WO2019061562A1 - Nucleic acid molecule which enhances antitumor activity of t cells - Google Patents

Abstract

Provided is a new chimeric antigen receptor (CAR) molecule containing a DAP10 protein intracellular segment, and a nucleic acid for encoding same, a cell expressing same, and a use thereof in the preparation of a drug for treating tumors. The CAR molecule is achieved by introducing an intracellular segment of DAP10 protein into a conventional second-generation CAR molecule, which activates the T cell DAP10 downstream signaling pathway by means of binding the CAR molecule to a target antigen, so as to enhance the killing effect of the corresponding CART cells on solid tumors in vivo and in vitro, thereby enhancing the ability of the molecule to proliferate and survive.

Description

A nucleic acid molecule which enhances anti-tumor activity of T cells Technical field

The present invention relates to the field of cellular immunotherapy technology for tumors, and in particular to a chimeric antigen receptor comprising an intracellular domain of DAP10 protein, a nucleic acid encoding the same, and a cell expressing the same, and the preparation thereof for treating tumors use.

Background technique

Chimeric Antigen Receptors (CAR) T cells are T cells that surface express a chimeric receptor that recognizes a specific antigen and can transmit signals. CAR T cells play an important role in anti-tumor by expressing chimeric antigen receptor (CAR) molecules, which usually include extracellular, transmembrane and intracellular segments: the extracellular segment is composed of antibody heavy and light chains The variable region is a single-chain variable region (ScFv) formed by ligation of a peptide segment; the intracellular segment is an intracellular segment chimera of various signaling molecules, including CD3zeta, CD28, OX-40, 4-1BB, etc. The transmembrane region is derived from the transmembrane region of other molecules such as CD8, CD4, CD28 and CD3zeta. The gene of the single-chain variable fragment portion is isolated, for example, from a hybridoma that produces a monoclonal antibody that recognizes the target antigen. The T cell expressing the CAR molecule directly recognizes the tumor cell surface antigen independently of the expression of the major histocompatibility antigen type I on the tumor cell, and simultaneously activates the T cell, and thus the T cell expressing the CAR can effectively kill the tumor cell. Briefly, CAR T cells recognize specific molecules on the surface of tumor cells through antigen-antibody recognition patterns, and then activate, proliferate and exert cell killing functions through their intracellular signaling.

The structural design of CAR molecules has undergone multiple generations of research and development. The structure of the first generation CAR molecule comprises a single-chain variable fragment (scFv) that recognizes a tumor cell surface antigen, a transmembrane domain, and an intracellular domain of a TCR complex CD3ζ that activates T cells. Since the intracellular segment of the first generation of CAR has only the CD3ζ signaling region and no co-stimulatory signal, the function of the first generation of CAR T cells is greatly deficient, and its expansion, persistence and effect function in the patient are all Showing a low level. In order to enhance the ability of first-generation CAR to activate T cells, a second-generation CAR has been developed. The second-generation CAR has intracellularly added costimulatory molecules (such as CD28, CD134 (OX-40), CD137 (4-1BB). ), etc.) The intracellular molecular signaling domain of the source. Clinical trials have shown that second-generation CAR T cells show better proliferation, persistence and effector function in patients. Most of the clinical trials of second-generation CAR T cells are anti-CD19 CAR T for the treatment of B-cell leukemia. Although CAR T cell clinical trials have achieved efficacy, there is room for further improvement. The third generation of CAR was developed to further enhance the efficacy of CAR T cell therapy. The intracellular segment of the third generation of CAR introduces a signal transduction region of two costimulatory molecules. Typically, one costimulatory signal is the intracellular region of CD28 and the other is the intracellular signaling region of CD134, CD137 or ICOS. Different combinations of costimulatory signals may affect the function and efficacy of CAR T cells. Studies have shown that not all third-generation CARs are better than the second generation.

DAP10 is an independent transmembrane adaptor protein expressed on hematopoietic cells. By forming a complex with the activating receptor NKG2D, it can recognize abnormally high expression of related ligands in tumorigenesis, infection and autoimmune response. DAP10 Signals have important physiological roles in maintaining autoimmune tolerance. According to the reported literature, the human NKG2D receptor is the only binding receptor for DAP10, which forms a dimer through the disulfide bond of the extracellular segment cysteine. The intracellular region of DAP10 contains a Tyr-Ile-Asn-Met motif that binds to the Src homolog 2 region of the regulatory subunit p85 of PI3K. Upon binding of DAP10 to p85, interaction with the signaling molecule Grb2 and Vav1 initiates activation and accumulation of the downstream Rho family of guanylate triphosphatase and C-γ2 phosphatase to initiate activation of immune cells. Binding to an NKG2D ligand initiates phosphorylation of four DAP10 molecules, amplifying signal transmission at low ligand concentrations.

The NKG2D-DAP10 receptor complex is expressed in NK, γδT, NKT and thymocytes and is up-regulated with innate immune stimulation. In humans, NKG2D interacts only with DAP10 to produce an immune effect, whereas in mouse cells, two NKG2D isoforms are expressed: NKG2D-1 and NKG2D-2, NKG2D-1 only interacts with DAP10, whereas NKG2D-2 can DAP10, DAP12 role. The ligand for the murine NKG2D-DAP10 receptor is a class I major histocompatibility complex (MHC I).

The study found that NKG2D-DAP10 plays an important role in monitoring tumorigenesis by knocking out NKG2D in C57BL/6 mice, which confirms that NKG2D-DAP10 plays an important role in monitoring tumorigenesis (NKG2D functionprotects the Host from tumor initiation. J Exp Med 2005; 202: 583-588.).

NKG2D also plays a role in autoimmune responses. Blocking the function of NKG2D will inhibit the proliferation of autoreactive CD8+ T cells in NOD mice and increase the survival rate of spontaneous diabetic NOD mice.

