Enhanced chimeric antigen receptor-T cell as well as preparation method and application thereof

Abstract

The invention relates to an enhanced chimeric antigen receptor-T cell and a preparation method and application thereof.A nucleotide sequence for coding a chimeric antigen receptor is exogenously inserted into a genome of the T cell, and the nucleotide sequence comprises at least one of the following genes: an NKG2D gene, a DNAM1 gene, an NKp30 gene, an NKp44 gene or an NKp46 gene, therefore, the expression level of the NKG2D protein, the DNAM1 protein, the NKp30 protein, the NKp44 protein or the NKp46 protein of the chimeric antigen receptor-T cell is higher than the expression level of the corresponding protein in the original T cell. The enhanced chimeric antigen receptor-T cell can more effectively attack tumor cells in a targeted manner, has high tumor killing rate, does not depend on specific tumor antigens and specific tumor mutation, is expected to break through antigen escape, and can be used for preparing anti-tumor products.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to an enhanced chimeric antigen receptor-T cell, its preparation method, and its uses. Background Technology

[0002] Chimeric antigen receptor T-cell therapy (CAR-T) has been the most extensively studied treatment for B-cell-derived malignancies, achieving encouraging efficacy. Phase I clinical trial results showed that CD19 CAR-T therapy could achieve a complete remission (CR) rate of 69%–90% in relapsed / refractory acute lymphoblastic leukemia (ALL). However, the success of CD19 CAR-T in ALL has not been replicated in acute myeloid leukemia (AML) and solid tumors.

[0003] Autologous CAR-T cells require each patient's own T cells to undergo a series of modifications, expansions, and quality checks in vitro, which presents challenges such as high production difficulty, high cost, and long patient waiting times, limiting large-scale clinical use. This urgently necessitates the advancement of off-the-shelf allogeneic CAR-T therapy. γδT cells are a subset of T cells, mainly found in tissues, accounting for 1% to 5% of circulating T cells. Their TCR is composed of γ and δ subunits. Unlike αβT cells, γδT cell activation is not affected by patient-specific MHC molecules, meaning they recognize tumor-associated antigens in an MHC-independent manner. Therefore, allogeneic γδT cells do not induce graft-versus-host disease (GVHD). Studies have shown that CD20 CAR γδT cells do not induce GVHD, while CD20 CAR αβT cell therapy does produce GVHD, leading to increased mortality. In 2015, the transcriptional profiles of tumor samples from 5872 patients with 25 types of malignant tumors were analyzed, and a correlation was found between γδT cells and overall survival [Gentles, AJ, et al., The prognostic landscape of genes and infiltrating immune cells across human cancers. Nat Med, 2015. 21(8): p. 938-945.]. In 2007, a study by KT Godder et al. found that patients with high levels of γδT cells after hematopoietic stem cell transplantation had a five-year survival rate of 54.4% and an overall survival rate of 70.8%, while patients with low levels of γδT cells had a five-year survival rate of 19.1% and an overall survival rate of 19.6% [Godder, KT, et al., Long term disease-free survival in acute leukemia patients recovering with increased gammadelta T cells after partially mismatched related donor bone marrow transplantation. Bone Marrow Transplant, 2007. 39(12): p. 751-7.]. Therefore, γδT plays an important role in disease control and overall survival.

[0004] Existing γδT cells have good safety profiles, but their efficacy needs improvement. Furthermore, current CAR-γδT therapy is target-dependent, and antigen escape is unavoidable during treatment, leading to a high tumor recurrence rate; therefore, overcoming antigen escape is necessary. Summary of the Invention

[0005] The purpose of this invention is to provide an enhanced chimeric antigen receptor-T cell, its preparation method, and its uses. This enhanced chimeric antigen receptor-T cell can more effectively target and attack tumor cells, has a high tumor killing rate, and is independent of specific tumor antigens and specific tumor mutations. It is expected to overcome antigen escape and can be used to prepare anti-tumor products.

[0006] To achieve the above objectives, the present invention provides an enhanced chimeric antigen receptor-T cell, wherein the genome of the T cell is exogenously inserted with a nucleotide sequence encoding a chimeric antigen receptor, said nucleotide sequence comprising at least one of the following genes:

[0007] NKG2D gene, DNAM1 gene, NKp30 gene, NKp44 gene, or NKp46 gene.

[0008] This results in the expression levels of NKG2D, DNAM1, NKp30, NKp44, or NKp46 proteins in chimeric antigen receptor-T cells being higher than the expression levels of the corresponding proteins in primitive T cells.

[0009] In some embodiments of the enhanced chimeric antigen receptor-T cells described above, the sequences of the NKG2D gene, DNAM1 gene, NKp30 gene, NKp44 gene, and NKp46 gene are shown as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively.

[0010] In some embodiments of the enhanced chimeric antigen receptor-T cell described above, the insertion sites of the nucleotide sequence encoding the chimeric antigen receptor in the T cell genome include: TRAC site, PD1 site, Regnase-1 site, Roquin-1 site, CISH site, and BCOR site.

[0011] Optionally, the insertion site of the nucleotide sequence encoding the chimeric antigen receptor in the genome of the T cell is the TRAC site;

[0012] Alternatively, the insertion site of the nucleotide sequence encoding the chimeric antigen receptor in the genome of the T cell is the TRAC site, and the Regnase-1 site in the genome of the T cell is knocked out.

[0013] In some embodiments of the enhanced chimeric antigen receptor-T cell described above, the chimeric antigen receptor includes an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain.

[0014] Optionally, the extracellular antigen recognition domain is one of the following proteins: NKG2D, DNAM1, NKp30, NKp44, or NKp46.

[0015] In some embodiments of the enhanced chimeric antigen receptor-T cell described above, the amino acid sequences of the NKG2D protein, DNAM1 protein, NKp30 protein, NKp44 protein, and NKp46 protein are shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively.

[0016] In some embodiments of the enhanced chimeric antigen receptor-T cell described above, the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α; optionally, the hinge region is derived from CD8α; more preferably, the amino acid sequence of the hinge region is as shown in SEQ ID NO: 11; and / or

[0017] The transmembrane region is derived from one or more of CD3z, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70; optionally, the transmembrane region is derived from CD8α or CD3z; more preferably, the amino acid sequence of the transmembrane region is as shown in SEQ ID NO: 12 or SEQ ID NO: 13.

[0018] In some embodiments of the enhanced chimeric antigen receptor-T cells described above, the intracellular domain includes an intracellular signal transduction region; optionally, it also includes a co-stimulatory signal transduction region.

[0019] Further optionally, the intracellular signal transduction region is derived from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CCD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk; optionally, the intracellular signal transduction region is derived from CD3ζ; more preferably, the amino acid sequence of the intracellular signal transduction region is as shown in SEQ ID NO: 14; and / or

[0020] Further optionally, the co-stimulatory signal transduction region is derived from one, two, or more of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, and MyD88; optionally, the co-stimulatory signal transduction region is derived from 4-1BB, CD28, or OX40; more preferably, the amino acid sequence of the co-stimulatory signal transduction region is as shown in SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

[0021] In some embodiments, the enhanced chimeric antigen receptor-T cell described above further includes a guide peptide located at the N-terminus of the chimeric antigen receptor amino acid sequence; optionally, the guide peptide is derived from CD8α; more preferably, the amino acid sequence of the guide peptide is as shown in SEQ ID NO: 18.

[0022] In some embodiments of the enhanced chimeric antigen receptor-T cell described above, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NOs: 19-30.

[0023] In some embodiments, the nucleotide sequences encoding the chimeric antigen receptor in the enhanced chimeric antigen receptor T cells described above are shown in SEQ ID NOs: 31-42, respectively.

[0024] In some embodiments, the enhanced chimeric antigen receptor-T cells described above also include the knockout of the CTLA-4 site, the Tigit site, or the NKG2A site in the genome of the T cells.

[0025] In some embodiments, the enhanced chimeric antigen receptor-T cells described above are chimeric antigen receptor-γδT cells.

[0026] This invention also provides a method for preparing enhanced chimeric antigen receptor-T cells, comprising:

[0027] 1. Preparation of T cells;

[0028] 2. Introducing a gene-editing tool and a donor template into T cells, wherein the gene-editing tool and the donor template enable the insertion of a nucleotide sequence encoding a chimeric antigen receptor into the genome of the T cells, said nucleotide sequence comprising at least one of the following genes:

[0029] NKG2D gene, DNAM1 gene, NKp30 gene, NKp44 gene, or NKp46 gene.

[0030] This results in the expression levels of NKG2D, DNAM1, NKp30, NKp44, or NKp46 proteins in chimeric antigen receptor-T cells being higher than the expression levels of the corresponding proteins in primitive T cells.

[0031] In some embodiments of the above preparation method, the gene editing tool is selected from one of the CRISPR / Cas system, zinc finger nuclease system, and transcription activator-like effector nuclease system;

[0032] Optionally, the gene-editing tool is selected from the CRISPR / Cas system;

[0033] Alternatively, the CRISPR / Cas system includes the Cas protein and sgRNA.

[0034] In some embodiments of the above preparation method, the Cas protein includes any one of the following: spCas9, AsCas12a, or LbCas12a;

[0035] The sgRNA includes any of the following insertion sites: TRAC site, PD1 site, Regnase-1 site, Roquin-1 site, CISH site, and BCOR site;

[0036] Optionally, the sgRNA sequence targeting the TRAC site is:

[0037] TRAC: 5'-AGAGTCTCTCAGCTGGTACACGG-3' (SEQ ID NO: 43);

[0038] The sgRNA sequence targeting the PD1 site is as follows:

[0039] PD1: 5'-CGACTGGCCAGGGGCGCCTGT-3' (SEQ ID NO: 44);

[0040] The sgRNA sequence targeting the Regnase-1 site is as follows:

[0041] Regnase-1: 5'-AAGGAGGTCTTCTCCTGCCG-3' (SEQ ID NO: 45);

[0042] The sgRNA sequence targeting the Roquin-1 site is as follows:

[0043] Roquin-1: 5'-TGAAGACACAAAGCATTATG-3' (SEQ ID NO: 46);

[0044] The sgRNA sequence targeting the CISH site is as follows:

[0045] CISH: 5'-GGCGCATCCTCCTTAGGCAT-3' (SEQ ID NO: 47);

[0046] The sgRNA sequence targeting the BCOR site is as follows:

[0047] BcoR: 5'-AGCACGGCCCATAGGATCGA-3' (SEQ ID NO: 48).

[0048] In some embodiments of the above preparation method, the donor template is plasmid DNA, dsDNA, or ssDNA;

[0049] Optionally, the donor template is dsDNA including the NKG2D gene, DNAM1 gene, NKp30 gene, NKp44 gene or NKp46 gene;

[0050] More preferably, the donor template includes, in the 5'-3' direction, a left homologous arm sequence, a polyA sequence, a marker gene sequence, a nucleotide sequence encoding an intracellular domain, a nucleotide sequence encoding a transmembrane region, a nucleotide sequence encoding a hinge region, a nucleotide sequence encoding an extracellular antigen recognition domain, a promoter sequence, and a right homologous arm sequence.

[0051] Further optionally, the donor template further comprises a nucleotide sequence encoding a signal peptide, and the nucleotide sequence encoding the signal peptide is located between the promoter sequence and the nucleotide sequence encoding an extracellular antigen recognition domain.

[0052] In some embodiments of the above preparation method, the promoter is selected from the SFFV promoter, CMV promoter, or EF1α promoter; optionally, the nucleotide sequence of the EF1α promoter is shown in SEQ ID NO: 49.

[0053] The marker gene sequence is an EGFP fluorescent protein gene or a GFP fluorescent protein gene; optionally, the nucleotide sequence of the GFP fluorescent protein gene is as shown in SEQ ID NO: 50.

[0054] The polyA is a BGH polyA signal sequence; optionally, the nucleotide sequence of the BGH polyA signal sequence is shown in SEQ ID NO: 51.

[0055] In some embodiments of the above preparation method, the nucleotide sequence encoding the extracellular antigen recognition domain includes: a nucleotide sequence encoding the NKG2D protein, a nucleotide sequence encoding the DNAM1 protein, a nucleotide sequence encoding the NKp30 protein, a nucleotide sequence encoding the NKp44 protein, or a nucleotide sequence encoding the NKp46 protein; and / or

[0056] The nucleotide sequence encoding the hinge region is the nucleotide sequence encoding CD8α; and / or

[0057] The nucleotide sequence encoding the transmembrane region is a nucleotide sequence encoding CD8α or CD3; and / or,

[0058] Nucleotide sequences encoding intracellular domains include those encoding CD3ζ and those encoding 4-1BB, CD28, or OX40.

[0059] In some embodiments of the above preparation method, the left homologous arm sequence and the right homologous arm sequence in the donor template are shown as SEQ ID NO: 52 and SEQ ID NO: 53, respectively.

[0060] In some embodiments of the above preparation method, in step 1, T cells are activated using phosphophosphate, antibody, or α-Glacer.

[0061] In some embodiments of the above preparation method, in step 1, IL2, IL7, IL10, IL15, IL12, and IL18 are also used to activate T cells;

[0062] Optionally, in step 1, a combination of phosphophosphate and IL2 / IL15 is used to activate T cells.

[0063] The present invention also provides an enhanced chimeric antigen receptor-T cell, which is obtained by the above preparation method.

[0064] This invention also provides the application of the above-mentioned enhanced chimeric antigen receptor-T cells in the preparation of antitumor drugs;

[0065] Optionally, the drug is used to treat acute T-lymphoblastic leukemia (T-ALL), non-small cell lung cancer (NSCLC), acute myeloid leukemia (AML), hepatocellular carcinoma (HCC), renal cell carcinoma (RCC), and acute promyelocytic leukemia.

[0066] In some embodiments of the above application, the drug is an intravenous injection.

[0067] The present invention also provides a drug combination comprising the above-described enhanced chimeric antigen receptor-T cells and a therapeutic agent;

[0068] Optionally, the therapeutic agent is a chemotherapeutic agent, a small molecule inhibitor, an antibody, or a cytokine;

[0069] Alternatively, the therapeutic agent may be Ara-C or etoposide.

[0070] The present invention also provides a pharmaceutical composition comprising the above-described enhanced chimeric antigen receptor-T cells and pharmaceutically acceptable excipients;

[0071] Optionally, pharmaceutically acceptable excipients include protective agents;

[0072] Alternatively, pharmaceutically acceptable excipients include cell cryopreservation solutions;

[0073] Alternatively, the pharmaceutical composition may be an intravenous injection.

