Q2).

[0237] A schematic diagram of the results of flow cytometry detection of TCR gene editing efficiency is shown in Figure 2.

[0238] As can be seen from Figures 3A-3J, CD8+ T cells with each nwTCR knocked in can bind to peptide-MHC complex (pMHC) tetramers; there are significant differences in the binding of CD4+ T cells with each TCR knocked in to peptide-MHC complex (pMHC) tetramers. This is because, as shown in Example 1, each nwTCR in the present invention is obtained by screening using CD8+ T cells. Therefore, when each nwTCR is knocked into CD8+ T cells, the CD8+ T cells expressing each nwTCR can specifically bind to the peptide-MHC complex (pMHC) tetramer; however, when each nwTCR is knocked into CD4+ T cells, the CD4+ T cells expressing each nwTCR have the following situation: Generally, when the affinity of the nwTCR and MHC is strong enough, the TCR can bind to the MHC molecule without the assistance of the CD8 molecule. For example, it is believed that nwTCR-5963 has a stronger binding ability to the MHC molecule than nwTCR-4536. Therefore, they will have different staining of pMHC tetramers on CD4+ T cells. Therefore, after each nwTCR is edited into CD4+ T cells and expressed, the nwTCR with strong affinity can still bind to MHC class I antigens, while the nwTCR with weak affinity also has a weak ability to bind to MHC class I antigens.

[0239] Example 3. In vitro functional study of KRAS_G12D mutant antigen-specific TCR-T cells

[0240] On Day 7, the KRAS_G12D mutant antigen-specific TCR-T cells of Example 2 were selectively activated using TransACT activator (Miltenyi catalog number: 130-111-160). TCR-T cells were cultured until Day 14. On Day 14, in vitro functional studies were performed on each TCR-T cell.

[0241] 3.1 Affinity detection of TCR-T cells binding to peptides

[0242] The TCR-T cell affinity detection method is implemented as follows. Antigen presenting cells (T2 cells or K562 cells overexpressing HLA-A*11:01) are collected, the cells are counted, and an appropriate amount of culture medium (such as RPMI-1640 culture medium, purchased from Gibco, catalog number: 22400089; FBS, purchased from Gibco, catalog number: 10099141C) is added to resuspend the cells to a cell density of 1E6 cells / mL, and 1 mL of the cell suspension is added to each well of a 24-well plate. The polypeptide solution to be tested (the polypeptide is the KRAS_G12D_7-16 peptide shown in SEQ ID NO: 239 and / or the KRAS_G12D_8-16 peptide shown in SEQ ID NO: 240) is gradiently diluted to 10 -12 -10 -5 M, and add 10ul of the diluted peptide solution to the corresponding wells of a 24-well plate. After incubation in an incubator (37°C, 5% CO2) for 2 hours, the incubated antigen-presenting cells were collected and washed, and 100ul of 1E6 / mL antigen-presenting cells were transferred to the corresponding wells of a 96-well plate. The nwTCR-T cells to be tested were collected, and an appropriate amount of T cell culture medium (purchased from STEMCELL, catalog number: 10981) was added to a cell density of 1E6 / mL. 100ul of cell suspension was added to the corresponding wells of a 96-well plate. After each nwTCR-T cell was co-cultured with the antigen-presenting cells (37°C, 5% CO2) for 16 hours, the cell supernatant was collected and the IFN-γ concentration was detected using an ELISA kit (purchased from Biolegend, catalog number: 430104). The binding affinity of T cells expressing each nwTCR to the short peptide represented by SEQ ID NO: 239 or SEQ ID NO: 240 presented by HLA-A*11:01 was detected by measuring the release level of IFN-γ.

[0243] Figures 4A and 4B show the results of binding affinity detection experiments of T cells expressing each nwTCR for the short peptide shown in SEQ ID NO: 239 or SEQ ID NO: 240 presented by HLA-A*11:01, wherein Figure 4A shows the binding affinity to the short peptide shown in SEQ ID NO: 239; Figure 4B shows the binding affinity to the short peptide shown in SEQ ID NO: 240.

[0244] The results in Figures 4A and 4B show that after T2 cells presenting peptide-MHC complexes were co-incubated with T cells expressing each nwTCR, specific binding of T cells expressing each nwTCR to the peptide-MHC complexes was detected, leading to IFN-γ release. Furthermore, considering that both 9-mer and 10-mer peptides can be presented by MHC when presenting peptides, the experimental results using 9-mer and 10-mer peptides in Figures 4A and 4B indicate that T cells expressing each nwTCR can specifically bind to pMHC complexes presenting 9-mer and 10-mer peptides, respectively.