A novel chimeric antigen receptor for NK cells, CAR NKG2D-DAP10-CD3ε, is described in a scientific paper entitled "A Chimeric Receptor with NKG2D Specificity Enhances Natural Killer Cell Activation and Killing of Tumor Cells". It enhances the anti-tumor function of NK cells in vitro and in vivo by enhancing the specific recognition and activation ability of tumor antigens; NKG2D specifically recognizes tumor-specific antigen MIC and RAET1/ULBP, and activates downstream signaling pathway through DAP10; DAP10 The primary role in this chimeric antigen receptor is to help localize the NKG2D receptor on the cell membrane.

At present, CART cells have achieved good results in preclinical and clinical trials of hematological tumors, but their efficacy for solid tumors is not clear. According to the basic principles of tumor immunology and tumor microenvironment, CART cells may be difficult to overcome the inhibition of the solid tumor microenvironment in vivo, and its effector functions (such as cell killing, cytokine secretion, etc.) are directly inhibited or difficult to perform due to depletion; In addition, the proliferation and survival ability of CART cells is also impaired by the tumor microenvironment, making the number of CART cells in the tumor unsustainable. Therefore, it is necessary to find ways and methods to effectively enhance the killing effect of CART cells on solid tumors in vivo and in vitro, and enhance their proliferation and survival ability.

Summary of the invention

It is an object of the present invention to provide a novel chimeric antigen receptor (CAR) molecule comprising an intracellular domain of the DAP10 protein, and a nucleic acid encoding the same, a cell expressing the same, and use thereof in the preparation of a medicament for treating a tumor. The invention is traditional The second generation of CAR molecules introduces the intracellular segment of DAP10 protein, which activates the downstream signaling pathway of T cell DAP10 by binding of CAR to target antigen, in order to enhance the killing effect of corresponding CART cells on solid tumors in vivo and in vitro, and enhance their The purpose of proliferation and viability.

The present invention achieves the above objects by the following technical solutions:

In a first aspect, the invention provides a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain and an intracellular domain capable of binding an antigen, wherein the intracellular domain comprises a cell of a DAP10 protein Inner segment sequence.

In the chimeric antigen receptor, the intracellular sequence of the DAP10 protein is: LCARPRRSPAQDGKVYINMPGRG (as shown in SEQ ID NO: 12), and the corresponding nucleotide sequence is: 5'-ctgtgcgcacgcccacgccgcagccccgcccaagaagatggcaaagtctacatcaacatgccaggcaggggc-3' (such as SEQ IDNO: 13).

Advantageously, said intracellular domain comprises an intracellular sequence of from 1 to 9, preferably from 1 to 3 DAP10 proteins;

Preferably, the intracellular domain comprises an intracellular segment sequence of two or more DAP10 proteins joined together in tandem.

In a specific embodiment, the above CAR molecule may have only one or more intracellular domain sequences of the DAP10 protein as its intracellular domain, and may also contain one or more sequences other than the DAP10 protein intracellular sequence (eg, 2 Or 3) other intracellular domains. For example, the intracellular domain may further comprise an intracellular costimulatory signaling domain and/or a CD3ζ signaling domain; preferably, the intracellular costimulatory signaling domain is selected from, but not limited to, functional signaling of a protein as follows Domain: CD28, 4-1BB, TLR1, TLR2, TLR3, TLR4, CD27, CD28, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CDS , ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6 , VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL , DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108 ), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, Any one or a combination of at least two of NKG2D or CD83 is preferably any one of TLR2, 4-1BB or CD28 or a combination of at least two.

Preferably, the extracellular domain capable of binding an antigen refers to a receptor extracellular domain of the antigen, or a single-chain variable fragment of a specific antibody to the antigen.

Preferably, the antigen is a tumor antigen and/or a tumor-associated helper cell antigen; the tumor antigen and/or tumor-associated helper cell antigen is selected from the group consisting of, but not limited to, 5T4, α5β1-integrin, 707-AP, AFP, ART-4, B7H4, BAGE, β-catenin/m, Bcr-abl, MN/C IX antibody, CA125, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22, CD25, CDC27/m , CD30, CD33, CD52, CD56, CD80, CDK4/m, CEA, CT, Cyp-B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE , HER-2/new, HLA-A*0201-R170I, HPV-E7, HSP70-2M, HST-2, hTERT (or hTRT), iCE, IGF-1R, IL-2R, IL-5, KIAA0205, LAGE , LDLR/FUT, MAGE, MART-1/melan-A, MART-2/Ski, MC1R, Mesothelin, myosin/m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, PAP , protease-3, p190minor bcr-abl, Pml/RARα, PRAME, PSA, PSM, PSMA, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, survival protein, TEL/AML1, TGFβ, TPI/m , TRP-1, TRP-2, TRP-2/INT2, VEGF, WT1, BCMA, CD123, PD-1, PD-L1/2, TIM3 LAG3, A2ARA2AR, B7H3, B7H4, BTLA, IDO, KIR, CD160, 2B4 (CD244), VISTA (C10orf54), TSHR, CD171, CS-1, CLL-1, GD3, TnAg, FLT3, CD38, CD44V6, B7- H3, CD117, IL-13Ra2, IL-11Ra, PRSS21, VEGFR2, Lewis (Y) antigen, CD24, PDGFR-β, SSEA-4, NCAM, CAIX, EphA2, GM1, sLe, TGS5, HMWMAA, OAcGD2, folic acid β, CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, lymphocyte antigen 6 complex, LY6K, OR51E2 TARP, ETS, SPA17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, ERG, androgen receptor, cyclin B1, SART3, CD79a, CD79b, CD72, LAIR1, FCAR Any one or a combination of at least two of LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, FCRL5, IGLL1, NY-Eso-1 or NY-Eso-B;

Furthermore, the antigen may also be an inflammatory cell surface molecule that occurs in an autoimmune disease or a TCR that causes autoimmunity.

Further preferably, the antigen is any one of PCSA, Mesothelin antigen or GPC3 antigen or a combination of at least two; more preferably, the extracellular domain is specifically recognized by the antigen Mesothelin, PSCA or GPC3 Extracellular segment and/or antibody.

Preferably, the transmembrane domain is a transmembrane domain corresponding to a transmembrane domain or an intracellular co-stimulatory signal molecule corresponding to an extracellular segment receptor, preferably a CD28 transmembrane domain.