[0074] Compared with the prior art, the present invention achieves the following beneficial effects:

[0075] This invention utilizes non-viral site-specific integration technology to prepare enhanced chimeric antigen receptor-T cells. This preparation method is simple and low-cost. The resulting enhanced chimeric antigen receptor-T cells can specifically recognize various tumor cells, more effectively target and attack tumor cells, exhibiting a high tumor-killing rate. Furthermore, it is independent of specific tumor antigens and mutations, showing promise in overcoming antigen escape. It can be used to prepare anti-tumor products and demonstrates good tumor-suppressing effects against various tumor cells. Compared to natural γδT cells, the enhanced chimeric antigen receptor-T cells prepared by this invention show approximately 10-fold increased NKG2D expression, thus significantly improving the tumor-killing efficiency of γδT cells. This invention holds promise for the preparation of anti-tumor products and can be combined with small molecule inhibitors, antibodies, cytokines, and other products to further enhance efficacy. It has particularly promising industrial application prospects for the preparation of drugs to treat various tumors such as acute leukemia, liver cancer, and lung cancer.

[0076] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0077] Figure 1 This is a schematic diagram of the donor DNA corresponding to NKG2D-8TM-28z-GFP of the present invention.

[0078] Figure 2 This is a schematic diagram of the donor DNA corresponding to NKG2D-3TM-z-GFP of the present invention;

[0079] Figure 3 This is a schematic diagram of the donor DNA corresponding to NKG2D-3TM-z-28-GFP of the present invention;

[0080] Figure 4 This is a schematic diagram illustrating the preparation of enhanced chimeric antigen receptor-T cells according to the present invention.

[0081] Figure 5 This diagram illustrates the cellular expression of multiple enhanced chimeric antigen receptor-T cells prepared according to the present invention.

[0082] Figure 6 This diagram illustrates the killing ability of multiple enhanced chimeric antigen receptor-T cells prepared in this invention against tumor cell lines. From left to right, they represent: UTD cells (untransduced CAR T cells), γδT cells containing the NKG2D-z structure, γδT cells containing the NKG2D-z-28 structure, γδT cells containing the NKG2D-z-BB structure, γδT cells containing the NKG2D-z-OX40 structure, γδT cells containing the NKG2D-8TM-28z structure, γδT cells containing the CLL1 CAR-8TM-BBz structure, γδT cells containing the NKG2D-z-28 structure with RKO knockout, and γδT cells containing the NKG2D-z structure with RKO.

[0083] Figure 7 This diagram illustrates the long-term killing ability of multiple enhanced chimeric antigen receptor-T cells prepared in this invention against tumor cell lines.

[0084] Figure 8 The diagram shows the killing ability of multiple enhanced chimeric antigen receptor-T cells prepared in this invention against tumor cell lines after multiple rounds of stimulation.

[0085] Figure 9 and Figure 10 This image shows the toxicity assay results of multiple enhanced chimeric antigen receptor-T cells prepared according to this invention against normal cells. Figure 9 In the diagram, each column represents, from top to bottom, a monocyte, an NK cell, a B cell, and a T cell.

[0086] Figure 11 This diagram illustrates the cytotoxic capabilities of multiple enhanced chimeric antigen receptor-T cells prepared in this invention against antigen-deficient cells. From left to right, they represent γδT cells, super-γδT cells, CLL1CARγδT cells, and CLL1CAR T cells, respectively.

[0087] Figure 12 This diagram illustrates the killing ability of the enhanced chimeric antigen receptor-T cells prepared in this invention against various tumor cells.

[0088] Figure 13 This diagram illustrates the anti-aging potential of the enhanced chimeric antigen receptor-T cells prepared according to the present invention.

[0089] Figure 14 This diagram illustrates the cytotoxic function of enhanced chimeric antigen receptor-T cells containing the extracellular domain of DNAM1 prepared according to the present invention. From left to right, they represent UTD cells (untransduced CAR T cells), γδT cells containing the DNAM1-z-28 structure, γδT cells containing the DNAM1-28 structure, γδT cells containing the DNAM1-28z structure, and γδT cells containing the DNAM1-8-28z structure.

[0090] Figure 15 The diagram illustrates the cytotoxic function of the enhanced chimeric antigen receptor-T cells containing the Nkp30 extracellular domain prepared according to this invention. Detailed Implementation

[0091] The following further describes this application: In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. Meanwhile, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0092] In this application, the terms "gene editing" and "gene editing technology" have their common meaning in the art, referring to a new technology for site-specific modification of the genome. This technology allows for precise targeting to a specific site on the genome, where target DNA fragments can be cut, or target gene fragments can be knocked in or knocked out. As a molecular biology technique, gene editing technology enables precise modification of chromosomes, thereby altering the existing functions of cells. Compared to cell lines commonly used in basic research, T cells, as primary cells, are not particularly unique except for their inability to proliferate long-term, and can also be edited using gene editing technology. Currently, there are three main gene editing technologies: zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN) technology, and RNA-guided CRISPR / Cas nuclease technology. Compared to traditional gene targeting techniques, this new gene editing technology retains the characteristic of site-specific modification, can be applied to more species and cells, is more efficient, has a shorter construction time, and is less costly.

[0093] In this application, the terms "CRISPR / Cas technology" and "CRISPR / Cas system" have their common meaning in the art. CRISPR stands for clustered regularly interspaced shortpalindromic repeats / CRISPR-associated proteins, an acquired immune system found in most bacteria and all archaea that can directionally cleave foreign gene fragments. Different types of CRISPR / Cas systems have been discovered, with the second type being relatively simple, consisting primarily of the Cas9 protein and guide RNA (gRNA). Compared to earlier ZFN and TALEN technologies, CRISPR / Cas offers advantages such as low off-target rates, high efficiency, affordability, and wide applicability. Furthermore, some literature reports that CRISPR hybrid RNA-DNA (chRDNA) guidance technology significantly improves the specificity of the Cas9 protein compared to whole RNA guidance technology, thereby achieving high levels of intended genome editing in cells and minimizing off-target time.

[0094] In this application, the terms "zinc finger nuclease technology" and "zinc finger nuclease system" have their common meaning in the art. The core design concept of zinc finger nuclease technology is the ingenious integration of two functional domains—a specific recognition module and a functional module. The most classic zinc finger nuclease fuses a non-specific endonuclease, Fok I, with a zinc finger-containing domain, which can recognize specific DNA sequences.

[0095] In this application, the terms "transcription activator-like effector nuclease technology" and "transcription activator-like effector nuclease system" have their common meaning in the art. TALE effectors were initially discovered as an invasion strategy for bacterial infection of plants. Researchers have created a powerful tool with specific gene-editing capabilities—TALEN proteins—by linking the Fok I nuclease to an artificial TALE with sequence-specific binding ability. A typical TALEN protein consists of an N-terminal domain containing a nuclear localization signal (NLS), a central domain containing a typical tandem TALE repeat sequence that recognizes a specific DNA sequence, and a C-terminal domain with Fok I endonuclease function. The core principle of TALEN technology is to achieve three distinct functions—guided entry into the cell nucleus, specific recognition of target DNA, and cleavage of target DNA—in an orderly manner on the same TALEN protein.

[0096] In this application, the term "Chimeric Antigen Receptor" (CAR) is a core component of CAR cell therapy drugs, which may include an extracellular antigen recognition domain (e.g., a portion that binds to tumor-associated antigens (TAAs)), a hinge region, a transmembrane region, and an intracellular domain. CAR-T (Chimeric Antigen Receptor T) cell immunotherapy is considered one of the most promising approaches to combating cancer. CAR-T cells utilize genetic modification to enable T cells to express CAR proteins. These CAR proteins are capable of recognizing intact proteins on the cell membrane surface without antigen presentation, thereby activating and functionally affecting T cells.

[0097] In this application, the term "extracellular antigen recognition domain" refers to the antigen recognition domain (ARD). CAR cell therapy products (such as CAR-T cells) rely on extracellular antigen recognition domains to specifically recognize and / or bind to target antigens expressed by tumor cells. To date, antigen recognition domains are derived from the single-chain variable fragment (scFv) of antibodies, or from receptor-ligand interactions, TCR mimics, and variable lymphocyte receptors (VLRs). The most common source to date is the antibody scFv segment, which includes the antibody heavy chain variable region (VH region) and light chain variable region (VL region) linked by a peptide chain, such as the 18-amino acid linker sequence GSTGSGSGKPGSGEGSTKG.

[0098] In this application, the term "hinge region" refers to the connecting segment that acts between the extracellular antigen recognition domain and the transmembrane domain. This region allows the CAR to recognize the antigen by providing a certain range of motion to the antigen recognition domain. Currently used hinge regions are mainly derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α. In addition, typical hinge regions also contain residues that participate in CAR dimerization, which helps to enhance antigen sensitivity.

[0099] In this application, "transmembrane region" refers to a transmembrane domain connecting the intracellular and extracellular components of the CAR structure. Different transmembrane domains can affect CAR expression and stability to some extent, but do not directly participate in signal transduction; however, they can enhance downstream signal transduction through interactions. The transmembrane region may be derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70.

[0100] In this application, the term "intracellular domain" includes intracellular signal transduction regions and may also include co-stimulatory signal transduction regions.

[0101] In this application, the term "intracellular signal transduction region" refers to the activation of at least one normal effector function of an immune effector cell responsible for expressing CAR. The intracellular signal transduction region may originate from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk.

[0102] In this application, the term "co-stimulatory signal transduction region" is used because, in addition to antigen-specific signal stimulation, many immune effector cells require co-stimulation to promote cell proliferation, differentiation, and survival, as well as to activate effector functions. In some embodiments, the CAR may further include one or more co-stimulatory signal transduction regions, wherein the co-stimulatory signal transduction regions may be derived from one, two, or more of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, and MyD88.

[0103] In this application, the term "guide peptide" refers to a short peptide preceding an extracellular antigen recognition domain (such as the scFv sequence), which guides the export of intracellularly synthesized recombinant proteins to the extracellular space. Commonly used guide peptides include the human CD8α signal peptide or the human GM-CSF receptor α signal peptide.

[0104] In this application, the term "immune effector cell" generally refers to a cell that participates in an immune response, such as promoting an immune effector response. Immune effector cells may be selected from one or more of the following groups: T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMCs), pluripotent stem cells, T lymphocytes differentiated from pluripotent stem cells, NK cells differentiated from pluripotent stem cells, induced pluripotent stem cells (iPSCs), T cells differentiated from induced pluripotent stem cells (iPSC-T), NK cells differentiated from induced pluripotent stem cells (iPSC-NK), and embryonic stem cells.

[0105] In this application, the term "chimeric antigen receptor-T cell" generally refers to CAR-T cells formed by transfecting chimeric antigen receptors (CARs) into T cells. These cells activate T cells by binding to specific antigens on the surface of tumor cells using an antigen-antibody binding mechanism, specifically recognizing and killing tumors (Jackson H et al., Nature Reviews Clinical Oncology, 2016, 13(6):370-383). CAR-T cells recognize tumor antigens without being restricted by human leukocyte antigens (HLA), effectively preventing immune escape by tumor cells through downregulation of major histocompatibility complex (MHC) molecule expression (Fesnak AD et al., Nature Reviews Cancer, 2016, 16(9):566-581).

[0106] In this application, the term "enhanced chimeric antigen receptor-T cell" refers to a CAR-T cell in which a nucleotide sequence encoding a chimeric antigen receptor is exogenously inserted, the nucleotide sequence comprising at least one of the following genes: NKG2D gene, DNAM1 gene, NKp30 gene, NKp44 gene, or NKp46 gene. The expression levels of NKG2D protein, DNAM1 protein, NKp30 protein, NKp44 protein, or NKp46 protein in the enhanced chimeric antigen receptor-T cell are higher than the expression levels of the corresponding proteins in the original T cell, thereby giving it a better anti-tumor effect than ordinary CAR-T cells (CAR-T cells that do not express the NKG2D gene, DNAM1 gene, NKp30 gene, NKp44 gene, or NKp46 gene) and the potential to overcome antigen escape.

[0107] In this application, the term "pharmaceutical composition" generally refers to a pharmaceutical composition suitable for administration to a patient, which may contain the immune effector cells described in this application, and may also contain one or more pharmaceutically acceptable excipients, such as: carriers, protectants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and preservatives. In some embodiments, pharmaceutically acceptable excipients include protectants, such as cell cryopreservation solutions. In some embodiments, the pharmaceutical composition of this application is a cell suspension or its cryopreserved cells.

[0108] In this application, the term "drug combination" includes the aforementioned enhanced chimeric antigen receptor-T cells and therapeutic agents; optionally, the therapeutic agent is a chemotherapeutic agent, a small molecule inhibitor, an antibody, or a cytokine; more preferably, the therapeutic agent is Ara-C (cytarabine) or etoposide. The drug combination of the present invention can improve the efficacy of enhanced chimeric antigen receptor-T cells without causing unacceptable adverse effects.

[0109] In this application, the term "comprising" generally means including the explicitly specified features, but does not exclude other elements.

[0110] In this application, the term "about" generally refers to a range of fluctuations acceptable to a person skilled in the art above or below a specified value, such as a variation within ±0.5% to 10%, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0111] This invention utilizes non-viral site-directed integration technology to prepare enhanced chimeric antigen receptor-T cells. Specifically, it employs the CRISPR-Cas9 gene knock-in method to site-directly knock exogenous groups into T cells.

[0112] CRISPR-Cas9 knock-in (KI) refers to the process where the Cas9 restriction enzyme cuts double-stranded DNA, simultaneously providing a DNA repair template highly homologous to the target gene. The organism then initiates the High-Homogeneous Recombination (HDR) repair pathway, inserting a foreign DNA fragment into the gene at a specific site. The foreign gene in KI can be a protein-coding gene, a DNA element involved in gene regulation, or a non-functional DNA sequence. This repair pathway requires the introduction of a DNA repair template highly homologous to the sequences immediately upstream and downstream of the target editing site, a specific gRNA, and the Cas9 nuclease into the cell. In the presence of a highly homologous DNA template, the HDR mechanism can precisely insert a DNA fragment into a specific genomic site through homologous recombination.

[0113] In this invention, Cas9 protein and sgRNA, along with a donor template containing exogenous groups (dsDNA containing the NKG2D gene, DNAM1 gene, NKp30 gene, NKp44 gene, or NKp46 gene), are co-transformed into T cells. The transcribed sgRNA guides the Cas9 protein to the insertion site on the T cell, cleaves the insertion site, and introduces a DNA double-strand break at the insertion site. Then, homologous recombination is used to knock the exogenous gene into the insertion site, thereby achieving site-specific knock-in of the exogenous gene into the T cell gene pool.

[0114] In this invention, the sgRNA is designed based on the insertion site sequence of T cells. In this invention, the insertion site refers to any nucleotide site in a gene that a person skilled in the art would like to knock in a foreign gene, such as the TRAC site, PD1 site, Regnase-1 site, or Roquin-1 site.