[0245] 3.2 Detection of target cell killing by each TCR-T cell by real-time fluorescence imaging

[0246] Target cells (CL-40 cell line (HLA-A*11:01+, KRAS G12D+; purchased from Nanjing Kebai Biotechnology Co., Ltd.) and SNU-601 cell line (HLA-A*11:01+, KRAS G12D+; purchased from Nanjing Kebai Biotechnology Co., Ltd.) were collected and counted. After resuspending each target cell in target cell culture medium (RPMI-1640 medium, purchased from Gibco, catalog number: 22400089, FBS, purchased from Gibco, catalog number: 10099141C) to a cell density of 1E6 cells / mL, 100 μL of 1E6 / mL antigen-presenting cells was transferred to a well of a 96-well plate.

[0247] Collect the nwTCR-T cells to be tested, add an appropriate amount of T cell culture medium (purchased from STEMCELL, catalog number: 10981) to a cell density of 1E6 / mL, add 100 μL of the nwTCR-T cell suspension to be tested to the wells of a 96-well plate, and mix with the target cells in the wells of the 96-well plate. Add 1 μL of ethidium bromide (1 mg / mL) solution to the cell mixture (i.e., ethidium bromide is added at 0 h), mix well, and place the cell culture plate in a real-time fluorescence imaging system (BioTek Lionheart) for cell killing characterization experiments. Target cells specifically recognized by T cells will be stained with ethidium bromide after entering a state of apoptosis, showing a red fluorescent signal.

[0248] Figures 5A-5C show the real-time fluorescence imaging results of T cells expressing nwTCR-0884-2, nwTCR-2462, or nwTCR-2918 specifically killing the CL-40 (KRAS G12D+) cell line (0 hours when the cells were co-incubated, and the killed target cells showed red fluorescence signals after 18 hours). "Blank" in the figure indicates that there are only target cells and no T cells expressing any nwTCR were added.

[0249] Figures 5D-5F show the real-time fluorescence imaging results of T cells expressing nwTCR-2985, nwTCR-4412, nwTCR-4536, nwTCR-5963, nwTCR-3178, nwTCR-6670-2, and nwTCR-6673 specifically killing the SNU-601 (KRAS G12D+) cell line (0 hours when the cells were co-incubated, and the killed target cells showed red fluorescence signals after 18 hours). "Blank" in the figure indicates that there are only target cells and no T cells expressing any nwTCR were added.

[0250] 3.3 Detection of target cell killing by each TCR-T cell using real-time cell analyzer

[0251] Target CL-40 and SNU-601 cell lines were collected and counted. After cell count, each target cell was resuspended in target cell culture medium (RPMI-1640 medium, purchased from Gibco, catalog number: 22400089, FBS, purchased from Gibco, catalog number: 10099141C) to a cell density of 1E6 cells / mL. An E-plate (available from Agilent, catalog number: 300600890) was prepared, and 100 μL of the mixed target cell suspension was added to the corresponding well. The plate was then placed in an RTCA real-time cell analyzer (available from Agilent, model: xCELLigence RTCA DP) for overnight detection.

[0252] Collect each TCR-T cell to be tested, count the cells, add an appropriate amount of T cell culture medium (purchased from STEMCELL, catalog number: 10981) to resuspend the cells. Take out the E-plate inoculated with target cells, add the T cell suspension, put the E-Plate back into the RTCA analyzer for detection, and obtain the cell index for 72 hours. Each independent experiment was performed three times. The interval slope was automatically calculated using RTCA software to evaluate the rate of change of the cell index. In order to demonstrate the effect of the treatment, the cell index was standardized to an equal value at the standardized time point. The in vitro killing results of target cells by each TCR-T cell are shown in Figures 6A-6F.

[0253] Figures 6A-6C show real-time analysis of CL-40 cell killing by T cells expressing various nwTCRs. The results demonstrate that gene-edited T cells have a specific killing effect on CL-40 cells. "Blank" in Figures 6A-6C indicates that only CL-40 cells were present, without the addition of T cells expressing any nwTCR.