In a preferred embodiment, the chimeric antigen receptor comprises, in sequence from the N-terminal side, a single-chain variable region of an antibody against an antigen as an extracellular domain or an extracellular portion of an antigen, CD28 molecule Transmembrane region, CD3 ζ chain, And one or more DAP10 intracellular sequence sequences linked in series.

In a second aspect, the invention provides a nucleic acid encoding a chimeric antigen receptor as described in the first aspect or a nucleic acid having at least 80% identity, preferably at least 90% identity thereto.

In the present invention, the nucleic acid having at least 80% identity, preferably at least 90% identity with the nucleic acid of the chimeric antigen receptor of the first aspect is a chimeric antigen receptor encoded by the chiral antigen receptor. Chimeric antigen receptors have the same or similar functions, and their base changes do not affect the structure and function of the chimeric antigen receptor.

In a third aspect, the present invention provides a chimeric antigen receptor-expressing cell, wherein the nucleic acid according to the second aspect is introduced; preferably, the cell is an immune effector cell, further preferably a T cell, a B cell, Any one or a combination of at least two of NK cells, NKT cells, dendritic cells, or macrophages.

In a fourth aspect, the present invention provides a method of producing a chimeric antigen receptor-expressing cell according to the third aspect, comprising the step of introducing a nucleic acid according to the second aspect into a cell;

Preferably, the nucleic acid is introduced into the cell by transduction mediated by a lentiviral vector;

Preferably, the cell is an immune effector cell, further preferably any one or a combination of at least two of T cells, B cells, NK cells, NKT cells, dendritic cells or macrophages.

In a fifth aspect, the present invention provides a recombinant vector comprising the nucleic acid of claim 3; preferably, the vector is any one of a recombinant cloning vector, a recombinant eukaryotic expression plasmid or a recombinant lentiviral vector or a combination of at least two, preferably a recombinant lentiviral vector;

Preferably, the recombinant cloning vector is selected from, but not limited to, any one of pUC18, pUC19, pMD19-T, pGM-T vector, pUC57, pMAX or pDC315 or a combination of at least two;

Preferably, the eukaryotic expression plasmid is selected from the group consisting of, but not limited to, a pCDNA3 series vector, a pCDNA4 series vector, a pCDNA5 series vector, a pCDNA6 series vector, a pRL series vector, a pUC57 vector, pMAX or pDC315, or at least two combination;

Preferably, the recombinant lentiviral vector is selected from, but not limited to, any one or at least two of a recombinant adenovirus vector, a recombinant adeno-associated virus vector, a recombinant retroviral vector, a recombinant herpes simplex virus vector or a recombinant vaccinia virus vector. The combination.

In the present invention, by recombining the nucleic acid construct of the chimeric antigen receptor with the vector, transfection of the recombinant vector into cells can be achieved to obtain T cells, and chimeric antigen receptors can be realized. Features.

In a sixth aspect, the present invention provides a recombinant virus comprising the recombinant virus obtained by co-transfecting a mammalian cell with the recombinant vector and the packaging helper plasmid according to the fifth aspect.

In a seventh aspect, the present invention provides a chimeric antigen receptor T cell, which comprises the recombinant virus according to the sixth aspect Transfection into T cells for expression.

The invention provides the chimeric antigen receptor of the first aspect, the nucleic acid of the second aspect, the recombinant vector of the fifth aspect, or the recombinant virus of the sixth aspect, for use in the eighth aspect, Amplify CAR T cells.

The ninth aspect, the present invention provides a pharmaceutical composition comprising the chimeric antigen receptor of the first aspect, the nucleic acid of the second aspect, the chimeric antigen receptor expressing cell of the third aspect Or a recombinant vector according to the fifth aspect, and optionally a pharmaceutically acceptable excipient.

In a tenth aspect, the invention provides a chimeric antigen receptor according to the first aspect, the nucleic acid of the second aspect, the chimeric antigen receptor expression cell of the third aspect or the fifth Use of the recombinant vector of the aspect in the manufacture of a medicament for the treatment of an autoimmune disease or tumor.

Preferably, the tumor is a solid tumor and/or a hematoma, and the tumor may be selected from, but not limited to, lung cancer, liver cancer, lymphoma, colon cancer, colon cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma. Any one or a combination of at least two of gallbladder cancer, esophageal cancer, renal cancer, glial carcinoma, melanoma, pancreatic cancer, prostate cancer, leukemia, multiple myeloma or malignant lymphoma.

In a specific embodiment of the use provided by the present invention, the tumor is leukemia, liver cancer or lung cancer.

In an eleventh aspect, the present invention provides a method of treating a subject having an autoimmune disease and/or a disease associated with expression of a tumor antigen, comprising administering to the subject an effective amount comprising an eighth a pharmaceutical composition of the pharmaceutical composition;

Preferably, the tumor is a solid tumor and/or a hematoma.

In a specific embodiment of the use provided by the present invention, the tumor is leukemia, liver cancer or lung cancer.

Notably, the CAR molecule of the invention is characterized in that it comprises the intracellular domain of DAP10 as its intracellular domain. The DAP10 intracellular segment includes variants thereof having the same function. The term "variant" refers to any variant comprising a substitution, deletion or addition of one or several to more amino acids, provided that the variant substantially retains the same function as the original sequence.

Beneficial effect

(1) The present invention can further couple other co-stimulatory signals by introducing an intracellular segment of DAP10 as a separate costimulatory signaling pathway based on a conventional second-generation CAR molecule, and can be coupled in series by Ways to further enhance the tumor immune effect;

(2) The anti-tumor effect of the CART cells integrating the intracellular signal domain of the DAP10 costimulatory molecule of the present invention is significantly improved, and the cytotoxicity of the cell to the tumor target cells is particularly persistent compared with the second generation CART of the unintegrated DAP10. The cytotoxicity was significantly enhanced, and the ability to secrete cytokines IL-2, IFN-y, Granzyme B, and GM-CSF was significantly improved. At the same time, it showed stronger tumor inhibition ability and better intratumoral proliferation ability in animal experiments;

(3) The DAP10 costimulatory signal molecule of the present invention is only 72 bp, and the fragment is very small, and does not cause a variety of transfection methods such as lentivirus, retrovirus, electroporation, liposome by aggravating the transfection vector load. The effect of decreased transfection efficiency, and the immune effect of CAR T cells integrated with DAP10 is significantly improved, and DAP10 costimulatory signaling molecules can increase the potential of T cell tumors while providing more potential for modification of CAR molecular carriers.