[0115] In this invention, the 5' and 3' ends of the donor template both contain homologous arm sequences, which are homologous to the T cell target gene. These homologous arm sequences mediate homologous recombination between the exogenous gene and the T cell target gene, thereby achieving the knock-in of the exogenous gene. In this invention, the exogenous gene can be selected from any gene fragment that can be knocked into T cells by those skilled in the art. It can be a single-gene fragment, or a multi-gene fragment such as a double-gene fragment, a triple-gene fragment, or a quadruple-gene fragment, for example, the NKG2D gene, the DNAM1 gene, the NKp30 gene, the NKp44 gene, or the NKp46 gene.

[0116] In this invention, the donor template is selected from plasmid templates, double-stranded DNA templates, linear single-stranded DNA templates, and circular single-stranded DNA templates. In other words, this invention does not have special requirements for the form of the donor template; any form of template can be selected according to specific experimental needs to achieve efficient gene knock-in.

[0117] In this invention, the donor template consists of the target gene to be introduced and homologous sequences (homologous arms) upstream and downstream of the target sequence. The length and position of the homologous arms are determined by the size of the edited sequence. The donor template can insert the target gene into the target site through homologous recombination, thereby precisely inserting a DNA sequence into a specific site.

[0118] The inventors of this invention unexpectedly discovered that T cells overexpressing the NKG2D gene, DNAM1 gene, NKp30 gene, NKp44 gene, or NKp46 gene have better anti-tumor effects, and are expected to provide a new, safer, more effective, scalable, and allogeneic cell preparation for various tumor cells, providing new treatment options for AML, various solid tumors, and metastatic tumors.

[0119] The present invention is further illustrated below by way of examples. These examples are for illustrative purposes only and not for limiting the invention. Simple modifications made to the present invention based on its essence are all within the scope of protection claimed by the present invention. The present invention is further described below with reference to examples:

[0120] Example

[0121] 1. Donor DNA preparation

[0122] The vector construction and preparation of donor DNA were synthesized by a CRO company. The following is relevant information about the donor DNA. Those skilled in the art can synthesize the following 12 types of donor DNA based on the following information:

[0123] (1) Donor DNA corresponding to NKG2D-8TM-28z-GFP;

[0124] (2) Donor DNA corresponding to NKG2D-3TM-z-GFP;

[0125] (3) Donor DNA corresponding to NKG2D-3TM-z-28-GFP;

[0126] (4) Donor DNA corresponding to NKG2D-3TM-z-BB-GFP;

[0127] (5) Donor DNA corresponding to NKG2D-3TM-z-OX40-GFP;

[0128] (6) Donor DNA corresponding to DNAM1-3TM-z-28-GFP;

[0129] (7) Donor DNA corresponding to DNAM1-3TM-28-GFP;

[0130] (8) Donor DNA corresponding to DNAM1-3TM-28z–GFP;

[0131] (9) Donor DNA corresponding to DNAM1-8TM-28z–GFP;

[0132] (10) Donor DNA corresponding to Nkp30-3TM-z-28-GFP;

[0133] (11) Donor DNA corresponding to Nkp44-3TM-z-28-GFP.

[0134] (12) Donor DNA corresponding to Nkp46-3TM-z-28-GFP;

[0135] For example, according to Figure 1 The donor DNA corresponding to NKG2D-8TM-28z-GFP was synthesized. The donor template included a left homologous arm sequence (SEQ ID NO: 52), a BGH polyA signal sequence (SEQ ID NO: 51), a nucleotide sequence encoding NKG2D-8TM-28z-GFP (SEQ ID NO: 55), an EF1α promoter sequence (SEQ ID NO: 49), and a right homologous arm sequence (SEQ ID NO: 53) in the 5'-3' direction.

[0136] according to Figure 2 The donor DNA corresponding to NKG2D-3TM-z-GFP was synthesized. The donor template included a left homologous arm sequence (SEQ ID NO: 52), a BGH polyA signal sequence (SEQ ID NO: 51), a nucleotide sequence encoding NKG2D-3TM-z-GFP (SEQ ID NO: 57), an EF1α promoter sequence (SEQ ID NO: 49), and a right homologous arm sequence (SEQ ID NO: 53) in the 5'-3' direction.

[0137] according to Figure 3 The donor DNA corresponding to NKG2D-3TM-z-28-GFP was synthesized. The donor template included a left homologous arm sequence (SEQ ID NO: 52), a BGH polyA signal sequence (SEQ ID NO: 51), a nucleotide sequence encoding NKG2D-3TM-z-28-GFP (SEQ ID NO: 59), an EF1α promoter sequence (SEQ ID NO: 49), and a right homologous arm sequence (SEQ ID NO: 53) in the 5'-3' direction.

[0138] The remaining nine donor DNAs were synthesized using a similar method.

[0139] The amino acid sequence of NKG2D-8TM-28z-GFP (containing T2A peptide and GFP) is as follows:

[0140] (The amino acid sequences of CD8 signal peptide, NKG2D extracellular sequence, CD8 transmembrane sequence, CD28+CD3Z intracellular sequence, T2A, and GFP are listed in order (SEQ ID NO: 54))

[0141] The nucleotide sequence encoding NKG2D-8TM-28z-GFP (containing the T2A peptide and GFP) is as follows:

[0142]

[0143] The amino acid sequence of NKG2D-3TM-z-GFP (containing T2A peptide and GFP) is as follows:

[0144] (The amino acid sequences of CD8 signal peptide, NKG2D extracellular sequence, CD3 transmembrane sequence, CD3Z intracellular sequence, T2A, and GFP, in that order (SEQ ID NO: 56)) The nucleotide sequence encoding NKG2D-3TM-z-GFP (containing T2A peptide and GFP) is as follows:

[0145]

[0146] The amino acid sequence of NKG2D-3TM-z-28-GFP (containing T2A peptide and GFP) is as follows:

[0147] (The amino acid sequence of CD8 signal peptide, NKG2D extracellular sequence, CD3 transmembrane sequence, CD3Z intracellular sequence + CD28 intracellular sequence, T2A, and GFP (SEQ ID NO: 58)) The nucleotide sequence encoding NKG2D-3TM-z-28-GFP (containing T2A peptide and GFP) is as follows:

[0148]

[0149] The amino acid sequence of NKG2D-3TM-z-BB-GFP (containing T2A peptide and GFP) is as follows:

[0150] (The amino acid sequences of CD8 signal peptide, NKG2D extracellular sequence, CD3 transmembrane sequence, CD3Z intracellular sequence +4-1BB intracellular sequence, T2A, and GFP, in that order (SEQ ID NO: 60)) The nucleotide sequence encoding NKG2D-3TM-z-BB-GFP (containing T2A peptide and GFP) is as follows:

[0151]

[0152] The amino acid sequence of NKG2D-3TM-z-OX40-GFP (containing T2A peptide and GFP) is as follows:

[0153] (The amino acid sequences of CD8 signal peptide, NKG2D extracellular sequence, CD3 transmembrane sequence, CD3Z intracellular sequence + OX40 intracellular sequence, T2A, and GFP, in that order (SEQ ID NO: 62))

[0154] The nucleotide sequence encoding NKG2D-3TM-z-OX40-GFP (containing T2A peptide and GFP) is as follows:

[0155]

[0156] The amino acid sequence of DNAM1-z-28-GFP (containing T2A peptide and GFP) is as follows:

[0157] (The amino acid sequences of CD8 signal peptide, DNAM1 extracellular sequence, CD3 transmembrane sequence, CD3Z intracellular sequence + CD28 intracellular sequence, T2A, and GFP are listed in order (SEQ ID NO: 64))

[0158] The nucleotide sequence encoding DNAM1-z-28-GFP (containing the T2A peptide and GFP) is as follows:

[0159]

[0160] The amino acid sequence of DNAM1-3TM-28-GFP (containing the T2A peptide and GFP) is as follows:

[0161] MALPVTALLLPLALLLHAARPEEVLWHTSVPFAENMSLECVYPSMGILTQVEWFKIGTQQDSIAIFSPTHGMVIRKPYAERVYFLNSTMASNNMTLFFRNASEDDVGYYSCSLYTYPQGTWQKVIQVVQSDSFEAAVPSNSHIVSEPGKNVTLTCQPQMTWPVQAVRWEKIQPRQIDLLTYCNLVHGRNFTSKFPRQIVSNCSHGRWSVIVIPDVTVSDSGLYRCYLQASAGENETFVMRLTVAEGKTDNQYTLFVATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSEGRGSLLTCGDVEENPGPMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO: 66)

[0162] The nucleotide sequence encoding DNAM1-3TM-28-GFP (containing the T2A peptide and GFP) is as follows:

[0163]

[0164] The amino acid sequence of DNAM1-3TM-28z–GFP (including the T2A peptide and GFP) is as follows:

[0165] MALPVTALLLPLALLLHAARPEEVLWHTSVPFAENMSLECVYPSMGILTQVEWFKIGTQQDSIAIFSPTHGMVIRKPYAERVYFLNSTMASNNMTLFFRNASEDDVGYYSCSLYTYPQGTWQKVIQVVQSDSFEAAVPSNSHIVSEPGKNVTLTCQPQMTWPVQAVRWEKIQPRQIDLLTYCNLVHGRNFTSKFPRQIVSNCSHGRWSVIVIPDVTVSDSGLYRCYLQASAGENETFVMRLTVAEGKTDNQYTLFVATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO: 68)

[0166] The nucleotide sequence encoding DNAM1-3TM-28z–GFP (including the T2A peptide and GFP) is as follows:

[0167]

[0168] The amino acid sequence of DNAM1-8TM-28z–GFP (including the T2A peptide and GFP) is as follows:

[0169] MALPVTALLLPLALLLHAARPEEVLWHTSVPFAENMSLECVYPSMGILTQVEWFKIGTQQDSIAIFSPTHGMVIRKPYAERVYFLNSTMASNNMTLFFRNASEDDVGYYSCSLYTYPQGTWQKVIQVVQSDSFEAAVPSNSHIVSEPGKNVTLTCQPQMTWPVQAVRWEKIQPRQIDLLTYCNLVHGRNFTSKFPRQIVSNCSHGRWSVIVIPDVTVSDSGLYRCYLQASAGENETFVMRLTVAEGKTDNQYTLFVATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO: 70) The nucleotide sequence encoding DNAM1-8TM-28z–GFP (including the T2A peptide and GFP) is as follows:

[0170]

[0171] The amino acid sequence of Nkp30-z-28-GFP (containing the T2A peptide and GFP) is as follows:

[0172] MALPVTALLLPLALLLHAARPLWVSQPPEIRTLEGSSAFLPCSFNASQGR

[0173] LAIGSVTWFRDEVVPGKEVRNGTPEFRGRLAPLASSRFLHDHQAELHIRDVR

[0174] GHDASIYVCRVEVLGLGVGTGNGTRLVVEKEHPQLGTTTPAPRPPTPAPTIAS

[0175] QPLSLRPEACRPAAGGAVHTRGLDFACDQSFGLLDPKLCYLLDGILFIYGVILTA

[0176] LFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP

[0177] RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY

[0178] DALHMQALPPRRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSE

[0179] GRGSLLTCGDVEENPGPMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEG

[0180] DATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMP

[0181] EGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEY

[0182] NYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLP

[0183] DNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (sequence of CD8 signal peptide, NKp30 extracellular sequence, CD3 transmembrane sequence, CD3Z intracellular sequence + CD28 intracellular sequence, T2A, GFP amino acid sequence (SEQ ID NO: 72))

[0184] The nucleotide sequence encoding Nkp30-z-28-GFP is as follows:

[0185] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCT

[0186] CCACGCCGCCAGGCCGctctgggtgtcccagcccctgagattcgtaccctggaaggatcctctgccttc

[0187] ctgccctgctccttcaatgccagccaagggagactggccattggctccgtcacgtggttccgagatgaggtggttccag

[0188] ggaaggaggtgaggaatggaaccccagttcaggggccgcctggccccacttgcttcttcccgtttcctccatgacc

[0189] accaggctgagctgcacatccgggacgtgcgaggccatgacgccagcatctacgtgtgcagagtggaggtgctggg

[0190] ccttggtgtcgggacagggaatgggactcggctggtggtggagaaagaacatcctcagctagggACCACGAC

[0191] GCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCC

[0192] CTGTCCCTGCGCCCAGAGGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGC

[0193] ACACGAGGGGGCTGGACTTCGCCTGTGATCAGAGCTTTGGCCTGCTGGAT

[0194] CCCAAACTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATT

[0195] CTCACTGCCTTGTTCCTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCC

[0196] CGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGA

[0197] CGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTG

[0198] AGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACA

[0199] ATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGAT

[0200] GAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGG

[0201] TCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCC

[0202] TGCCCCCTCGCAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACAT

[0203] GAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCT

[0204] ATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCGAGGGCAGAGGAAG

[0205] TCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTATGGTGAGC

[0206] AAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGG

[0207] ACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGG

[0208] CGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCA

[0209] AGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTG

[0210] CAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAA

[0211] GTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGG

[0212] ACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACAC

[0213] CCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGC

[0214] AACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCT

[0215] ATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGAT

[0216] CCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAG

[0217] CAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACT

[0218] ACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGA

[0219] TCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCA

[0220] TGGACGAGCTGTACAAG (encoding CD8 signal peptide, NKp30 extracellular sequence, CD3, etc., respectively)

[0221] Transmembrane sequence, CD3Z intracellular sequence + CD28 intracellular sequence, T2A, GFP nucleotide sequence (SEQ ID NO: 73)

[0222] The amino acid sequence of Nkp44-z-28-GFP (containing T2A peptide and GFP) is as follows:

[0223] MALPVTALLLPLALLLHAARPQSKAQVLQSVAGQTLTVRCQYPPTGSLYEK

[0224] KGWCKEASALVCIRLVTSSKPRTMAWTSRFTIWDDPDAGFFTVTMTDLREEDS

[0225] GHYWCRIYRPSDNSVSKSVRFYLVVSPASASTQTSWTPRDLVSSQTQTQSCVPP

[0226] TAGARQAPESPSTIPVPSQPQNSTLRPGPAAPIATTTPAPRPPTPAPTIASQPLSLRP

[0227] EACRPAAGGAVHTRGLDFACDQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKF

[0228] SRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE

[0229] GLYNELQKDKMAEAAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ

[0230] ALPPRRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSEGRGSLLT

[0231] CGDVEENPGPMMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKL

[0232] TLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQE

[0233] RTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN

[0234] VYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLS

[0235] TQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (sequence of CD8 signal peptide, NKp44 extracellular sequence, CD3 transmembrane sequence, CD3Z intracellular sequence + CD28 intracellular sequence, T2A, GFP amino acid sequence (SEQ ID NO: 74))