[0254] Figures 6D-6F show real-time analysis of T cells expressing various nwTCRs killing SNU-601 cells. The results demonstrate that gene-edited T cells have a specific killing effect on SNU-601 cells. "Blank" in Figures 6D-6F indicates only SNU-601 cells, without the addition of T cells expressing any nwTCR.

[0255] Therefore, each TCR-T cell showed significant in vitro killing effect on HLA-A*11:01+, KRAS G12D+ target cells.

[0256] Example 4. Co-expression of exogenous TCR and CD8 molecules

[0257] This example describes the use of CRISPR / Cas9 and homologous recombination technology to co-express exogenous TCR and CD8 molecules on the surface of CD4+ T cells, thereby redirecting CD4+ T cells. Furthermore, this gene editing method can enhance the binding of TCR-T cells to pMHC molecules.

[0258] 4.1 T cell sorting and activation

[0259] T cells can be obtained commercially (eg, frozen human peripheral blood CD4+CD45RA+ T cells, Stem Cell Technology, catalog number 70029) or prepared from leukocyte aliquots (Day 0).

[0260] For the preparation of T cells from leukocyte aliquots, CD4 / CD8 T cells were enriched and sorted from leukocyte aliquots. The enriched and sorted CD4 / CD8 T cells were aliquoted and frozen (5x10 6 cells / cryotube) for future use.

[0261] 4.2 Targeting strategy and targeting vector preparation

[0262] The TCR targeting strategy and targeting vector are the same as those in Example 2.2 above.

[0263] When the TCR sequence used was nwTCR-2985, as can be seen from Figure 8A, the binding of CD8+ T cells expressing nwTCR-2985 to the pMHC molecule of the KRAS G12D amino acid sequence from position 7 to 16 (KRAS_G12D_7-16 peptide) was stronger than the binding of CD4+ T cells expressing nwTCR-2985 to the pMHC molecule presenting the KRAS_G12D_7-16 peptide.

[0264] When the TCR sequence used was nwTCR-4536, as shown in Figure 8C, CD8+ T cells expressing nwTCR-4536 could specifically bind to the pMHC molecules of the KRAS G12D amino acid sequence from position 7 to 16 (KRAS_G12D_7-16 peptide), but CD4+ T cells expressing nwTCR-4536 hardly bound to the pMHC molecules presenting the KRAS_G12D_7-16 peptide due to the lack of CD8 molecule assistance.

[0265] When the TCR sequence used was nwTCR-6670-2, it can be seen from Figure 8E that the binding of CD8+ T cells expressing nwTCR-6670-2 to the pMHC molecules of the KRAS G12D amino acid sequence from positions 7 to 16 (KRAS_G12D_7-16 peptide) was stronger than the binding of CD4+ T cells expressing nwTCR-6670-2 to the pMHC molecules presenting the KRAS_G12D_7-16 peptide.

[0266] To redirect CD4+ T cells, the following constructs were further constructed and inserted into the pUC57-HA targeting vector (also known as the HDR vector):

[0267] The KI construct of nwTCR-4536-CD8ab is: 2A or its variant-CD8a-2A or its variant-CD8b-2A or its variant-SP-TCR β-2A or its variant-SP-TRAV-TRAJ, and its amino acid sequence and nucleotide sequence are shown in SEQ ID NO: 261 and SEQ ID NO: 262, respectively.

[0268] The KI construct of nwTCR-2985-CD8ab is: 2A or its variant-CD8a-2A or its variant-CD8b-2A or its variant-SP-TCR β-2A or its variant-SP-TRAV-TRAJ, and its construction method is similar to the construction method of the KI construct of nwTCR-4536-CD8ab, but wherein TCR β, TRAV, TRAJ use the TCR β, TRAV, TRAJ of nwTCR-2985 given in the sequence list.

[0269] The K construct of nwTCR-6670-2-CD8ab is: 2A or its variant-CD8a-2A or its variant-CD8b-2A or its variant-SP-TCR β-2A or its variant-SP-TRAV-TRAJ. Its construction method is similar to that of the KI construct of nwTCR-4536-CD8ab, but the TCR β, TRAV, and TRAJ used are those of nwTCR-6670-2 given in the sequence listing.

[0270] The targeting strategy diagram of nwTCR-CD8ab is shown in Figure 7 .

[0271] 4.3. Electroporation transfection (Day 2)

[0272] The sgRNA designed and synthesized in Example 2.2 was thoroughly mixed with the Cas9 enzyme and incubated at room temperature for 10 min to prepare RNP.