DRAWINGS

Figure 1 shows a series of CAR molecules constructed in the examples of the present invention including CAR-Mes, CAR-Mes-41BB, CAR-Mes-DAP10, CAR-Mes-DAP10*3, CAR-Mes-DAP10*9, CAR- Mes-41BB-CD28, CAR-Mes-41BB-DAP10, CAR-PSCA, CAR-PSCA-DAP10, CAR-PSCA-DAP10*3, CAR-GPC3 (control group), CAR-GPC3-41BB, CAR-GPC3- Schematic diagram of the molecular structure of DAP10, CAR-GPC3-41BB-CD28, CAR-GPC3-41BB-DAP10.

Figure 2 shows the in vitro tumor killing effect of CAR-Mes T, CAR-Mes-DAP10 T, CAR-Mes-DAP10*3 T cells on A549GL cells (A) and histogram (B), and CAR-Mes, The efficiency of transfection of T cells with the CAR-Mes-DAP10 lentiviral vector.

Figure 3 shows the in vitro tumor killing effect of CAR-Mes T, CAR-Mes-DAP10 T, CAR-Mes-DAP10*9T cells on A549GL cells (A) and histogram (B), and CAR-Mes T, The expression of relevant T cell activation surface markers CD25(C), CD69(D) and CD107a(E) was correlated with CAR-Mes-DAP10 T cells three times after co-culture with tumor cells.

Figure 4 shows a graph (A) and a histogram (B) of the in vitro tumor killing effect of CAR-PSCA T, CAR-PSCA-DAP10 T cells on A549 GL cells.

Figure 5 shows the in vitro tumor killing effect of CAR-Mes T, CAR-Mes-41BB-CD28 T, CAR-Mes-DAP10 T cells on A549GL (A) and H460 Mes + GL (B) cells, respectively.

Figure 6 shows the in vitro tumor killing effect of CAR-GPC3 T, CAR-GPC3-41BB-CD28 T, CAR-GPC3-DAP10 T cells on HepG2GL (A) and HC04-GL (B) cells, respectively.

Figure 7 shows ELISA detection of cytokine IL after incubated with GFP T, CAR-MesT, CAR-Mes-DAP10 T, CAR-Mes-DAP10*3 T or CAR-Mes-DAP10*9 T cells and A549-GL cells for 18 h. -2 (A), IFN-γ (B), GZMB (C) and GM-CSF (D) secretion.

Figure 8 shows tumor weight (A) and volume (A) and volume of A549-GL cells formed in immunodeficient mice by NC, GFP T, CAR-Mes T or CAR-Mes-41BB-CD28, CAR-Mes-DAP10 T cells. )Impact.

Figure 9 is a bar graph showing the proportion of tumor infiltrating T cells after treatment of lung cancer tumor xenograft models with CAR-Mes T or CAR-Mes-DAP10 T cells in vivo.

Figure 10 is a graph showing the in vitro tumor killing effect of CAR-Mes-41BB T, CAR-Mes-41BB-DAP10 T cells on A549-GL (A), H460+Mes-GL (B) cells, and CAR-Mes- A graph of three consecutive killing effects of CD28 T, CAR-Mes-41BB T, and CAR-Mes-DAP10 T cells on A549-GL cells in vitro (C).

Figure 11 is a graph showing the in vitro tumor killing effect of CAR-GPC3-41BB T, CAR-GPC3-DAP10 T cells on Hepg2-GL (A), HC04-GL (B) cells.

Figure 12 is a graph showing the in vitro tumor killing effect of CAR-GPC3 T, CAR-GPC3-DAP10 T cells on HC04-GL cells.

Detailed ways

To facilitate an understanding of the invention, the invention is set forth below. It should be understood by those skilled in the art that the present invention is not to be construed as limited.

Example 1. Construction of CAR plasmid

1) Synthesis of CAR-Mes (control group), CAR-Mes-DAP10, CAR-Mes-41BB, CAR-Mes-DAP10*3, CAR-Mes-DAP10*9, CAR-Mes-41BB- by gene synthesis CD28, CAR-Mes-41BB-DAP10, CAR-PSCA (control group), CAR-PSCA-DAP10, CAR-PSCA-DAP10*3, CAR-GPC3 (control group), CAR-GPC3-41BB, CAR-GPC3- DAP10, CAR-GPC3-41BB-CD28, CAR-GPC3-41BB-DAP10 molecules (the structure of each molecule is shown in Figure 1), the C-terminus of the synthesized gene contains restriction endonuclease Pme1 cleavage site and its protective base The base and N-terminus contain a restriction endonuclease AsiSI cleavage site and a protected base thereof; wherein "41BB" means that the molecule contains the intracellular signal domain 4-1BB commonly used in the prior art.

For the gene sequence of each molecule synthesized, see SEQ ID NO: 2 (CAR-Mes), SEQ ID NO: 3 (CAR-Mes-DAP10), SEQ ID NO: 4 (CAR-Mes-41BB), SEQ in the Sequence Listing. ID NO: 5 (CAR-Mes-DAP10*3), SEQ ID NO: 6 (CAR-Mes-DAP10*9), SEQ ID NO: 7 (CAR-Mes-41BB-CD28), SEQ ID NO: 8 ( CAR-Mes-41BB-DAP10), SEQ ID NO: 9 (CAR-PSCA), SEQ ID NO: 10 (CAR-PSCA-DAP10), SEQ ID NO: 11 (CAR-PSCA-DAP10*3), SEQ ID NO: 13 (CAR-GPC3 (control)), SEQ ID NO: 14 (CAR-GPC3-41BB), SEQ ID NO: 15 (CAR-GPC3-DAP10) and SEQ ID NO: 16 (CAR-GPC3-41BB - CD28), SEQ ID NO: 17 (CAR-GPC3-41BB-DAP10).