[0236] The nucleotide sequence encoding Nkp44-z-28-GFP is as follows:

[0237] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCT

[0238] CCACGCCGCCAGGCCGCAATCCAAGGCTCAGGTACTTCAAAGTGTGGCA

[0239] GGGCAGACGCTAACCGTGAGATGCCAGTACCCGCCCACGGGCAGTCTCT

[0240] ACGAGAAGAAAGGCTGGTGTAAGGAGGCTTCAGCACTTGTGTGCATCAG

[0241] GTTAGTCACCAGCTCCAAGCCCAGGACGATGGCTTGGACCTCTCGATTCA

[0242] CAATCTGGGACGACCCTGATGCTGGCTTCTTCACTGTCACCATGACTGAT

[0243] CTGAGAGAGGAAGACTCAGGACATTACTGGTGTAGAATCTACCGCCCTT

[0244] CTGACAACTCTGTCTCTAAGTCCGTCAGATTCTATCTGGTGGTATCTCCA

[0245] GCCTCTGCCTCCACACAGACCTCCTGGACTCCCCGCGACCTGGTCTCTTC

[0246] ACAGACCCAGACCCAGAGCTGTGTGCCTCCCACTGCAGGAGCCAGACAA

[0247] GCCCCTGAGTCTCCATCTACCATCCCTGTCCCTTCACAGCCACAGAACTC

[0248] CACGCTCCGCCCTGGCCCTGCAGCCCCCATTGCCACCACGACGCCAGCGC

[0249] CGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTG

[0250] CGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGG

[0251] GGGCTGGACTTCGCCTGTGATCAGAGCTTTGGCCTGCTGGATCCCAAACT

[0252] CTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGC

[0253] CTTGTTCCTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTAC

[0254] CAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAG

[0255] AGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGG

[0256] GGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACT

[0257] GCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGG

[0258] CGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGT

[0259] ACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCC

[0260] CTCGCAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACAT

[0261] GACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCC

[0262] CACCACGCGACTTCGCAGCCTATCGCTCCGAGGGCAGAGGAAGTCTTCTA

[0263] ACATGCGGTGACGTGGAGGAGAATCCCGGCCCTATGGTGAGCAAGGGCG

[0264] AGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGA

[0265] CGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCC

[0266] ACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCC

[0267] CGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCT

[0268] TCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCC

[0269] ATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACG

[0270] GCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGT

[0271] GAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATC

[0272] CTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCA

[0273] TGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCA

[0274] CAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAAC

[0275] ACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAG

[0276] CACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATG

[0277] GTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGA

[0278] GCTGTACAAG (the nucleotide sequence encoding CD8 signal peptide, NKp44 extracellular sequence, CD3 transmembrane sequence, CD3Z intracellular sequence + CD28 intracellular sequence, T2A, GFP (SEQ ID NO: 75))

[0279] The amino acid sequence of Nkp46-z-28-GFP (containing T2A peptide and GFP) is as follows:

[0280] MALPVTALLLPLALLLHAARPQQQTLPKPFIWAEPHFMVPKEKQVTICCQG

[0281] NYGAVEYQLHFEGSLFAVDRPKPPERINKVQFYIPDMNSRMAGQYSCIYRVGEL

[0282] WSEPSNLLDLVVTEMYDTPTLSVHPGPEVISGEKVTFYCRLDTATSMFLLLKEG

[0283] RSSHVQRGYGKVQAEFPLGPVTTAHRGTYRCFGSYNNHAWSFPSEPVKLLVTG

[0284] DIENTSLAPEDPTFPADTWGTYLLTTETGLQKDHALWDHTAQNLLRTTTPAPRP

[0285] PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDQSFGLLDPKLCYLLDGIL

[0286] FIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRD

[0287] PEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS

[0288] TATKDTYDALHMQALPPRRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRD

[0289] FAAYRSEGRGSLLTCGDVEENPGPMMVSKGEELFTGVVPILVELDGDVNGHKFSV

[0290] SGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDF

[0291] FKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNIL

[0292] GHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGD

[0293] GPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (sequence of CD8 signal peptide, NKp46 extracellular sequence, CD3 transmembrane sequence, CD3Z intracellular sequence + CD28 intracellular sequence, T2A, GFP amino acid sequence (SEQ ID NO: 76))

[0294] The nucleotide sequence encoding Nkp46-z-28-GFP is as follows:

[0295] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCT

[0296] CCACGCCGCCAGGCCGCAGCAGCAGACTCTCCCAAAACCGTTCATCTGG

[0297] GCCGAGCCCCATTTCATGGTTCCAAAGGAAAAGCAAGTGACCATCTGTTG

[0298] CCAGGGAAATTATGGGGCTGTTGAATACCAGCTGCACTTTGAAGGAAGC

[0299] CTTTTTGCCGTGGACAGACCAAAACCCCCTGAGCGGATTAACAAAGTCCA

[0300] ATTCTACATCCCGGACATGAACTCCCGCATGGCAGGGCAATACAGCTGC

[0301] ATCTATCGGGTTGGGGAGCTCTGGTCAGAGCCCAGCAACTTGCTGGATCT

[0302] GGTGGTAACAGAAATGTATGACACACCCACCCTCTCGGTTCATCCTGGAC

[0303] CCGAAGTGATCTCGGGAGAGAAGGTGACCTTCTACTGCCGTCTAGACACT

[0304] GCAACAAGCATGTTCTTACTGCTCAAGGAGGGAAGATCCAGCCACGTAC

[0305] AGCGCGGATACGGGAAGGTCCAGGCGGAGTTCCCCCTGGGCCCTGTGAC

[0306] CACAGCCCACAGAGGGACATACCGATGTTTTGGCTCCTATAACAACCATG

[0307] CCTGGTCTTTCCCCAGTGAGCCAGTGAAGCTCCTGGTCACAGGCGACATT

[0308] GAGAACACCAGCCTTGCACCTGAAGACCCCACCTTTCCTGCAGACACTTG

[0309] GGGCACCTACCTTTTAACCACAGAGACGGGACTCCAGAAAGACCATGCC

[0310] CTCTGGGATCACACTGCCCAGAATCTCCTTCGGACCACGACGCCAGCGCC

[0311] GCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGC

[0312] GCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGG

[0313] GGCTGGACTTCGCCTGTGATCAGAGCTTTGGCCTGCTGGATCCCAAACTC

[0314] TGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCC

[0315] TTGTTCCTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACC

[0316] AGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGA

[0317] GGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGG

[0318] GGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTG

[0319] CAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGC

[0320] GAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTA

[0321] CAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCT

[0322] CGCAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGA

[0323] CTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCA

[0324] CCACGCGACTTCGCAGCCTATCGCTCCGAGGGCAGAGGAAGTCTTCTAAC

[0325] ATGCGGTGACGTGGAGGAGAATCCCGGCCCTATGGTGAGCAAGGGCGAG

[0326] GAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACG

[0327] TAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCAC

[0328] CTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCG

[0329] TGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTC

[0330] AGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCAT

[0331] GCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGC

[0332] AACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGA

[0333] ACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCT

[0334] GGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATG

[0335] GCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACA

[0336] ACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACAC

[0337] CCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCA

[0338] CCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGT

[0339] CCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGC

[0340] TGTACAAG (encoding CD8 signal peptide, NKp46 extracellular sequence, CD3 transmembrane sequence, CD3Z, etc.)

[0341] Intracellular sequence + CD28 intracellular sequence, T2A, GFP nucleotide sequence (SEQ ID NO: 77)

[0342] The amino acid sequence of the extracellular domain of NKG2D is as follows:

[0343] The nucleotide sequence encoding the NKG2D extracellular domain of IWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYES QASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILS PNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV (SEQ ID NO: 6) is as follows:

[0344] ATATGGAGTGCTGTATTCCTAAACTCATTATTCAACCAAGAAGTTCAAAT

[0345] TCCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAAAACTGGATATGTTACA

[0346] AAAATAACTGCTACCAATTTTTTGATGAGAGTAAAAACTGGTATGAGAGCCA

[0347] GGCTTCTTGTATGTCTCAAAATGCCAGCCTTCTGAAAGTATACAGCAAAGAG

[0348] GACCAGGATTTACTTAAACTGGTGAAGTCATATCATTGGATGGGACTAGTACA

[0349] CATTCCAACAAATGGATCTTGGCAGTGGGAAGATGGCTCCATTCTCTCACCC

[0350] AACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGTGCACTCTATGCCT

[0351] CGAGCTTTAAAGGCTATATAGAAAACTGTTCAACTCCAAATACGTACATCTGC

[0352] ATGCAAAGGACTGTG(SEQ ID NO:1)

[0353] The amino acid sequence of the extracellular domain of DNAM1 is as follows:

[0354] EEVLWHTSVPFAENMSLECVYPSMGILTQVEWFKIGTQQDSIAIFSPTHGMVIRKPYAERVYFLNSTMASNNMTLFFRNASEDDVGYYSCSLYTYPQGTWQKVIQVVQSDSFEAAVPSNSHIVSEPGKNVTLTCQPQMTWPVQAVRWEKIQPRQIDLLTYCNLVHGRNFTSKFPRQIVSNCSHGRWSVIVIPDVTVSDSGLYRCYLQASAGENETFVMRLTVAEGKTDNQYTLFVA(SEQ ID NO:7)

[0355] The nucleotide sequence encoding the extracellular domain of DNAM1 is as follows:

[0356] GAAGAGGTGCTTTGGCATACATCAGTTCCCTTTGCCGAGAACATGTCTCT

[0357] AGAATGTGTGTATCCATCAATGGGCATCTTAACACAGGTGGAGTGGTTCAAG

[0358] ATCGGGACCCAGCAGGATTCCATAGCCATTTTCAGCCCTACTCATGGCATGGT

[0359] CATAAGGAAGCCCTATGCTGAGAGGGTTTACTTTTTGAATTCAACGATGGCTT

[0360] CCAATAACATGACTCTTTTCTTTCGGAATGCCTCTGAAGATGATGTTGGCTAC

[0361] TATTCCTGCTCTCTTTACACTTACCCACAGGGAACTTGGCAGAAGGTGATACA

[0362] GGTGGTTCAGTCAGATAGTTTTGAGGCAGCTGTGCCATCAAATAGCCACATT

[0363] GTTTCGGAACCTGGAAAGAATGTCACACTCACTTGTCAGCCTCAGATGACGT

[0364] GGCCTGTGCAGGCAGTGAGGTGGGAAAAGATCCAGCCCCGTCAGATCGACC

[0365] TCTTAACTTACTGCAACTTGGTCCATGGCAGAAATTTCACCTCCAAGTTCCCA

[0366] AGACAAATAGTGAGCAACTGCAGCCACGGAAGGTGGAGCGTCATCGTCATC

[0367] CCCGATGTCACAGTCTCAGACTCGGGGCTTTACCGCTGCTACTTGCAGGCCA

[0368] GCGCAGGAGAAAACGAAACCTTCGTGATGAGATTGACTGTAGCCGAGGGTA

[0369] AAACCGATAACCAATATACCCTCTTTGTGGCT(SEQ ID NO:2)

[0370] The amino acid sequence of the extracellular domain of NKp30 is as follows:

[0371] LWVSQPPEIRTLEGSSAFLPCSFNASQGRLAIGSVTWFRDEVVPGKEVRNG TPEFRGRLAPLASSRFLHDHQAELHIRDVRGHDASIYVCRVEVLGLGVGTGNGT RLVVEKEHPQLG(SEQ ID NO:8)

[0372] The nucleotide sequence encoding the extracellular domain of NKp30 is as follows:

[0373] CTCTGGGTGTCCCAGCCCCCTGAGATTCGTACCCTGGAAGGATCCTCTG

[0374] CCTTCCTGCCCTGCTCCTTCAATGCCAGCCAAGGGAGACTGGCCATTGGCTC

[0375] CGTCACGTGGTTCCGAGATGAGGTGGTTCCAGGGAAGGAGGTGAGGAATGG

[0376] AACCCCAGAGTTCAGGGGCCGCCTGGCCCCACTTGCTTCTTCCCGTTTCCTC

[0377] CATGACCACCAGGCTGAGCTGCACATCCGGGACGTGCGAGGCCATGACGCC

[0378] AGCATCTACGTGTGCAGAGTGGAGGTGCTGGGCCTTGGTGTCGGGACAGGG

[0379] AATGGGACTCGGCTGGTGGTGGAGAAAGAACATCCTCAGCTAGGG(SEQ ID NO:3)

[0380] The amino acid sequence of the extracellular domain of NKp44 is as follows:

[0381] QSKAQVLQSVAGQTLTVRCQYPPTGSLYEKKGWCKEASALVCIRLVTSSKP

[0382] RTMAWTSRFTIWDDPDAGFFTVTMTDLREEDSGHYWCRIYRPSDNSVSKSVRF

[0383] YLVVSPASASTQTSWTPRDLVSSQTQTQSCVPPTAGARQAPESPSTIPVPSQPQNS

[0384] TLRPGPAAPIA(SEQ ID NO: 9)

[0385] The nucleotide sequence encoding the extracellular domain of NKp44 is as follows:

[0386] CAATCCAAGGCTCAGGTACTTCAAAGTGTGGCAGGGCAGACGCTAACCGTGAGATGCCAGTACCCGCCCACGGGCAGTCTCTACGAGAAGAAAGGCTGGTGTAAGGAGGCTTCAGCACTTGTGTGCATCAGGTTAGTCACCAGCTCCAAGCCCAGGACGATGGCTTGGACCTCTCGATTCACAATCTGGGACGACCCTGATGCTGGCTTCTTCACTGTCACCATGACTGATCTGAGAGAGGAAGACTCAGGACATTACTGGTGTAGAATCTACCGCCCTTCTGACAACTCTGTCTCTAAGTCCGTCAGATTCTATCTGGTGGTATCTCCAGCCTCTGCCTCCACACAGACCTCCTGGACTCCCCGCGACCTGGTCTCTTCACAGACCCAGACCCAGAGCTGTGTGCCTCCCACTGCAGGAGCCAGACAAGCCCCTGAGTCTCCATCTACCATCCCTGTCCCTTCACAGCCACAGAACTCCACGCTCCGCCCTGGCCCTGCAGCCCCCATTGCC(SEQ ID NO: 4)

[0387] The amino acid sequence of the extracellular domain of NKp46 is as follows:

[0388] QQQTLPKPFIWAEPHFMVPKEKQVTICCQGNYGAVEYQLHFEGSLFAVDRP

[0389] KPPERINKVQFYIPDMNSRMAGQYSCIYRVGELWSEPSNLLDLVVTEMYDTPTL

[0390] SVHPGPEVISGEKVTFYCRLDTATSMFLLLKEGRSSHVQRGYGKVQAEFPLGPV

[0391] TTAHRGTYRCFGSYNNHAWSFPSEPVKLLVTGDIENTSLAPEDPTFPADTWGTY

[0392] LLTTETGLQKDHALWDHTAQNLLR(SEQ ID NO: 10)

[0393] The nucleotide sequence encoding the extracellular domain of NKp46 is as follows:

[0394] CAGCAGCAGACTCTCCCAAAACCGTTCATCTGGGCCGAGCCCCATTTCATGGTTCCAAAGGAAAAGCAAGTGACCATCTGTTGCCAGGGAAATTATGGGGCTGTTGAATACCAGCTGCACTTTGAAGGAAGCCTTTTTGCCGTGGACAGACCAAAACCCCCTGAGCGGATTAACAAAGTCCAATTCTACATCCCGGACATGAACTCCCGCATGGCAGGGCAATACAGCTGCATCTATCGGGTTGGGGAGCTCTGGTCAGAGCCCAGCAACTTGCTGGATCTGGTGGTAACAGAAATGTATGACACACCCACCCTCTCGGTTCATCCTGGACCCGAAGTGATCTCGGGAGAGAAGGTGACCTTCTACTGCCGTCTAGACACTGCAACAAGCATGTTCTTACTGCTCAAGGAGGGAAGATCCAGCCACGTACAGCGCGGATACGGGAAGGTCCAGGCGGAGTTCCCCCTGGGCCCTGTGACCACAGCCCACAGAGGGACATACCGATGTTTTGGCTCCTATAACAACCATGCCTGGTCTTTCCCCAGTGAGCCAGTGAAGCTCCTGGTCACAGGCGACATTGAGAACACCAGCCTTGCACCTGAAGACCCCACCTTTCCTGCAGACACTTGGGGCACCTACCTTTTAACCACAGAGACGGGACTCCAGAAAGACCATGCCCTCTGGGATCACACTGCCCAGAATCTCCTTCGG(SEQ ID NO: 5)

[0395] The amino acid sequence of the hinge region of CD8α is as follows:

[0396] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(SEQ ID NO:11)

[0397] The amino acid sequence of the transmembrane region CD8α is as follows:

[0398] IYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO: 12)

[0399] The amino acid sequence of the transmembrane region CD3z is as follows:

[0400] LCYLLDGILFIYGVILTALFL (SEQ ID NO: 13)

[0401] The amino acid sequence of the intracellular signal transduction region derived from CD3ζ is as follows:

[0402] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT KDTYDALHMQALPPR (SEQ ID NO: 14)

[0403] The amino acid sequence of the co-stimulatory signal transduction region 4-1BB is as follows:

[0404] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 15)

[0405] The amino acid sequence of CD28 in the co-stimulatory signal transduction region is as follows:

[0406] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO: 16)

[0407] The amino acid sequence of the co-stimulatory signal transduction region OX40 is as follows:

[0408] RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI(SEQ ID NO: 17)

[0409] The amino acid sequence of the guide peptide CD8α is as follows:

[0410] MALPVTALLLPLALLLHAARP (SEQ ID NO: 18)

[0411] The amino acid sequence of T2A is as follows:

[0412] EGRGSLLTCGDVEENPGP (SEQ ID NO: 78)

[0413] T2A is a self-cleaving polypeptide that allows a single dsDNA to simultaneously express multiple genes in a cell.

[0414] The nucleotide sequence encoding T2A is as follows:

[0415] GAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATC CCGGCCCT (SEQ ID NO: 79)

[0416] The amino acid sequence of the GFP peptide is as follows:

[0417] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTT

[0418] GKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD

[0419] GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADK

[0420] QKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKD

[0421] PNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO: 80)

[0422] The nucleotide sequence encoding the GFP peptide is as follows:

[0423] The amino acid sequence of the chimeric antigen receptor NKG2D-8TM-28z is as follows:

[0424] MALPVTALLLPLALLLHAARPIWSAVFLNSLFNQEVQIPLTESYCGPCPKN

[0425] WICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYH

[0426] WMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNT

[0427] YICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI

[0428] WAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPP

[0429] RDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE

[0430] MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA

[0431] TKDTYDALHMQALPPR(SEQ ID NO:19)

[0432] The nucleotide sequence encoding the chimeric antigen receptor NKG2D-8TM-28z is as follows:

[0433]

[0434] The amino acid sequence of the chimeric antigen receptor NKG2D-3TM-z is as follows:

[0435] MALPVTALLLPLALLLHAARPIWSAVFLNSLFNQEVQIPLTESYCGPCPKN

[0436] WICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYH

[0437] WMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNT

[0438] YICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDQSF

[0439] GLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGR

[0440] REEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER

[0441] RRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 20)

[0442] The nucleotide sequence encoding the chimeric antigen receptor NKG2D-3TM-z is as follows:

[0443]

[0444] The amino acid sequence of the chimeric antigen receptor NKG2D-3TM-z-28 is as follows:

[0445] MALPVTALLLPLALLLHAARPIWSAVFLNSLFNQEVQIPLTESYCGPCPKN

[0446] WICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYH

[0447] WMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNT

[0448] YICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDQSF

[0449] GLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGR

[0450] REEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER

[0451] RRGKGHDGLYQGLSTATKDTYDALHMQALPPRRSKRSRLLHSDYMNMTPRRP

[0452] GPTRKHYQPYAPPRDFAAYRS(SEQ ID NO: 21)

[0453] The nucleotide sequence encoding the chimeric antigen receptor NKG2D-3TM-z-28 is as follows:

[0454]

[0455] Amino acid sequence of chimeric antigen receptor NKG2D-3TM-z-BB:

[0456] MALPVTALLLPLALLLHAARPIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 22)

[0457] The nucleotide sequence encoding chimeric antigen receptor NKG2D-3TM-z-BB is as follows:

[0458] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCC

[0459] ACGCCGCCAGGCCGATATGGAGTGCTGTATTCCTAAACTCATTATTCAACCAA

[0460] GAAGTTCAAATTCCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAAAACT

[0461] GGATATGTTACAAAAATAACTGCTACCAATTTTTTGATGAGAGTAAAAACTGG

[0462] TATGAGAGCCAGGCTTCTTGTATGTCTCAAAATGCCAGCCTTCTGAAAGTATA

[0463] CAGCAAAGAGGACCAGGATTACTTAAACTGGTGAAGTCATATCATTGGATG

[0464] GGACTAGTACACATTCCAACAAATGGATCTTGGCAGTGGGAAGATGGCTCCA

[0465] TTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGTGC

[0466] ACTCTATGCCTCGAGCTTTAAAGGCTATATAGAAAACTGTTCAACTCCAAATA

[0467] CGTACATCTGCATGCAAAGGACTGTGACCACGACGCCAGCGCCGCGACCAC

[0468] CAACACCGGCGCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGG

[0469] CGTGCCGGCCAGCGGCGGGGGCGCAGTGCACACGAGGGGGCTGGACTTC

[0470] GCCTGTGATCAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGG

[0471] ATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGAGAGTG

[0472] AAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCA

[0473] GCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGTACGATGTTTTGGAC

[0474] AAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGGAAGGAAGAA

[0475] CCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGC

[0476] CTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACG

[0477] ATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCT

[0478] TCACATGCAGGCCCTGCCCCCTCGCAAACGGGGCAGAAAGAAACTCCTGTAT

[0479] ATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATG

[0480] GCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG(SEQ ID NO:34)

[0481] Amino acid sequence of chimeric antigen receptor NKG2D-3TM-z-OX40:

[0482] MALPVTALLLPLALLLHAARPIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTVTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI(SEQ ID NO:23)

[0483] The nucleotide sequence encoding chimeric antigen receptor NKG2D-3TM-z-OX40 is as follows:

[0484] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCC

[0485] ACGCCGCCAGGCCGATATGGAGTGCTGTATTCCTAAACTCATTATTCAACCAA

[0486] GAAGTTCAAATTCCCTTGACCGAAAGTTACTGTGGCCCATGTCCTAAAAACT

[0487] GGATATGTTACAAAAATAACTGCTACCAATTTTTTGATGAGAGTAAAAACTGG

[0488] TATGAGAGCCAGGCTTCTTGTATGTCTCAAAATGCCAGCCTTCTGAAAGTATA

[0489] CAGCAAAGAGGACCAGGATTTACTTAAACTGGTGAAGTCATATCATTGGATG

[0490] GGACTAGTACACATTCCAACAAATGGATCTTGGCAGTGGGAAGATGGCTCCA

[0491] TTCTCTCACCCAACCTACTAACAATAATTGAAATGCAGAAGGGAGACTGTGC

[0492] ACTCTATGCCTCGAGCTTTAAAGGCTATATAGAAAACTGTTCAACTCCAAATA

[0493] CGTACATCTGCATGCAAAGGACTGTGACCACGACGCCAGCGCCGCGACCAC

[0494] CAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGG

[0495] CGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTC

[0496] GCCTGTGATCAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTACCTGCTGG

[0497] ATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTGAGAGTG

[0498] AAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCA

[0499] GCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGAC

[0500] AAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAA

[0501] CCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGC

[0502] CTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACG

[0503] ATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCT

[0504] TCACATGCAGGCCCTGCCCCCTCGCCGGAGGGACCAGAGGCTGCCCCCCGA

[0505] TGCCCACAAGCCCCCTGGGGGAGGCAGTTTCCGGACCCCCATCCAAGAGGA

[0506] GCAGGCCGACGCCCACTCCACCCTGGCCAAGATC(SEQ ID NO:35)

[0507] The amino acid sequence of the chimeric antigen receptor DNAM1-z-28 is as follows:

[0508] MALPVTALLLPLALLLHAARPEEVLWHTSVPFAENMSLECVYPSMGILTQVEWFKIGTQQDSIAIFSPTHGMVIRKPYAERVYFLNSTMASNNMTLFFRNASEDDVGYYSCSLYTYPQGTWQKVIQVVQSDSFEAAVPSNSHIVSEPGKNVTLTCQPQMTWPVQAVRWEKIQPRQIDLLTYCNLVHGRNFTSKFPRQIVSNCSHGRWSVIVIPDVTVSDSGLYRCYLQASAGENETFVMRLTVAEGKTDNQYTLFVATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO: 24)

[0509] The nucleotide sequence encoding chimeric antigen receptor DNAM1-z-28 is as follows:

[0510] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCT

[0511] CCACGCCGCCAGGCCGGAAGAGGTGCTTTGGCATACATCAGTTCCCTTTG

[0512] CCGAGAACATGTCTCTAGAATGTGTGTATCCATCAATGGGCATCTTAACA

[0513] CAGGTGGAGTGGTTCAAGATCGGGACCCAGCAGGATTCCATAGCCATTTT

[0514] CAGCCCTACTCATGGCATGGTCATAAGGAAGCCCTATGCTGAGAGGGTTT

[0515] ACTTTTTGAATTCAACGATGGCTTCCAATAACATGACTCTTTTCTTTCGGA

[0516] ATGCCTCTGAAGATGATGTTGGCTACTATTCCTGCTCTCTTTACACTTACC

[0517] CACAGGGAACTTGGCAGAAGGTGATACAGGTGGTTCAGTCAGATAGTTT

[0518] TGAGGCAGCTGTGCCATCAAATAGCCACATTGTTTCGGAACCTGGAAAG

[0519] AATGTCACACTCACTTGTCAGCCTCAGATGACGTGGCCTGTGCAGGCAGT

[0520] GAGGTGGGAAAAGATCCAGCCCCGTCAGATCGACCTCTTAACTTACTGC

[0521] AACTTGGTCCATGGCAGAAATTTCACCTCCAAGTTCCCAAGACAAATAGT

[0522] GAGCAACTGCAGCCACGGAAGGTGGAGCGTCATCGTCATCCCCGATGTC

[0523] ACAGTCTCAGACTCGGGGCTTTACCGCTGCTACTTGCAGGCCAGCGCAGG

[0524] AGAAAACGAAACCTTCGTGATGAGATTGACTGTAGCCGAGGGTAAAACC

[0525] GATAACCAATATACCCTCTTTGTGGCTACCACGACGCCAGCGCCGCGACC

[0526] ACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAG

[0527] AGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGG

[0528] ACTTCGCCTGTGATCAGAGCTTTGGCCTGCTGGATCCCAAACTCTGCTAC

[0529] CTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTC

[0530] CTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGG

[0531] GCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTA

[0532] CGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAG

[0533] CCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAA

[0534] GATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCC

[0535] GGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCAC

[0536] CAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCAGG

[0537] AGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCC

[0538] GCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACG

[0539] CGACTTCGCAGCCTATCGCTCC(SEQ ID NO:36)

[0540] The amino acid sequence of the chimeric antigen receptor DNA M1-3-28 is as follows:

[0541] MALPVTALLLPLALLLHAARPEEVLWHTSVPFAENMSLECVYPSMGILTQVEWFKIGTQQDSIAIFSPTHGMVIRKPYAERVYFLNSTMASNNMTLFFRNASEDDVGYYSCSLYTYPQGTWQKVIQVVQSDSFEAAVPSNSHIVSEPGKNVTLTCQPQMTWPVQAVRWEKIQPRQIDLLTYCNLVHGRNFTSKFPRQIVSNCSHGRWSVIVIPDVTVSDSGLYRCYLQASAGENETFVMRLTVAEGKTDNQYTLFVATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO: 25)

[0542] The nucleotide sequence encoding the chimeric antigen receptor DNAM1-3-28 is as follows:

[0543] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCC

[0544] ACGCCGCCAGGCCGGAAGAGGTGCTTTGGCATACATCAGTTCCCTTTGCCGA

[0545] GAACATGTCTCTAGAATGTGTGTATCCATCAATGGGCATCTTAACACAGGTGG

[0546] AGTGGTTCAAGATCGGGACCCAGCAGGATTCCATAGCCATTTTCAGCCCTAC

[0547] TCATGGCATGGTCATAAGGAAGCCCTATGCTGAGAGGGTTTACTTTTTGAATT

[0548] CAACGATGGCTTCCAATAACATGACTCTTTTCTTTCGGAATGCCTCTGAAGAT

[0549] GATGTTGGCTACTATTCCTGCTCTCTTTACACTTACCCACAGGGAACTTGGCA

[0550] GAAGGTGATACAGGTGGTTCAGTCAGATAGTTTTGAGGCAGCTGTGCCATCA

[0551] AATAGCCACATTGTTTCGGAACCTGGAAAGAATGTCACACTCACTTGTCAGC

[0552] CTCAGATGACGTGGCCTGTGCAGGCAGTGAGGTGGGAAAAGATCCAGCCCC

[0553] GTCAGATCGACCTCTTAACTTACTGCAACTTGGTCCATGGCAGAAATTTCACC

[0554] TCCAAGTTCCCAAGACAAATAGTGAGCAACTGCAGCCACGGAAGGTGGAGC

[0555] GTCATCGTCATCCCCGATGTCACAGTCTCAGACTCGGGGCTTTACCGCTGCTA

[0556] CTTGCAGGCCAGCGCAGGAGAAAACGAAACCTTCGTGATGAGATTGACTGT

[0557] AGCCGAGGGTAAAACCGATAACCAATATACCCTCTTTGTGGCTACCACGACG

[0558] CCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTG

[0559] TCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACAC

[0560] GAGGGGGCTGGACTTCGCCTGTGATCAGAGCTTTGGCCTGCTGGATCCCAAA

[0561] CTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCC

[0562] TTGTTCCTGAGAGTGAAGaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccg

[0563] ccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctcc(SEQ ID NO:37)

[0564] The amino acid sequence of the chimeric antigen receptor DNA M1-3-28z is as follows:

[0565] MALPVTALLLPLALLLHAARPEEVLWHTSVPFAENMSLECVYPSMGILTQV

[0566] EWFKIGTQQDSIAIFSPTHGMVIRKPYAERVYFLNSTMASNNMTLFFRNASEDD

[0567] VGYYSCSLYTYPQGTWQKVIQVVQSDSFEAAVPSNSHIVSEPGKNVTLTCQPQM

[0568] TWPVQAVRWEKIQPRQIDLLTYCNLVHGRNFTSKFPRQIVSNCSHGRWSVIVIPD

[0569] VTVSDSGLYRCYLQASAGENETFVMRLTVAEGKTDNQYTLFVATTTPAPRPPTP

[0570] APTIASQPLSLRPEACRPAAGGAVHTRGLDFACDQSFGLLDPKLCYLLDGILFIY

[0571] GVILTALFLRVKRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSR

[0572] VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN

[0573] PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH

[0574] MQALPPR(SEQ ID NO:26)

[0575] The nucleotide sequence encoding the chimeric antigen receptor DNA M1-3-28z is as follows:

[0576]

[0577] The amino acid sequence of the chimeric antigen receptor DNA M1-8-28z is as follows:

[0578] MALPVTALLLPLALLLHAARPEEVLWHTSVPFAENMSLECVYPSMGILTQV

[0579] EWFKIGTQQDSIAIFSPTHGMVIRKPYAERVYFLNSTMASNNMTLFFRNASEDD

[0580] VGYYSCSLYTYPQGTWQKVIQVVQSDSFEAAVPSNSHIVSEPGKNVTLTCQPQM

[0581] TWPVQAVRWEKIQPRQIDLLTYCNLVHGRNFTSKFPRQIVSNCSHGRWSVIVIPD

[0582] VTVSDSGLYRCYLQASAGENETFVMRLTVAEGKTDNQYTLFVATTTPAPRPPTP

[0583] APTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITL

[0584] YCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADA

[0585] PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL

[0586] QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0587] (SEQ ID NO: 27)

[0588] The nucleotide sequence encoding the chimeric antigen receptor DNA M1-8-28z is as follows:

[0589]

[0590] The amino acid sequence of the chimeric antigen receptor Nkp30-z-28 is as follows:

[0591] MALPVTALLLPLALLLHAARPLWVSQPPEIRTLEGSSAFLPCSFNASQGR

[0592] LAIGSVTWFRDEVVPGKEVRNGTPEFRGRLAPLASSRFLHDHQAELHIRDVR

[0593] GHDASIYVCRVEVLGLGVGTGNGTRLVVEKEHPQLGTTTPAPRPPTPAPTIAS

[0594] QPLSLRPEACRPAAGGAVHTRGLDFACDQSFGLLDPKLCYLLDGILFIYGVILTA

[0595] LFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP

[0596] RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY

[0597] DALHMQALPPRRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0598] (SEQ ID NO: 28)

[0599] The nucleotide sequence encoding the chimeric antigen receptor Nkp30-z-28 is as follows:

[0600]

[0601] MALPVTALLLPLALLLHAARPQSKAQVLQSVAGQTLTVRCQYPPTGSLYEK

[0602] KGWCKEASALVCIRLVTSSKPRTMAWTSRFTIWDDPDAGFFTVTMTDLREEDS

[0603] GHYWCRIYRPSDNSVSKSVRFYLVVSPASASTQTSWTPRDLVSSQTQTQSCVPP

[0604] TAGARQAPESPSTIPVPSQPQNSTLRPGPAAPIATTTPAPRPPTPAPTIASQPLSLRP

[0605] EACRPAAGGAVHTRGLDFACDQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKF

[0606] SRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE

[0607] GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ

[0608] ALPPRRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO:29)

[0609] The nucleotide sequence encoding the chimeric antigen receptor Nkp44-z-28 is as follows:

[0610] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCT

[0611] CCACGCCGCCAGGCCGCAATCCAAGGCTCAGGTACTTCAAAGTGTGGCA

[0612] GGGCAGACGCTAACCGTGAGATGCCAGTACCCGCCCACGGGCAGTCTCT

[0613] ACGAGAAGAAAGGCTGGTGTAAGGAGGCTTCAGCACTTGTGTGCATCAG

[0614] GTTAGTCACCAGCTCCAAGCCCAGGACGATGGCTTGGACCTCTCGATTCA

[0615] CAATCTGGGACGACCCTGATGCTGGCTTCTTCACTGTCACCATGACTGAT

[0616] CTGAGAGAGGAAGACTCAGGACATTACTGGTGTAGAATCTACCGCCCTT

[0617] CTGACAACTCTGTCTCTAAGTCCGTCAGATTCTATCTGGTGGTATCTCCA

[0618] GCCTCTGCCTCCACACAGACCTCCTGGACTCCCCGCGACCTGGTCTCTTC

[0619] ACAGACCCAGACCCAGAGCTGTGTGCCTCCCACTGCAGGAGCCAGACAA

[0620] GCCCCTGAGTCTCCATCTACCATCCCTGTCCCTTCACAGCCACAGAACTC

[0621] CACGCTCCGCCCTGGCCCTGCAGCCCCCATTGCCACCACGACGCCAGCGC

[0622] CGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTG

[0623] CGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGG

[0624] GGGCTGGACTTCGCCTGTGATCAGAGCTTTGGCCTGCTGGATCCCAAACT

[0625] CTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGC

[0626] CTTGTTCCTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTAC

[0627] CAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAG

[0628] AGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGG

[0629] GGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACT

[0630] GCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGG

[0631] CGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGT

[0632] ACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCC

[0633] CTCGCAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACAT

[0634] GACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCC

[0635] CACCACGCGACTTCGCAGCCTATCGCTCC(SEQ ID NO:41)

[0636] The amino acid sequence of the chimeric antigen receptor Nkp46-z-28 is as follows:

[0637] MALPVTALLLPLALLLHAARPQQQTLPKPFIWAEPHFMVPKEKQVTICCQGNYGAVEYQLHFEGSLFAVDRPKPPERINKVQFYIPDMNSRMAGQYSCIYRVGELWSEPSNLLDLVVTEMYDTPTLSVHPGPEVISGEKVTFYCRLDTATSMFLLLKEGRSSHVQRGYGKVQAEFPLGPVTTAHRGTYRCFGSYNNHAWSFPSEPVKLLVTGDIENTSLAPEDPTFPADTWGTYLLTTETGLQKDHALWDHTAQNLLRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO: 30)

[0638] The nucleotide sequence encoding the chimeric antigen receptor Nkp46-z-28 is as follows:

[0639] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCT

[0640] CCACGCCGCCAGGCCGCAGCAGCAGACTCTCCCAAAACCGTTCATCTGG

[0641] GCCGAGCCCCATTTCATGGTTCCAAAGGAAAAGCAAGTGACCATCTGTTG

[0642] CCAGGGAAATTATGGGGCTGTTGAATACCAGCTGCACTTTGAAGGAAGC

[0643] CTTTTTGCCGTGGACAGACCAAAACCCCCTGAGCGGATTAACAAAGTCCA

[0644] ATTCTACATCCCGGACATGAACTCCCGCATGGCAGGGCAATACAGCTGC

[0645] ATCTATCGGGTTGGGGAGCTCTGGTCAGAGCCCAGCAACTTGCTGGATCT

[0646] GGTGGTAACAGAAATGTATGACACACCCACCCTCTCGGTTCATCCTGGAC

[0647] CCGAAGTGATCTCGGGAGAGAAGGTGACCTTCTACTGCCGTCTAGACACT

[0648] GCAACAAGCATGTTCTTACTGCTCAAGGAGGGAAGATCCAGCCACGTAC

[0649] AGCGCGGATACGGGAAGGTCCAGGCGGAGTTCCCCCTGGGCCCTGTGAC

[0650] CACAGCCCACAGAGGGACATACCGATGTTTTGGCTCCTATAACAACCATG

[0651] CCTGGTCTTTCCCCAGTGAGCCAGTGAAGCTCCTGGTCACAGGCGACATT

[0652] GAGAACACCAGCCTTGCACCTGAAGACCCCACCTTTCCTGCAGACACTTG

[0653] GGGCACCTACCTTTTAACCACAGAGACGGGACTCCAGAAAGACCATGCC

[0654] CTCTGGGATCACACTGCCCAGAATCTCCTTCGGACCACGACGCCAGCGCC

[0655] GCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGC

[0656] GCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGG

[0657] GGCTGGACTTCGCCTGTGATCAGAGCTTTGGCCTGCTGGATCCCAAACTC

[0658] TGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCC

[0659] TTGTTCCTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACC

[0660] AGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGA

[0661] GGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGG

[0662] GGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTG

[0663] CAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGC

[0664] GAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTA

[0665] CAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCT

[0666] CGCAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGA

[0667] CTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCA

[0668] CCACGCGACTTCGCAGCCTATCGCTCC(SEQ ID NO:42)

[0669] EF1α promoter sequence:

[0670] GGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAG(SEQ IDNO:49)

[0671] BGH polyA signal sequence:

[0672] CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG(SEQ ID NO:51)

[0673] Left homologous arm sequence for the TRAC locus:

[0674] AACATACCATAAACCTCCCATTCTGCTAATGCCCAGCCTAAGTTGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTGCTTTGCTGGGCCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGTTTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCCTGCCGTG(SEQ ID NO:52)

[0675] Right homologous arm sequence for the TRAC locus:

[0676] ACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCTATTCACCGATTGATTCTCAAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAACTGTGCTAGACA TGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATGACTTTGCATGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGG GCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTGGTCAATGTCTAAAACTCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAA ACCCTCTTTTTACTAAAACAGTGAGCCTTGTTCTGGCAGTCCAGAGAATGACACGGGAAAAAAGCAGATGAAGAGAAGGTGGCAGGAGAGGGCACGTGGCCCAGCCTCAGTCTCTCCAAC(SEQ ID NO:53)

[0677] Guide RNA N20:

[0678] TRAC:5'-AGAGTCTCTCAGCTGGTACACGG-3'(SEQ ID NO:43)

[0679] PD1:5'-CGACTGGCCAGGGCGCCTGT-3'(SEQ ID NO:44)

[0680] Regnase-1:5'-AAGGAGGTCTTCTCCTGCCG-3'(SEQ ID NO:45)

[0681] Roquin-1:5'-TGAAGACACAAAGCATTATG-3'(SEQ ID NO:46)

[0682] CISH:5'-GGCGCATCCTCCTTAGGCAT-3':(SEQ ID NO:47)

[0683] BcoR:5'-AGCACGGCCCATAGGATCGA-3'(SEQ ID NO:48)

[0684] 2. Cell culture:

[0685] according to Figure 4 The preparation of enhanced chimeric antigen receptor-T cells included: sorting and activation; KI; expansion; and harvesting. On Day 0, T cells were sorted and activated in basal medium: OpTmizer + 2.6% supplement + 5% ISR + 1% GlutaMax (commercially available). IL-2 100-500 IU / mL was added, followed by 5-10 μM ZOL (zoretinoic acid). After electroporation, Dasatinib was added on Day 3 to obtain enhanced chimeric antigen receptor-T cells.

[0686] exist Figure 4 In this context, "No EP" represents cells that have not been electroporated; "NKG2D-z" represents γδT cells containing the chimeric antigen receptor NKG2D-3TM-z, which are prepared by transfecting γδT cells with the donor template NKG2D-3TM-z-GFP; "NKG2D-z-28" represents γδT cells containing the chimeric antigen receptor NKG2D-3TM-z-28, which are prepared by transfecting γδT cells with the donor template NKG2D-3TM-z-28-GFP; "NKG2D-z-BB" represents γδT cells containing the chimeric antigen receptor NKG2D-3TM-z-BB, which are prepared by transfecting γδT cells with the donor template NKG2D-3TM-z-BB-GFP; NKG2D-z -OX40 represents γδT cells containing the chimeric antigen receptor NKG2D-3TM-z-OX40, prepared by transfecting γδT cells with the donor template NKG2D-3TM-z-OX40-GFP; NKG2D-8-28z represents γδT cells containing the chimeric antigen receptor NKG2D-8TM-28z, prepared by transfecting γδT cells with the donor template NKG2D-8TM-28z-GFP; NKG2D-z-28+RKO represents γδT cells containing the chimeric antigen receptor NKG2D-3TM-z-28 and with Regnase-1 knockout, prepared by transfecting γδT cells with the donor template NKG2D-3TM-z-28-GFP and using SEQ γδT cells were prepared by transfecting γδT cells with the Guide RNA N20 sequence shown in SEQ ID NO: 39; NKG2D-z+RKO represents γδT cells containing the chimeric antigen receptor NKG2D-3TM-z and with Regnase-1 knockout, which were prepared by transfecting γδT cells with the donor template NKG2D-3TM-z-GFP and the Guide RNA N20 sequence shown in SEQ ID NO: 39.

[0687] The Regnase-1 site knockout was performed by electroporation of cells using the Guide RNA N20 sequence (100 μM) shown in SEQ ID NO: 39, P3 buffer, and Cas9 (10 mg / mL) (for example, using steps 2.2-2.4, but without adding the donor template).

[0688] 2.1 Cell sorting activation

[0689] 1. Cell sorting: Take the frozen or freshly isolated PBMCs out of the liquid nitrogen tank, quickly transfer them through the transfer window to the cell chamber, and immediately place them in a 37°C water bath to thaw;

[0690] 2. Take a 50ml centrifuge tube, gently mix the melted cell suspension, mix the cells, and take a sample for counting;

[0691] 3. Collect cells, sample for flow cytometry analysis, centrifuge at 300g for 10 minutes, and remove the supernatant;

[0692] 4. Resuspend cells in Miltenyi sorting buffer, and add sorting beads according to the reference dosage of CD4 and CD8 magnetic beads or αβT sorting kit.