[0273] The targeting vectors prepared in Example 4.2, i.e., the targeting vectors used to knock in nwTCR-2985-CD8ab, nwTCR-4536-CD8ab and nwTCR-6670-2-CD8ab, respectively, were thoroughly mixed with the incubated RNPs and the specified concentration of T cells (1.25E6 / electric shock tube) prepared in Example 4.1 to knock out (KO) endogenous TCR and knock in (KI) exogenous TCR and CD8ab.

[0274] The mixed solution was loaded into an electroporation transfection instrument (Celetrix; catalog number: CTX-1500ALE) for cell electroporation transfection. The electroporation transfection conditions were 480-560 V, 20 ms.

[0275] After the electroporation transfection, the cells were allowed to stand for 15 minutes before being taken out and transferred to pre-warmed culture medium (ImmunoCult TM -XF T Cell Expansion Medium, Stemcell catalog number: 10981). After culturing the cells for 5 days, flow cytometry analysis was performed on Day 7.

[0276] Flow Cytometric Analysis of nwTCR Expression (Day 7)

[0277] The cell suspension obtained in Example 4.3 was thoroughly mixed, and an appropriate amount of cells was collected after cell counting and stained with the labeled peptide-MHC (HLA-A11:01) tetramer (VVVGADGVGK-HLA-A11:01).

[0278] Prepare the peptide-MHC tetramer staining solution and LIVE / DEAD containing specific antigen in advance. TM Fixable Near-IR was purchased from Invitrogen, catalog number: L10119; CD4-FITC was purchased from BioLegend, catalog number: 357406; CD8-PerCP-cy5.5 was purchased from BioLegend, catalog number: 344710; anti-human TCRα / β-BV510 antibody was purchased from BioLegend, catalog number: 306734.

[0279] The collected cells were stained with a labeled peptide-MHC tetramer stain, washed, and characterized by flow cytometry. The results are shown below. In the flow cytometric graph, cells on Day 7 were divided into three groups:

[0280] 1) Wild-type T cells that have not undergone gene editing (Q3);

[0281] 2) Completed KO cells with endogenous TCR knockout (Q4);

[0282] 3) KO and KI were completed, and the cell population expressing nwTCR was identified (Q2).

[0283] As can be seen from Figures 8B, 8D and 8F, when the CD8ab molecule is co-edited with nwTCR-2985, nwTCR-4536 or nwTCR-6670-2 into primary T cells (the primary T cells contain CD4+T cells and CD8+T cells), flow cytometry characterization shows that the proportion of CD8+CD4+T cells increases. These cells are CD4+T cells that express exogenous CD8 molecules. These CD4+T cells can specifically bind to the labeled peptide-MHC (HLA-A11:01) tetramer (VVVGADGVGK-HLA-A11:01) with the assistance of exogenous CD8 molecules.

[0284] In this example, the selection of pMHC tetramers that hardly bind to CD4+ T cells expressing nwTCR-4536 after gene editing is intended to show that even for such nwTCR, by introducing CD8 molecules into CD4 T cells, the binding of CD4 T cells to pMHC molecules after gene editing of nwTCR can be enhanced. Therefore, the specific nwTCR used in this example can be replaced with any other nwTCR of the present invention and can achieve the effect of enhancing the binding of CD4 T cells to pMHC molecules after gene editing of nwTCR. This example also further exemplifies the results after nwTCR-2985 and nwTCR-6670-2 were co-edited with CD8ab molecules into primary T cells.

[0285] At the same time, thanks to the ability of this method to redirect CD4+ T cells, the overall gene editing efficiency of T cells is improved (Table 3), where the gene editing efficiency (GE%) is calculated as follows: the percentage of cells expressing only CD8+ in the viable T cell population × the percentage of cells expressing only CD8+ in tetramer staining + the percentage of cells expressing only CD4+ in the viable T cell population × the percentage of cells expressing only CD4+ in tetramer staining + the percentage of cells expressing both CD8+ and CD4+ in the viable T cell population × the percentage of cells expressing both CD8+ and CD4+ in tetramer staining.

[0286] Table 3. Gene editing efficiency of each cell population in nwTCR and nwTCR-CD8ab cells

[0287] While the exemplary embodiments of the present invention have been described above, it should be understood by those skilled in the art that these disclosures are merely exemplary and that various other substitutions, adaptations, and modifications may be made within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments listed herein.