2) Digested with restriction endonucleases Pme1 and AsiSI, respectively, to obtain synthetic DNA fragments containing sticky ends (CAR-Mes, CAR-Mes-DAP10, CAR-Mes-41BB, CAR-Mes-DAP10*3, CAR -Mes-DAP10*9, CAR-Mes-41BB-CD28, CAR-Mes-41BB-DAP10, CAR-PSCA, CAR-PSCA-DAP10, CAR-PSCA-DAP10*3, CAR-GPC3 (control group), CAR -GPC3-41BB, CAR-GPC3-DAP10, Linearized DNA of CAR-GPC3-41BB-CD28, CAR-GPC3-41BB-DAP10) and LEGO-2A-eGFP vector (containing sticky ends, the sequence of which is shown in SEQ ID NO: 1).

3) Recombinant DNA fragments with sticky ends were obtained by agarose gel electrophoresis (CAR-Mes, CAR-Mes-DAP10, CAR-Mes-41BB, CAR-Mes-DAP10*3, CAR-Mes-DAP10*) 9. CAR-Mes-41BB-CD28, CAR-Mes-41BB-DAP10, CAR-PSCA, CAR-PSCA-DAP10, CAR-PSCA-DAP10*3, CAR-GPC3 (control group), CAR-GPC3-41BB, CAR-GPC3-DAP10, CAR-GPC3-41BB-CD28, CAR-GPC3-41BB-DAP10) and linearized LEGO-2A-eGFP vector DNA fragment.

4) Linearized LEGO-2A-eGFP and synthetic DNA fragments with sticky ends by T4 DNA ligase (InVitrogent) (CAR-Mes, CAR-Mes-DAP10, CAR-Mes-41BB, CAR-Mes -DAP10*3, CAR-Mes-DAP10*9, CAR-Mes-41BB-CD28, CAR-Mes-41BB-DAP10, CAR-PSCA, CAR-PSCA-DAP10, CAR-PSCA-DAP10*3, CAR-GPC3 (control group), CAR-GPC3-41BB, CAR-GPC3-DAP10, CAR-GPC3-41BB-CD28, CAR-GPC3-41BB-DAP10) ligated to obtain CAR plasmid transformation vector (LEGO-CAR-Mes-2A-EGFP) , LEGO-CAR-Mes-41BB-2A-EGFP, LEGO-CAR-Mes-DAP10-2A-EGFP, LEGO-CAR-Mes-DAP10*3-2A-EGFP, LEGO-CAR-Mes-DAP10*9-2A -EGFP, LEGO-CAR-Mes-41BB-CD28-2A-EGFP, LEGO-CAR-Mes-41BB-DAP10-2A-EGFP, LEGO-CAR-PSCA-2A-EGFP, LEGO-CAR-PSCA-DAP10-2A -EGFP, LEGO-CAR-PSCA-DAP10*3-2A-EGFPLEGO-CAR-GPC3-2A-EGFP, LEGO-CAR-GPC3-41BB-2A-EGFP, LEGO-CAR-GPC3-DAP10-2A-EGFP, LEGO - CAR-GPC3-41BB-CD28-2A-EGFP, LEGO-CAR-GPC3-41BB-DAP10-2A-EGFP).

Example 2. Lentiviral packaging of CAR plasmid

The CAR plasmid of the present invention prepared in Example 1 and the related control plasmid were subjected to lentiviral packaging, and the specific steps were as follows:

1) 293T cells were cultured in a 10 cm culture dish, and the medium was: DMEM high glucose medium + 10% FBS (fetal calf serum) + 1% double antibody (100 x penicillin-streptomycin mixed solution);

2) When the density of 293T cells in a 150 mm culture dish reaches 80-90%, the medium is changed: DMEM high sugar medium + 1% FBS + 1% double antibody;

3) After changing the culture medium for 2-6 hours, the LEGO-CAR-EGFP plasmid (LEGO-CAR-Mes-2A-EGFP, LEGO-CAR-Mes-41BB-2A-EGFP, LEGO-CAR-Mes, respectively) was separated by PEI. -DAP10-2A-EGFP, LEGO-CAR-Mes-DAP10*3-2A-EGFP, LEGO-CAR-Mes-DAP10*9-2A-EGFP, LEGO-CAR-Mes-41BB-CD28-2A-EGFP, LEGO-CAR-Mes-41BB-DAP10-2A-EGFP, LEGO-CAR-PSCA-2A- EGFP, LEGO-CAR-PSCA-DAP10-2A-EGFP, LEGO-CAR-PSCA-DAP10*3-2A-EGFP, LEGO-CAR-GPC3-2A-EGFP, LEGO-CAR-GPC3-41BB-2A-EGFP, LEGO-CAR-GPC3-DAP10-2A-EGFP, LEGO-CAR-GPC3-41BB-CD28-2A-EGFP, LEGO-CAR-GPC3-41BB-DAP10-2A-EGFP) or blank control plasmid LEGO-EGFP and slow The virus packaging helper plasmids psPAX2 and pMD2.G were co-transformed into 293T cells, and the reagents and dosages were as follows:

4) The culture supernatant was collected at 24, 48 and 72 hours after the transformation, respectively, and fresh medium (DMEM high sugar medium + 1% FBS + 1% double antibody) was added;

5) After the supernatant of the culture medium is collected, the supernatant is centrifuged at 2500 g for 0.5 hour (optional step);

6) taking the supernatant, filtering with a 0.45 um filter, and centrifuging at 28000 rpm for 1.5 hours using an ultracentrifuge (optional step);

7) After ultra-high speed centrifugation, gently remove the supernatant, add 200 ul of PBS, and dissolve at 4 degrees for 12-16 hours to obtain CAR lentivirus or blank control GFP lentivirus (optional step);

8) After the virus is dissolved, the collected virus solution is dispensed into a PCR tube and stored frozen at -80 ° C until use.