[0693] 5. After mixing, incubate in a refrigerator at 2-8℃ for 15 minutes;

[0694] 6. Add Buffer ( Resuspend the cells in Running Buffer, centrifuge at 300g for 10 minutes, and remove the supernatant;

[0695] 7. Follow the 0.5mL buffer / 10 8 Cells are added to the sorting solution ( Resuspend cells in Running Buffer; (add 500 μL if less than 500 μL);

[0696] 8. Separate negative and positive cells using LS columns and magnetic racks;

[0697] Centrifuge at 300g for 8 minutes and discard the supernatant;

[0698] 10. Count the isolated negative and positive cells separately, according to (2-3) × 10⁻⁶. 6Cells were resuspended at a density of / ml by adding appropriate amounts of complete T-cell culture medium / γδT-cell culture medium (OpTmizer CTS + 2.6% OpTmizer additive + 5% ISR + 1% GlutaMax-1 + 5-20 ng / mL IL7 / IL15; OpTmizer CTS + 2.6% OpTmizer additive + 5% ISR + 1% GlutaMax-1 + 100-500 U / mL IL2) and cultured in a CO2 incubator. CTSDynabeads CD3 / CD28 magnetic beads were added to the T-cell culture medium for activation; 5-10 μM beryllium phosphate was added to the γδT-cell culture medium for activation.

[0699] 2.2 Cell electroporation:

[0700] Three to four days after cell activation, the cells were pipetted and counted. An electroporation mixture was prepared according to the LONZA electroporation system, at a concentration of (0.8–1) × 10⁶ cells / cell. 6 The electroporation system corresponding to each cell is as follows:

[0701]

[0702] Different sgRNAs are selected depending on the insertion site. For example, if the insertion site is a TRAC site, the selected sgRNA is: AGAGTCTCTCAGCTGGTACACGG.

[0703] After preparation, incubate at room temperature for 15 minutes;

[0704] According to the cell volume required for each group in the RNP preparation table, add 0.8M to 1M cells to each group (resuspended in 10μL with P3 buffer);

[0705] Transfer to the electroplating strips, avoiding the formation of air bubbles, and complete the electroplating using the LONZA EH100 electroplating instrument program.

[0706] Prepare γδT complete medium containing 1–5 μM M3814 (an effective and selective inhibitor of DNA-dependent protein kinase (DNA-PK), commercially available), 10% FBS or HPL (human platelet lysis buffer), add 100 μL to each well, and incubate at 37°C for 15 min.

[0707] After incubation, transfer to a 96-well plate and add γδT complete medium containing 5 μM M3814, 10% FBS or HPL to each well.

[0708] 2.3 Cell Culture:

[0709] (1) 24 hours after electroporation, fresh culture medium was added and the cells were transferred to 48-well or 24-well plates according to the cell density;

[0710] (2) On Day 3 after electroporation, 10-30 nM Dasatinib and 10-50 μM Vitamin C were added to the culture medium. The cells were passaged according to the cell density to maintain the cell density at 0.5 E6 to 3 E6 / mL.

[0711] (3) During the culture process, the cell density, viability, positive rate and purity are detected.

[0712] 2.4 Cell harvesting: Cells were harvested on Day 10-Day 14 after electroporation.

[0713] This invention uses the above method to prepare γδT cells containing the NKG2D-z structure (corresponding to the chimeric antigen receptor NKG2D-3TM-z), γδT cells containing the NKG2D-z-28 structure (corresponding to the chimeric antigen receptor NKG2D-3TM-z-28), γδT cells containing the NKG2D-z-BB structure (corresponding to the chimeric antigen receptor NKG2D-3TM-z-BB), and γδT cells containing the NKG2D-z-OX40 structure (corresponding to the chimeric antigen receptor NKG2D-3TM-OX40). γδT cells containing M-z-OX40, γδT cells containing the NKG2D-8-28z structure (corresponding to the chimeric antigen receptor NKG2D-8TM-28z), γδT cells containing the NKG2D-z-28 structure (corresponding to the chimeric antigen receptor NKG2D-3TM-z-28) and with Regnase-1 knockout, and γδT cells containing the NKG2D-z structure (corresponding to the chimeric antigen receptor NKG2D-3TM-z) and with Regnase-1 knockout.

[0714] The present invention uses the above method to prepare γδT cells containing the DNAM1-z-28 structure (corresponding to the chimeric antigen receptor DNAM1-3TM-z-28), γδT cells containing the DNAM1-28 structure (corresponding to the chimeric antigen receptor DNAM1-3TM-28), γδT cells containing the DNAM1-28z structure (corresponding to the chimeric antigen receptor DNAM1-3TM-28z), and γδT cells containing the DNAM1-8-28z structure (corresponding to the chimeric antigen receptor DNAM1-8TM-28z).

[0715] The present invention uses the above method to prepare γδT cells containing the nkp30-z-28 structure (corresponding to the chimeric antigen receptor nkp30-3TM-z-28).

[0716] 3. Activity detection:

[0717] (1) Phenotypic detection:

[0718] During cell culture, a certain amount of cells are taken for testing, and antibodies include anti-NKG2D, anti-Vd2, and anti-CD3.

[0719] After incubation with flow cytometry antibody, the cells were washed with FACS buffer and the expression of cell surface markers was detected by flow cytometry.

[0720] (2) External killing (short-term):

[0721] 1. Target cell plate

[0722] 1.1 Target cell preparation:

[0723] 1.1.1 The target cells are in good logarithmic growth phase with a cell viability of over 85%.

[0724] 1.1.2 Count the target cells according to standard cell counting procedures, and take 2 × 10⁻⁶ cells. 6 Transfer the target cells to a 15ml centrifuge tube, centrifuge at 300g for 8 minutes, remove the supernatant, and resuspend the target cells in T0 medium (X-Vivo medium + 5% inactivated FBS + 1% GlutaMax) until the cell density is 2×10⁻⁶ cells / mL. 5 per ml.

[0725] 1.1.3 Carefully transfer the diluted target cells into a sterile sample loading tray. Following the plate preparation plan, use a pipette to add the target cells into a black, transparent, flat-bottomed 96-well plate at a rate of 50 μL / well.

[0726] 1.2 Effector cell plate

[0727] 1.2.1 Effector Cell Preparation: Calculate the cell volume according to the experimental plan. Count the effector cells, take the required number of enhanced γδT cell samples into a 15ml centrifuge tube, centrifuge at 300g for 8min, and discard the supernatant.

[0728] 1.2.2 Resuspend γδT cells in T0 medium (X-Vivo medium + 5% inactivated FBS + 1% GlutaMax) and adjust the effector cell density according to the effector-target ratio.

[0729] 1.2.3 According to the plate preparation plan, add effector cells of different dilutions to the well plates, 50 μL / well.

[0730] 1.3 Setting up the target cell control group

[0731] Add 50 μL of target cells and 50 μL of T0 medium (X-Vivo medium + 5% inactivated FBS + 1% GlutaMax) to the wells of the target cell control group to make the total culture volume of the target cell control group consistent with the total culture volume of the co-culture experimental group.

[0732] 2. Detect luc value

[0733] 2.1 Cell co-culture: Place the 96-well plate with the added samples in a CO2 incubator and co-culture for 18-24 hours.

[0734] 2.2 Adding test reagents: According to the plan, take steadyglo luciferase (commercially available) and melt it at 4°C or room temperature in the dark. Dilute the steadyglo required for the experiment with PBS 3 times and mix well. Add 50 μL to each well and place it on a 96-well plate shaker. Shake at 100 rpm for 15 min.

[0735] 2.3 Instrumental Detection: Turn on the multi-mode microplate reader and computer software. Place the shaken 96-well plate into the multi-mode microplate reader and detect the luciferase fluorescence intensity. Remove the 96-well plate and turn off the instrument and computer.

[0736] 3. Calculation of cell killing activity

[0737] After obtaining the luc values ​​of all wells, calculate the cytotoxic activity using the following formula.

[0738] Cell-killing activity = (average luc value of target cells alone - luc value of co-culture wells) / average luc value of target cells alone × 100%

[0739] (3) External killing (long duration):

[0740] Similar to short-term killing assays, groups with different time points were set during plate preparation, and the luc values ​​of cells were detected at different time points, and the results were analyzed and statistically analyzed using an ELISA reader.

[0741] (4) In vitro killing (flow-based assay)

[0742] Target cells and effector cells were counted, and AO / PI staining confirmed that cell viability was above 85%.

[0743] Based on the effector-to-target ratio of effector cells and target cells in the in vitro killing experiment, add the corresponding target cells and effector cells;

[0744] At the planned time points after killing the target cells (e.g., 8h / 24h / 48h), the cells were homogenized, and 100μL was collected for flow cytometry analysis to calculate the residual target cells.

[0745] Cell-killing activity = (Number of viable target cells in the isolated target cell group - Number of viable target cells in the co-culture group) / Number of viable target cells in the isolated target cell group × 100%

[0746] (5) Cytokine detection

[0747] Target cells and effector cells were counted, and the cell viability was over 85%.

[0748] Centrifuge the cell culture at 300g for 8 minutes and remove the culture supernatant;

[0749] After resuspending the cells in PBS, bring the volume to 7 ml, centrifuge at 300 g for 8 min, remove the supernatant, and resuspend in T0 medium (X-Vivo medium + 5% inactivated FBS + 1% GlutaMax).

[0750] On a V-bottom 96-well plate, arrange the plates according to the plate design (designed effector-to-target ratio), add 100 μL of effector cells and 100 μL of target cells to each well, and add 100 μL of T0 medium (X-Vivo medium + 5% inactivated FBS + 1% GlutaMax) to each well containing target cells and T cells, for a total volume of 200 μL / well.

[0751] The surrounding holes were filled with an equal volume of PBS and placed in an incubator for a total of 24 hours.

[0752] Centrifuge and collect the supernatant;

[0753] Seal the 96-well plate containing the co-cultured material with sealing film, place it in a centrifuge flat plate rotor, and centrifuge at 1000g for 10 minutes.

[0754] Carefully transfer 150 μL of supernatant into a new well and store in a -80°C freezer.

[0755] Perform the cytokine assays according to the operating procedures referenced for the Biolegend cytokine assay kit.

[0756] (6) Multi-round stimulation

[0757] Target cells and effector cells were counted, and the cell viability was over 85%.

[0758] Based on the effector-to-target ratio of effector cells and target cells in multiple rounds, effector cells and target cells were co-incubated in 24-well plates.

[0759] Every 48 hours, the cells were homogenized, counted using a cell counter, and analyzed by flow cytometry to calculate target cell residue and effector cell expansion.

[0760] Based on the effector-to-target ratio of effector cells and target cells in multiple rounds of stimulation, add the corresponding target cells in each round;

[0761] Testing will continue after 48 hours.

[0762] (7) Detection of toxicity to normal cells

[0763] Effector cells were labeled using CFSE (carboxyfluorescein diacetate succinimide ester);

[0764] Effector cells and target cells were counted.

[0765] Effector cells and target cells were co-incubated in T0 medium (X-Vivo medium + 5% inactivated FBS + 1% GlutaMax) (at a certain E:T ratio);

[0766] Cells were harvested for staining and flow cytometry analysis after co-culturing for 8h, 24h, 48h, and 72h.

[0767] Analyze changes in cell composition.

[0768] 4. Experimental Results

[0769] 4.1 Level of Expression

[0770] like Figure 5 As shown, on Day 10 after electroporation, the positive expression levels of enhanced chimeric antigen receptor-T cells (NKG2D-z, NKG2D-z-28, NKG2D-z-BB, NKG2D-z-OX40, NKG2D-8-28z, NKG2D-z-28+RKO, NKG2D-z+RKO) in each group ranged from 10% to 75%.

[0771] 4.2 External killing effect (short-term)

[0772] like Figure 6 As shown, there is a significant improvement compared to natural γδT (blue). In terms of short-term killing, the enhanced chimeric antigen receptor T cells (NKG2D-z, NKG2D-z-28, NKG2D-z-BB, NKG2D-z-OX40, NKG2D-8-28z, NKG2D-z-28+RKO, NKG2D-z+RKO) in each group are similar in function to CLL1 CAR-γδT (CAR-γδT containing the extracellular antigen recognition domain CLL-1 (human C-type lectin-like molecule 1)) (dark blue).

[0773] 4.3 External killing effect (long duration)

[0774] like Figure 7 As shown, the target cells in the left image are THP1 cells (human monocytic leukemia cells), with effector-to-target ratios of 3:1 or 1:1; the target cells in the right image are HL60 cells (human promyelocytic leukemia cells), with effector-to-target ratios of 3:1 or 1:1; UTD represents untransduced CAR T cells.

[0775] from Figure 7 It can be seen from this:

[0776] 1. The first addition of target cells showed significant inhibitory and killing effects, except for NKG2D-8TM-28z, the killing functions of each group were comparable;

[0777] 2. The second group was added with target cells. After 24 hours, the results were similar across groups. After 48 hours, the NKG2D-z+RKO group showed enhanced inhibition in THP1 3:1, HL60 3:1 and 1:1.

[0778] 3. The NKG2D-z series cells exert their function on target cells mainly through high expression of NKG2D;

[0779] 4. Regnase KO can enhance the function of NKG2D-z, which may be related to improving cell persistence.

[0780] In summary, long-term killing assays showed a significant improvement over natural γδT (red), and after repeated antigen stimulation, the enhanced chimeric antigen receptor-T cells (NKG2D-z, NKG2D-z+RKO, NKG2D-z-28, NKG2D-z-28+RKO, NKG2D-8-28z) in each group still maintained strong killing ability.

[0781] 4.4 Multiple rounds of stimulation

[0782] like Figure 8 As shown, from Figure 8 It can be seen from this:

[0783] 1. After two rounds of target cell stimulation, the proportion of CD3- cells increased, indicating CD3- cell expansion;

[0784] 2. After two rounds of target cell stimulation, the NKG2D-z+RKO group had the most remaining cells, mainly CD3+Vd2 cells.

[0785] In summary, in multi-round stimulation experiments, it was found that the targeted insertion of the NKG2D-z structure (NKG2D-3TM-z structure) maintained good killing function after two rounds of stimulation, and Regnase-1KO further enhanced the survival ability of NKG2D-z.