[0288] Sequence Listing

Claims

1. An isolated or purified T cell receptor, also referred to as TCR, characterized in that It specifically binds to the KRAS_G12D mutant antigen, the TCR comprises an α chain and a β chain, wherein the α chain and the β chain each comprise three complementarity determining regions, which are also referred to as CDRs, and the amino acid sequence of CDR3 of the α chain is selected from SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30 and variants having 1 or 2 amino acid residues changed with the sequence, and the amino acid sequence of CDR3 of the β chain is selected from SEQ ID NO: 113, 116, 119, 122, 125, 128, 131, 134, 137, 140 and variants having 1 or 2 amino acid residues changed with the sequence.

2. The TCR according to claim 1, wherein: 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 set forth in SEQ ID NO: 3, or a variant thereof having 1 or 2 amino acid residues altered therefrom; and the β chain CDR3 amino acid sequence set forth in SEQ ID NO: 113, or a variant thereof having 1 or 2 amino acid residues altered therefrom; (ii) the α-chain CDR3 amino acid sequence set forth in SEQ ID NO: 6, or a variant thereof having 1 or 2 amino acid residues altered therefrom; and the β-chain CDR3 amino acid sequence set forth in SEQ ID NO: 116, or a variant thereof having 1 or 2 amino acid residues altered therefrom; (iii) the α chain CDR3 amino acid sequence set forth in SEQ ID NO: 9, or a variant thereof having 1 or 2 amino acid residues altered therefrom; and the β chain CDR3 amino acid sequence set forth in SEQ ID NO: 119, or a variant thereof having 1 or 2 amino acid residues altered therefrom; (iv) the α chain CDR3 amino acid sequence set forth in SEQ ID NO: 12, or a variant thereof having 1 or 2 amino acid residues altered therefrom; and the β chain CDR3 amino acid sequence set forth in SEQ ID NO: 122, or a variant thereof having 1 or 2 amino acid residues altered therefrom; (v) the α chain CDR3 amino acid sequence set forth in SEQ ID NO: 15, or a variant thereof having 1 or 2 amino acid residues altered therefrom; and the β chain CDR3 amino acid sequence set forth in SEQ ID NO: 125, or a variant thereof having 1 or 2 amino acid residues altered therefrom; (vi) the α chain CDR3 amino acid sequence set forth in SEQ ID NO: 18, or a variant thereof having 1 or 2 amino acid residues altered therefrom; and the β chain CDR3 amino acid sequence set forth in SEQ ID NO: 128, or a variant thereof having 1 or 2 amino acid residues altered therefrom; (vii) the α chain CDR3 amino acid sequence set forth in SEQ ID NO:21, or a variant thereof having 1 or 2 amino acid residues altered therefrom; and the β chain CDR3 amino acid sequence set forth in SEQ ID NO:131, or a variant thereof having 1 or 2 amino acid residues altered therefrom; (viii) the α chain CDR3 amino acid sequence set forth in SEQ ID NO:24, or a variant thereof having 1 or 2 amino acid residues altered therefrom; and the β chain CDR3 amino acid sequence set forth in SEQ ID NO:134, or a variant thereof having 1 or 2 amino acid residues altered therefrom; (ix) the α chain CDR3 amino acid sequence set forth in SEQ ID NO:27, or a variant thereof having 1 or 2 amino acid residues altered therefrom; and the β chain CDR3 amino acid sequence set forth in SEQ ID NO:137, or a variant thereof having 1 or 2 amino acid residues altered therefrom; (x) the α chain CDR3 amino acid sequence set forth in SEQ ID NO:30, or a variant thereof having 1 or 2 amino acid residues altered therefrom; and the β chain CDR3 amino acid sequence set forth in SEQ ID NO:140, or a variant thereof having 1 or 2 amino acid residues altered therefrom; Preferably, 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; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 113; (ii) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 6; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 116; (iii) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 9; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 119; (iv) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 12; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 122; (v) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 15; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 125; (vi) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 18; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 128; (vii) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 21; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 131; (viii) the α chain CDR3 amino acid sequence set forth in SEQ ID NO:24; and the β chain CDR3 amino acid sequence set forth in SEQ ID NO:134; (ix) the α chain CDR3 amino acid sequence shown in SEQ ID NO: 27; and the β chain CDR3 amino acid sequence shown in SEQ ID NO: 137; (x) the α chain CDR3 amino acid sequence shown in SEQ ID NO:30; and the β chain CDR3 amino acid sequence shown in SEQ ID NO:

140.