Example 3, T cell activation and CAR lentivirus infection

Isolation and purification of 1T cells: Mononuclear cells in the blood were separated by Ficoll density gradient method, and red blood cells were lysed by lysis of red blood cell lysate, and then T cells were sorted by MACS Pan-T magnetic beads;

2 sorted T cells were diluted with medium (AIM-V medium + 5% FBS + penicillin 100 U / ml + streptomycin 0.1 mg / ml to a cell concentration of 2.5 × 10 ⁶ / ml for use;

3 Stimulate T cells by magnetic beads coated with CD2, CD3, CD28 antibody (product source: German scorpio), ie, coated magnetic beads and T cells mixed in a ratio of 1:2, the final density of T cells should be 5×10 ⁶ / ml / cm2. After mixing, the cells were incubated at 37 ° C in a 5% CO ₂ incubator for 48 hours;

4 lentivirus transfected T cells: the magnetic beads in the activated T cell-bead mixture were removed by magnetic field, centrifuged at 300g for 5min, the supernatant was removed, resuspended in fresh medium, and added to express CAR and GFP (blank After the control) lentivirus (virus addition amount is MOI=10), 8 μg/ml of polybrene and 300 IU/ml of IL-2 were added. After being cultured at 37 ° C for 24 h in a 5% CO ₂ incubator, centrifugation at 300 g for 5 min, the supernatant was removed, and resuspended in fresh medium containing 300 IU/ml IL-2 to obtain T cells expressing the CAR plasmid;

5CART cell expansion: The CART cell density was maintained at about 1×10 ⁶ /ml, and a half-time change was performed every 2-3 days. After two weeks, the number of CAR T cells can be amplified by a factor of 100. The GFP-positive cells were successfully transfected cells, and the GFP-positive ratio was detected by flow, that is, CAR T cells were obtained, which are respectively referred to as CAR-Mes (also referred to as M28z in the drawing) and CAR-Mes-41BB (also in the figure). Referred to as MBBz), CAR-Mes-DAP10 (also referred to as M28z10 in the drawing), CAR-Mes-DAP10*3 (also referred to as M28z10*3 in the drawing), CAR-Mes-DAP10*9 (also referred to as M28z10 in the drawing) *9), CAR-Mes-41BB-CD28 (also referred to as MBB28z in the drawing), CAR-Mes-41BB-DAP10 (also referred to as MBBz10 in the drawing), CAR-PSCA (also referred to as P28z in the drawing), CAR- PSCA-DAP10 (also referred to as P28z10 in the drawing), CAR-PSCA-DAP10*3 (also referred to as P28z10*3 in the drawing), CAR-GPC3 (also referred to as G28z in the drawing), CAR-GPC3-41BB (drawings Also referred to as GBBz), CAR-GPC3-DAP10 (also referred to as G28z10 in the drawing), CAR-GPC3-41BB-CD28 (also referred to as GBB28z in the drawing), CAR-GPC3-41BB-DAP10 (also referred to as GBBz10 in the drawing) Or the ratio of blank control T cells (GFP-T).

The efficiency of transfection of T cells by CAR-Mes and CAR-Mes-DAP10 lentiviral vectors was detected by flow cytometry (Fig. 2C). The results showed that the integration of DAP10 did not affect the transfection efficiency of CAR T cells.

Example 4: Effect of CAR-Mes-DAP10 T cells on killing tumors in vitro

Comparison of GFP T (blank control), CAR-Mes T (negative control), CAR-Mes-DAP10, CAR-Mes-DAP10*3, CAR-Mes-DAP10*9 T cells in vitro to identify and kill lung cancer cells, tumor The cells were selected from human lung adenocarcinoma cell lines with A549-GL luciferase.

The GFP T (blank control), CAR-Mes T (negative control), CAR-Mes-DAP10 T, CAR-Mes-DAP10*3 T, CAR-Mes-DAP10*9 T cells prepared in Example 3 were respectively associated with 1×10 ⁴ tumor cells A549-GL were mixed at a ratio of 2:1, 1:1, 1:2, ^1:4 , 1:8 (E:T ratio), and added to a 96-well U-shaped plate, each set of 3 The duplicate wells were centrifuged at 250 g for 5 min, and then co-cultured in a 37 ° C, 5% CO ₂ incubator.

Luciferase quantitative killing efficiency evaluation method: 18 hours after CAR T cells were co-cultured with tumor cells (the experimental control group was cultured with tumor cells alone), 100 ul/well of luciferase bottom was added to a 96-well cell culture plate. (1×), the cells were resuspended and mixed, and the RLU (relative light unit) was immediately measured by a multi-function microplate reader, and the measurement time was set to 1 second. Killing ratio calculation formula: 100% × (control hole reading - experimental hole reading) / control hole reading (no blank cell group reading can be ignored); the results are shown in Figure 2 (A) - (B), Figure 3 (A )-(B).

CAR-Mes-DAP10 and CAR-Mes T cells were mixed and co-cultured with A549-GL tumor cells three times with E:T=1:2, and the total co-culture time was 24 hours. After 72 hours, the flow was passed. Cell-based technique to detect the proportion of T cell activation surface markers CD25 (C), CD69 (D) and T cell degranulation surface marker CD107a (E) positive cells in CAR-Mes-DAP10 and CAR-Mes T cells, respectively. 3 (C), 3 (D) and 3 (E).

The results showed that the in vitro killing efficiency of CAR-MES-DAP10 T, CAR-MES-DAP10*3, CAR-MES-DAP10*9 T cells on tumor cell A549-GL was significantly higher than that of CAR-MES T cells. When T (ie, the ratio of Effector T cells to Target target cells) is small, CAR-MES-DAP10, CAR-MES-DAP10*3, and CAR-MES-DAP10*9 T cells can also exhibit strong tumors. The killing activity was significantly higher than that of CAR-MES T cells, while the killing ability of CAR-MES-DAP10*3 and CAR-MES-DAP10*9 T cells was slightly higher than that of CAR-MES-DAP10 T cells, respectively; CAR-Mes- DAP10 cells were significantly up-regulated on the surface of CD25, CD69 and CD107a after activation by target cells, suggesting that DAP10 costimulatory molecules can significantly increase the activation level and cytotoxicity of CART cells.