[0786] 4.5 Detection of toxicity to normal cells

[0787] like Figure 9 and Figure 10As shown, compared with CLL1 CAR-T and CLL1 CARγδT, the enhanced chimeric antigen receptor-T cells (NKG2D-z-γδT, NKG2D-z-28-γδT) in each group did not kill CLL1+ monocytes and had no effect on cell composition.

[0788] 4.6 Killing of antigen-deficient cells (THP1 CLL1 KO)

[0789] The experimental method employed an in vitro killing (flow-based assay) approach, and the results are as follows: Figure 11 As shown.

[0790] from Figure 11 It can be seen from this:

[0791] • E:T = 1:3, the trends over 8 hours and 24 hours are basically the same:

[0792] CLL1 CARγδT and NKG2D-z-28γδT (super-γδT) both showed significantly enhanced killing effects on two types of target cells.

[0793] • CLL1 CAR-T cells can significantly kill CLL1+ target cells, but have a very weak killing ability against CLL1 KO target cells.

[0794] 4.7 Killing effect on various tumor cells

[0795] The experimental method employed an in vitro (short-term) killing method, and the results were as follows: Figure 12 As shown.

[0796] Eleven types of tumor cells (Jurkat (human T-lymphocytic leukemia cells), CCRF (human acute lymphoblastic leukemia cells), A549 (human non-small cell lung cancer cells), H226 (human lung cancer cells), HL60 (human promyelocytic leukemia cells), THP1 (human monocytic leukemia cells), HepG2 (human liver cancer cells), Huh7 (human liver cancer cells), ACHN (human kidney cancer cells), 786-0 (human clear cell adenocarcinoma cells of the kidney), and OVCAR3 (human ovarian cancer cells)) were tested in vitro, and it was found that the super-γδT (NKG2D-z-28γδT) in this invention enhanced the anti-tumor function of natural γδT to varying degrees in all tumor cells.

[0797] 4.8 The anti-aging potential of Super-γδT

[0798] The experimental method employed an in vitro killing (flow-based assay) approach, and the results are as follows: Figure 13 As shown.

[0799] Senescence of two types of lung fibroblasts was induced in vitro using etoposide. Senescent cells highly expressed NKG2DLs (as shown in the left figure). When senescent cells were co-incubated with γδT or super-γδT (NKG2D-z-28γδT), it was found that the γδT we prepared could specifically clear senescent cells in vitro. Moreover, super-γδT (NKG2D-z-28γδT) had a stronger ability to clear senescent cells and was almost non-toxic to normal cells.

[0800] 4.9 In vitro killing (short-term) of chimeric antigen receptor-T cells containing the extracellular domain DNAM1

[0801] like Figure 14 As shown, in Figure 14 In this context, UTD represents untransduced CAR T cells; DNAM1-z-28 represents γδT cells containing the chimeric antigen receptor DNAM1-3TM-z-28, which are prepared by transfecting γδT cells with the donor template DNAM1-3TM-z-28-GFP; DNAM1-28 represents γδT cells containing the chimeric antigen receptor DNAM1-3TM-28, which are prepared by transfecting γδT cells with the donor template DNAM1-3TM-28-GFP; DNAM1-28z represents γδT cells containing the chimeric antigen receptor DNAM1-3TM-28z, which are prepared by transfecting γδT cells with the donor template DNAM1-3TM-28z-GFP; and DNAM1-8-28z represents γδT cells containing the chimeric antigen receptor DNAM1-8TM-28z, which are prepared by transfecting γδT cells with the donor template DNAM1-8TM-28z-GFP.

[0802] from Figure 14 As can be seen, the extracellular domain is DNAM1, and each group of enhanced chimeric antigen receptor-T cells (DNAM1-z-28, DNAM1-28, DNAM1-28z, DNAM1-8-28z) has a significantly enhanced killing function against HL60.

[0803] 4.10 In vitro killing (long-term) of chimeric antigen receptor-T cells containing the extracellular domain Nkp30

[0804] like Figure 15 As shown, in Figure 15 In this context, UTD represents untransduced CAR T cells; nkp30-z-28 represents γδT cells containing the chimeric antigen receptor nkp30-3TM-z-28, which are prepared by transfecting γδT cells with the donor template nkp30-3TM-z-28-GFP.

[0805] from Figure 15As can be seen, the extracellular domain is Nkp30, and the enhanced chimeric antigen receptor T cells (nkp30-z-28) have a significantly enhanced killing function against THP1.

[0806] While the above descriptions are merely examples of specific embodiments of the present invention, those skilled in the art should understand that these are only illustrative, and the scope of protection of the present invention is defined by the appended claims. Those skilled in the art can make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, and all such changes or modifications shall fall within the scope of protection of the present invention.

Claims

1. An enhanced chimeric antigen receptor-T cell, wherein a nucleotide sequence encoding a chimeric antigen receptor is exogenously inserted into the genome of the T cell, said nucleotide sequence comprising at least one of the following genes: NKG2D gene, DNAM1 gene, NKp30 gene, NKp44 gene, or NKp46 gene. This results in the expression levels of NKG2D, DNAM1, NKp30, NKp44, or NKp46 proteins in chimeric antigen receptor-T cells being higher than the expression levels of the corresponding proteins in primitive T cells.

2. The enhanced chimeric antigen receptor-T cell according to claim 1, wherein the sequences of the NKG2D gene, DNAM1 gene, NKp30 gene, NKp44 gene and NKp46 gene are as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively.

3. The enhanced chimeric antigen receptor-T cell according to claim 1 or 2, wherein the insertion site of the nucleotide sequence encoding the chimeric antigen receptor in the genome of the T cell comprises: TRAC site, PD1 site, Regnase-1 site, Roquin-1 site, CISH site, and BCOR site; Optionally, the insertion site of the nucleotide sequence encoding the chimeric antigen receptor in the genome of the T cell is the TRAC site; Alternatively, the insertion site of the nucleotide sequence encoding the chimeric antigen receptor in the genome of the T cell is the TRAC site, and the Regnase-1 site in the genome of the T cell is knocked out.

4. The enhanced chimeric antigen receptor-T cell according to any one of claims 1-3, wherein the chimeric antigen receptor comprises an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain; Optionally, the extracellular antigen recognition domain is one of the following proteins: NKG2D, DNAM1, NKp30, NKp44, or NKp46.

5. The enhanced chimeric antigen receptor-T cell according to claim 4, wherein the amino acid sequences of the NKG2D protein, DNAM1 protein, NKp30 protein, NKp44 protein and NKp46 protein are as shown in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

6. The enhanced chimeric antigen receptor-T cell according to claim 4, wherein the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α; optionally, the hinge region is derived from CD8α; more preferably, the amino acid sequence of the hinge region is as shown in SEQ ID NO: 11; and / or The transmembrane region is derived from one or more of CD3z, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70; optionally, the transmembrane region is derived from CD8α or CD3z; more preferably, the amino acid sequence of the transmembrane region is as shown in SEQ ID NO: 12 or SEQ ID NO:

13.

7. The enhanced chimeric antigen receptor-T cell of claim 4, wherein the intracellular domain comprises an intracellular signal transduction region; optionally, it further comprises a co-stimulatory signal transduction region; Further optionally, the intracellular signal transduction region is derived from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CCD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk; optionally, the intracellular signal transduction region is derived from CD3ζ; more preferably, the amino acid sequence of the intracellular signal transduction region is as shown in SEQ ID NO: 14; and / or Further optionally, the co-stimulatory signal transduction region is derived from one, two, or more of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, and MyD88; optionally, the co-stimulatory signal transduction region is derived from 4-1BB, CD28, or OX40; more preferably, the amino acid sequence of the co-stimulatory signal transduction region is as shown in SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO:

17.

8. The enhanced chimeric antigen receptor-T cell according to any one of claims 1-7, further comprising a guide peptide located at the N-terminus of the chimeric antigen receptor amino acid sequence; optionally, wherein the guide peptide is derived from CD8α; more preferably, the amino acid sequence of the guide peptide is as shown in SEQ ID NO:

18.

9. The enhanced chimeric antigen receptor-T cell according to any one of claims 1-8, wherein the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NOs: 19-30.

10. The enhanced chimeric antigen receptor-T cell according to claim 9, wherein the nucleotide sequences encoding the chimeric antigen receptor are shown in SEQ ID NOs: 31-42, respectively.

11. The enhanced chimeric antigen receptor-T cell according to any one of claims 1-10, wherein, The T cell genome also includes the knockout of the CTLA-4 site, Tigit site, or NKG2A site.

12. The enhanced chimeric antigen receptor-T cell according to any one of claims 1-11, wherein the chimeric antigen receptor-T cell is a chimeric antigen receptor-γδT cell.

13. A method for preparing enhanced chimeric antigen receptor-T cells, comprising:

1. Preparation of T cells; 2. Introducing a gene-editing tool and a donor template into T cells, wherein the gene-editing tool and the donor template enable the insertion of a nucleotide sequence encoding a chimeric antigen receptor into the genome of the T cells, said nucleotide sequence comprising at least one of the following genes: NKG2D gene, DNAM1 gene, NKp30 gene, NKp44 gene, or NKp46 gene. This results in the expression levels of NKG2D, DNAM1, NKp30, NKp44, or NKp46 proteins in chimeric antigen receptor-T cells being higher than the expression levels of the corresponding proteins in primitive T cells.

14. The preparation method according to claim 13, wherein the gene editing tool is selected from one of the CRISPR / Cas system, zinc finger nuclease system, and transcription activator-like effector nuclease system; Optionally, the gene-editing tool is selected from the CRISPR / Cas system; Alternatively, the CRISPR / Cas system includes the Cas protein and sgRNA.

15. The preparation method according to claim 14, wherein the Cas protein comprises any one of the following: spCas9, AsCas12a, or LbCas12a; The sgRNA includes any of the following insertion sites: TRAC site, PD1 site, Regnase-1 site, Roquin-1 site, CISH site, and BCOR site; Optionally, the sgRNA sequence targeting the TRAC site is: TRAC: 5'-AGAGTCTCTCAGCTGGTACACGG-3' (SEQ ID NO: 43); The sgRNA sequence targeting the PD1 site is as follows: PD1: 5'-CGACTGGCCAGGGGCGCCTGT-3' (SEQ ID NO: 44); The sgRNA sequence targeting the Regnase-1 site is as follows: Regnase-1: 5'-AAGGAGGTCTTCTCCTGCCG-3' (SEQ ID NO: 45); The sgRNA sequence targeting the Roquin-1 site is as follows: Roquin-1: 5'-TGAAGACACAAAGCATTATG-3' (SEQ ID NO: 46); The sgRNA sequence targeting the CISH site is as follows: CISH: 5'-GGCGCATCCTCCTTAGGCAT-3' (SEQ ID NO: 47); The sgRNA sequence targeting the BCOR site is as follows: BcoR: 5'-AGCACGGCCCATAGGATCGA-3' (SEQ ID NO: 48).

16. The preparation method according to any one of claims 13-15, wherein the donor template is plasmid DNA, dsDNA or ssDNA; Optionally, the donor template is dsDNA including the NKG2D gene, DNAM1 gene, NKp30 gene, NKp44 gene or NKp46 gene; More preferably, the donor template includes, in the 5'-3' direction, a left homologous arm sequence, a polyA sequence, a marker gene sequence, a nucleotide sequence encoding an intracellular domain, a nucleotide sequence encoding a transmembrane region, a nucleotide sequence encoding a hinge region, a nucleotide sequence encoding an extracellular antigen recognition domain, a promoter sequence, and a right homologous arm sequence. Further optionally, the donor template further comprises a nucleotide sequence encoding a signal peptide, and the nucleotide sequence encoding the signal peptide is located between the promoter sequence and the nucleotide sequence encoding an extracellular antigen recognition domain.

17. The preparation method according to claim 16, wherein the promoter is selected from the SFFV promoter, the CMV promoter, or the EF1α promoter; optionally, the nucleotide sequence of the EF1α promoter is shown in SEQ ID NO: 49; The marker gene sequence is an EGFP fluorescent protein gene or a GFP fluorescent protein gene; optionally, the nucleotide sequence of the GFP fluorescent protein gene is as shown in SEQ ID NO:

50. The polyA is a BGH polyA signal sequence; optionally, the nucleotide sequence of the BGH polyA signal sequence is shown in SEQ ID NO:

51.

18. The preparation method according to claim 16, wherein the nucleotide sequence encoding the extracellular antigen recognition domain comprises: The nucleotide sequence encoding the NKG2D protein, the nucleotide sequence encoding the DNAM1 protein, the nucleotide sequence encoding the NKp30 protein, the nucleotide sequence encoding the NKp44 protein, or the nucleotide sequence encoding the NKp46 protein; and / or The nucleotide sequence encoding the hinge region is the nucleotide sequence encoding CD8α; and / or The nucleotide sequence encoding the transmembrane region is a nucleotide sequence encoding CD8α or CD3; and / or, Nucleotide sequences encoding intracellular domains include those encoding CD3ζ and those encoding 4-1BB, CD28, or OX40.

19. The preparation method according to any one of claims 13-18, wherein the left homologous arm sequence and the right homologous arm sequence in the donor template are as shown in SEQ ID NO: 52 and SEQ ID NO: 53, respectively.

20. The preparation method according to any one of claims 13-19, wherein in step 1, T cells are activated using phosphophosphate, antibody, or α-Glacer.

21. The preparation method according to claim 20, wherein in step 1, IL2, IL7, IL10, IL15, IL12, and IL18 are further used to activate T cells; Optionally, in step 1, a combination of phosphophosphate and IL2 / IL15 is used to activate T cells.

22. An enhanced chimeric antigen receptor-T cell, obtained by any one of claims 13-21.

23. The use of the enhanced chimeric antigen receptor-T cells according to any one of claims 1-12 and 22 in the preparation of antitumor drugs; Optionally, the drug is used to treat acute T-lymphoblastic leukemia (T-ALL), non-small cell lung cancer (NSCLC), acute myeloid leukemia (AML), hepatocellular carcinoma (HCC), renal cell carcinoma (RCC), and acute promyelocytic leukemia.

24. The application according to claim 23, wherein the drug is an intravenous injection.

25. A pharmaceutical combination comprising the enhanced chimeric antigen receptor-T cell and the therapeutic agent as described in any one of claims 1-12 and 22; Optionally, the therapeutic agent is a chemotherapeutic agent, a small molecule inhibitor, an antibody, or a cytokine; Alternatively, the therapeutic agent may be Ara-C or etoposide.

26. A pharmaceutical composition comprising any one of claims 1-12 and 22, an enhanced chimeric antigen receptor-T cell, and a pharmaceutically acceptable excipient; Optionally, pharmaceutically acceptable excipients include protective agents; Alternatively, pharmaceutically acceptable excipients include cell cryopreservation solutions; Alternatively, the pharmaceutical composition may be an intravenous injection.

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