3. The TCR according to claim 2, wherein: The amino acid sequences of the three CDRs contained in the α chain and the amino acid sequences of the three CDRs contained in the β chain are: (i) the α-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, or variants thereof having 1 or 2 amino acid residues changed therefrom, respectively; and the β-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 111, 112, and 113, or variants thereof having 1 or 2 amino acid residues changed therefrom, respectively; (ii) the α-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, or variants thereof having 1 or 2 amino acid residues altered therefrom, respectively; and the β-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 114, 115, and 116, or variants thereof having 1 or 2 amino acid residues altered therefrom, respectively; (iii) the α-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, or variants thereof having 1 or 2 amino acid residues altered therefrom, respectively; and the β-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 117, 118, and 119, or variants thereof having 1 or 2 amino acid residues altered therefrom, respectively; (iv) the α chain CDR1, CDR2, CDR3 amino acid sequences set forth in SEQ ID NOs: 10, 11, 12, or variants thereof having 1 or 2 amino acid residues altered therefrom, respectively; and the β chain CDR1, CDR2, CDR3 amino acid sequences set forth in SEQ ID NOs: 120, 121, 122, or variants thereof having 1 or 2 amino acid residues altered therefrom, respectively; (v) the α chain CDR1, CDR2, CDR3 amino acid sequences set forth in SEQ ID NOs: 13, 14, 15, or variants thereof having 1 or 2 amino acid residues altered therefrom, respectively; and the β chain CDR1, CDR2, CDR3 amino acid sequences set forth in SEQ ID NOs: 123, 124, 125, or variants thereof having 1 or 2 amino acid residues altered therefrom, respectively; (vi) the α-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, or variants thereof having 1 or 2 amino acid residues altered therefrom, respectively; and the β-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 126, 127, and 128, or variants thereof having 1 or 2 amino acid residues altered therefrom, respectively; (vii) the α-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, or variants thereof having 1 or 2 amino acid residues altered therefrom, respectively; and the β-chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 129, 130, and 131, or variants thereof having 1 or 2 amino acid residues altered therefrom, respectively; (viii) the α chain CDR1, CDR2, CDR3 amino acid sequences set forth in SEQ ID NOs: 22, 23, 24, or variants thereof having 1 or 2 amino acid residues altered therefrom, respectively; and the β chain CDR1, CDR2, CDR3 amino acid sequences set forth in SEQ ID NOs: 132, 133, 134, or variants thereof having 1 or 2 amino acid residues altered therefrom, respectively; (ix) the α chain CDR1, CDR2, CDR3 amino acid sequences set forth in SEQ ID NOs: 25, 26, 27, or variants thereof having 1 or 2 amino acid residues altered therefrom, respectively; and the β chain CDR1, CDR2, CDR3 amino acid sequences set forth in SEQ ID NOs: 135, 136, 137, or variants thereof having 1 or 2 amino acid residues altered therefrom, respectively; (x) the α chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, or variants thereof having 1 or 2 amino acid residues altered therefrom, respectively; and the β chain CDR1, CDR2, and CDR3 amino acid sequences set forth in SEQ ID NOs: 138, 139, and 140, or variants thereof having 1 or 2 amino acid residues altered therefrom, respectively; Preferably, the amino acid sequence of the three CDRs contained in the α chain and the amino acid sequence of the three CDRs contained in the β chain are: (i) the α-chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 1, 2, and 3; and the β-chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 111, 112, and 113; (ii) the α chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 4, 5, and 6; and the β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 114, 115, and 116; (iii) the α chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 7, 8, and 9; and the β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 117, 118, and 119; (iv) the α-chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 10, 11, and 12; and the β-chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 120, 121, and 122; (v) the α chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 13, 14, and 15; and the β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 123, 124, and 125; (vi) the α chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 16, 17, and 18; and the β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 126, 127, and 128; (vii) the α chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 19, 20, and 21; and the β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 129, 130, and 131; (viii) the α chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 22, 23, and 24; and the β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 132, 133, and 134; (ix) the α chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 25, 26, and 27; and the β chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 135, 136, and 137; (x) the α-chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 28, 29, and 30; and the β-chain CDR1, CDR2, and CDR3 amino acid sequences shown in SEQ ID NOs: 138, 139, and 140.