Example 5: Effect of CAR-PSCA-DAP10 T cells on killing tumors in vitro

In vitro comparison of GFP T (blank control), CAR-PSCA T (negative control), CAR-PSCA-DAP10 T cells for recognition and killing of lung cancer cells, and tumor cells using A549-GL luciferase-containing human lung adenocarcinoma cell line . The experimental method is as described in Example 4.

The results showed that the in vitro killing efficiency of CAR-PSCA-DAP10 T on tumor cell A549-GL was significantly higher than that of CAR-PSCA T cells, and the E:T (ie, the ratio of Effector T cells to Target target cells) was small. , CAR-PSCA-DAP10 cells also showed strong tumor killing activity, significantly higher than CAR-PSCA T cells (see Figure 4 (A) and Figure 4 (B)), indicating: DAP10 - intracellular domain Significant enhancement of the killing function of CAR T cells is not limited to the single tumor target Mesothelin.

Example 6. Comparison of CAR-Mes-DAP10 T cells and CAR-Mes-41BB-CD28 in vitro identification of killing tumors

Comparison of GFP T (blank control), CAR-Mes T (negative control), CAR-Mes-DAP10 T (abbreviated as PSCA-DAP10), and CAR-Mes-41BB-CD28 T cells in vitro to identify and kill lung cancer cells, tumor The cells were selected from human lung adenocarcinoma cell line with A549-GL luciferase and H460+Mes-GL human lung cancer cell line overexpressing Mesothelin antigen. The experimental method is as described in Example 4.

The results showed that the killing function of CAR-Mes-DAP10 T and CAR-Mes-41BB-CD28 T cells was significantly higher than that of CAR-Mes T cells in both cell lines, while CAR-Mes-DAP10 T and CAR-Mes The killing efficiency of -41BB-CD28 T cells is basically the same, but because the DAP10 gene fragment is small, it does not affect the transfection efficiency and the plasticity of the CAR vector. DAP10 costimulatory molecules have higher application value in CAR T cell applications than 41BB costimulatory molecules. (Fig. 5(A) and Fig. 5(B))

Example 7 In vitro killing effect and comparison of CAR-GPC3-DAP10 cells on hepatoma cells

In vitro comparison of GFP T (blank control), CAR-GPC3 T (negative control), CAR-GPC3-DAP10 T, CAR-GPC3-41BB-CD28 T cells for the recognition and killing function of hepatoma cells, tumor cells were selected for luciferase Human hepatocellular carcinoma cell line and HC04 human liver cell line of HepG2-GL and HC04-GL. The experimental method is as described in Example 4.

The results showed that the killing function of CAR-GPC3-DAP10 T and CAR-GPC3-41BB-CD28 T cells was significantly higher than that of CAR-GPC3 T cells in both cell lines (CAR-GPC3-CD28- in the prior art) CD3ζT cells, abbreviated as G28z), while the killing efficiency of CAR-GPC3-DAP10 T and CAR-GPC3-41BB-CD28 T cells is basically equivalent, further confirming that Example 6: DAP10 costimulatory molecule is more than 41BB costimulatory molecule in CAR T cells The application has higher application value. (Fig. 6(A) and Fig. 6(B))

Example 8. Secretion of cytokines after co-culture of CAR-Mes-DAP10 T cells with tumor cells

1) Inoculate 2 × 10 ⁵ tumor cells A549-GL into each well in a 24-well culture plate, overnight adherent culture, and then the number of positive cells reached 1 × 10 ⁵ GFP T (blank control), CAR-Mes T (negative control), CAR-Mes-DAP10 T, CAR-Mes-DAP10*3 T, CAR-Mes-DAP10*9 T construct cells (prepared as in Example 3) and A549-GL cells with E:T=1 : 2 mixed, placed in a 37 ° C, 5% CO ₂ incubator co-culture;

2) After co-culture for 18 hours, the medium in which GFPT, CAR-Mes T, CAR-Mes-DAP10 T, CAR-Mes-DAP10*3 T, CAR-Mes-DAP10*9 T cells were co-cultured with tumor cells, respectively The supernatant was assayed for interleukin 2 (IL-2), interferon gamma (IFN-γ), granzyme B (GZMB) and granulocyte-macrophage colony-stimulating factor (GM-CSF) levels by ELISA. Figures 7(A), 7(B), 7(C) and 7(D);

3) CAR-Mes-DAP10, CAR-Mes-DAP10*3, CAR-Mes-DAP10*9 T cells secrete immune effector interleukin-2, interferon gamma, granzyme B, GM-CSF (CAR-Mes-DAP10) The level of DAP10 was significantly higher than that of CAR-Mes T cells, which indicated that the intracellular domain of DAP10 could significantly increase the secretion of interleukin-2, interferon gamma, granzyme B and GM-CSF in CART cells, which could significantly promote T cell proliferation and tumor killing.

Example 9. Recognition of killing tumors by CAR-Mes-DAP10 T cells in vivo

1) 5×10 ⁵ A549-GL cells were transplanted into the inguinal space of NOD/SCID IL2rg-/-immuno deficient mice to construct a mouse model of lung cancer;

2) Three days after tumor transplantation, the number of GFP-positive cells in the lung cancer mouse model was 3×10 ⁶ GFP T, CAR-Mes T, CAR-Mes-41BB-CD28 T, CAR-Mes-DAP10 T. Cells, plus NC blank control group without injection of cells, a total of five experimental groups, each set of four replicates;

3) All the mice were sacrificed on the 36th day after the tumor transplantation, the tumor mass was removed, and the tumor weight and volume were recorded and analyzed (Fig. 8(A) and Fig. 8(B), respectively), and the tumor block was ground and sieved. A single cell suspension was used to detect the proportion of tumor infiltrating T cells by draining cells (Fig. 9).