4. The TCR according to any one of claims 1 to 3, wherein: The TCR further comprises a constant region, for example, the constant region is a mouse constant region; Preferably, the TCR comprises an alpha chain sequence as set forth in SEQ ID NO: 91, 93, 95, 97, 99, 101, 103, 105, 107 or 109, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereof; and a beta chain sequence as set forth in SEQ ID NO: 219, 221, 223, 225, 227, 229, 231, 233, 235 or 237, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereof; More preferably, the TCR comprises the α chain sequence shown in SEQ ID NO: 91 and the β chain sequence shown in SEQ ID NO: 219; The TCR comprises an α chain sequence shown in SEQ ID NO: 93 and a β chain sequence shown in SEQ ID NO: 221; The TCR comprises an α chain sequence shown in SEQ ID NO: 95 and a β chain sequence shown in SEQ ID NO: 223; The TCR comprises an α chain sequence shown in SEQ ID NO: 97 and a β chain sequence shown in SEQ ID NO: 225; The TCR comprises an α chain sequence shown in SEQ ID NO: 99 and a β chain sequence shown in SEQ ID NO: 227; The TCR comprises an α chain sequence shown in SEQ ID NO: 101 and a β chain sequence shown in SEQ ID NO: 229; The TCR comprises an α chain sequence shown in SEQ ID NO: 103 and a β chain sequence shown in SEQ ID NO: 231; The TCR comprises an α chain sequence shown in SEQ ID NO: 105 and a β chain sequence shown in SEQ ID NO: 233; The TCR comprises an α chain sequence shown in SEQ ID NO: 107 and a β chain sequence shown in SEQ ID NO: 235; The TCR comprises the α chain sequence shown in SEQ ID NO: 109 and the β chain sequence shown in SEQ ID NO:

237.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the TCR according to any one of claims 1 to 4, preferably, the nucleic acid molecule is a codon-optimized nucleotide sequence encoding the TCR according to any one of claims 1 to 4.

6. A carrier, characterized in that Comprising the nucleic acid molecule of claim 5, the vector is preferably a plasmid, a shuttle plasmid, a phagemid, a cosmid, an expression vector; more preferably a homologous recombination repair (HDR) vector or a viral vector, for example, a lentiviral vector, an adenoviral vector, an adeno-associated virus (AAV) vector, a herpes virus vector, a retroviral vector, a baculovirus vector.

7. A T cell receptor fusion protein or a T cell receptor conjugate comprising the TCR described in any one of claims 1 to 4 and other biologically active molecules, wherein the other biologically active molecules are, for example, antibodies, cytokines, cytotoxic agents, enzymes, radioactive substances, detectable markers, and wherein there is or is no linker between the TCR and the other biologically active molecules.

8. An engineered cell, characterized in that Expressing the TCR described in any one of claims 1 to 4, preferably, the engineered cells are engineered T cells, engineered NK cells; or the engineered cells are engineered stem cells, for example, the engineered cells are engineered human CD4+T cells or engineered human CD8+T cells, or are a mixed cell population of engineered human CD4+T cells and engineered human CD8+T cells; or, the engineered cells are engineered hematopoietic stem cells.

9. An engineered human CD4+T cell and / or an engineered human CD8+T cell, characterized in that: Expressing the TCR according to any one of claims 1 to 4 and expressing exogenous CD8a or CD8ab.

10. A method for producing the T cells of claim 9, comprising the step of transfecting CD4+T cells and / or CD8+T cells with exogenous CD8a or CD8ab; and the TCR of any one of claims 1-4, for example, the exogenous CD8a or CD8ab and the TCR of any one of claims 1-4 are expressed in the CD4+T cells and / or CD8+T cells from the same vector, for example, wherein the construct expressing the exogenous CD8a or CD8ab and the construct expressing the TCR of any one of claims 1-4 are separated by a 2A element or an IRES element.

11. A pharmaceutical composition, characterized in that Comprising the engineered cells of claim 8 and / or the engineered human CD4+T cells and / or engineered human CD8+T cells of claim 9.

12. Use of the TCR according to any one of claims 1 to 4, the engineered cell according to claim 8 or 9, or the pharmaceutical composition according to claim 11, characterized in that: Used for preparing a drug for treating a disease (e.g., a tumor) having a KRAS_G12D mutation.

Images