The results showed that CAR-Mes-DAP10 T cells and CAR-Mes-41BB-CD28 T cells significantly reduced tumor volume in lung cancer tumor xenograft models, suggesting that CAR-Mes-DAP10 T cells have higher killing efficiency against tumor cells. And can overcome the inhibition of T cells by the tumor microenvironment. In contrast, CAR-Mes T cells were difficult to shrink tumors in lung cancer tumor xenograft models (relative to NC and GFPT controls). Analysis of tumor-infiltrating T cells showed that CAR-Mes T cells were very rare in tumors (total T cell ratio was less than 1%), while CAR-Mes-DAP10 T cells were significantly increased (total T cell ratio was greater than 3). %, Figure 9); These results indicate that the intracellular domain of DAP10 can enhance the proliferation and survival ability of T cells in the tumor microenvironment, overcome the inhibition of tumor microenvironment, and enhance the tumor killing effect of CAR T cells in vivo.

Example 10: Comparison of CAR-Mes-41BB-DAP10 T and CAR-Mes-41BB T cells in vitro to identify killing tumors

In vitro comparison of GFP T (blank control), CAR-Mes-41BB T (negative control), CAR-Mes-41BB-DAP10 T cells for lung cancer cell identification and killing function, tumor cells using A549-GL and H460+Mes-GL bands Luciferase of human lung adenocarcinoma cell line. The experimental method was as described in Example 4, and the results are shown in Figures 10(A) and 10(B).

The continuous tumor killing effect of GFP T (blank control), CAR-Mes T, CAR-Mes-41BB T, and CAR-Mes-DAP10 T cells was compared by continuous killing in vitro. The tumor cell line was selected from A549-GL with luciferase. Human lung adenocarcinoma cell line. After GFP T, CAR-Mes T, CAR-Mes-41BB T, and CAR-Mes-DAP10 T cells were co-cultured with tumor cells for three times at E:T = 1:2, respectively, each time was detected by a microplate reader. The percentage of CAR T cells killing tumor cells after co-culture, as shown in Figure 10 (C) (luciferase assay as described in Example 4).

The results showed that the in vitro killing efficiency of CAR-Mes-41BB-DAP10 T and CAR-Mes-DAP10 T cells on tumor cell A549-GL was significantly higher than that of CAR-Mes T, CAR-Mes-41BB T cells, and continuous killing in vitro. In the experiment, CAR-Mes-DAP10 T has a stronger sustained killing ability. This indicates that DAP10-containing CAR T cells can further enhance the tumor immune killing effect of CAR T cells, especially long-term and multiple sustained killing effects, indicating that DAP10-containing CAR T cells have higher application in tumor killing in humans. value.

Example 11 Comparison of in vitro killing effect of CAR-GPC3-41BB-DAP10 T and CAR-GPC3-41BB T cells on hepatoma cells

Comparison of GFP T (blank control), CAR-GPC3-41BB T (negative control), CAR-GPC3-41BB-DAP10 in vitro T cells recognize and kill liver cancer cells, and the tumor cells adopt HepG2-G L and HC04-GL human hepatocellular carcinoma cell lines with luciferase labeling. The experimental method is as described in Example 4.

The results showed that the killing function of CAR-GPC3-41BB-DAP10 T cells was significantly higher than that of CAR-GPC3-41BB T cells in both cell lines (Fig. 11(A) and Fig. 11(B)), which further proved Integration of DAP10 costimulatory molecules can effectively enhance the tumor killing effect of CAR T cells.

Example 12, CAR-GPC3-DAP10 T cells recognize killing tumors in vivo

1) 5×10 ⁵ HepG2-GL cells were transplanted into the inguinal space of NOD/SCID IL2rg-/-immuno deficient mice to construct a mouse model of liver cancer;

2) Three days after tumor transplantation, 3×10 ⁶ GFP T, CAR-GPC3 T, CAR-GPC3-DAP10 T cells were intravenously injected into the liver cancer mouse model, respectively, for a total of three experimental groups. Group set four repetitions;

3) All mice were sacrificed on the 36th day after tumor transplantation, the tumor mass was removed, and the tumor volume was recorded and analyzed (Fig. 12).

The results showed that CAR-GPC3 T, CAR-GPC3-DAP10 T cells significantly reduced tumor volume in lung cancer tumor xenograft models, while CAR-GPC3-DAP10 T cells were better than CAR-GPC3 T cells, suggesting CAR-Mes -DAP10 T cells have high killing efficiency against tumor cells such as liver cancer.

The Applicant declares that the present invention describes the products, uses, and uses thereof of the present invention by the above embodiments, but the present invention is not limited to the above detailed uses and modes of use, that is, does not mean that the present invention must rely on the above detailed uses and uses. The way can be implemented. It should be apparent to those skilled in the art that any modifications of the present invention, equivalent substitution of the various materials of the products of the present invention, addition of auxiliary components, selection of specific means, and the like, are all within the scope of the present invention.

Claims (10)

A chimeric antigen receptor comprising an extracellular domain, a transmembrane domain and an intracellular domain capable of binding an antigen, wherein the intracellular domain comprises an intracellular domain sequence of a DAP10 protein. The chimeric antigen receptor according to claim 1, wherein the intracellular sequence of the DAP10 protein is as shown in SEQ ID NO: 12; Preferably, the intracellular domain comprises an intracellular sequence of 1 to 9, preferably 1 to 3 DAP10 proteins; Preferably, the intracellular domain comprises an intracellular segment sequence of two or more DAP10 proteins joined together in tandem; Preferably, the intracellular domain further comprises an intracellular costimulatory signaling domain and/or a CD3ζ signaling domain; Preferably, the intracellular costimulatory signaling domain is selected from the functional signaling domains of proteins: CD28, 4-1BB, TLR1, TLR2, TLR3, TLR4, CD27, CD28, OX40, CD30, CD40, PD- 1. ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, CDS, ICAM-1, GITR, BAFFR, HVEM, SLAMF7, NKp80, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, Ly9, CD160, PSGL1, CD100, CD69, SLAMF6, SLAM, BLAME, SELPLG, LTBR, LAT, Any one or a combination of at least two of GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D or CD83, preferably any one or at least two of TLR2, 4-1BB or CD28 combination; Preferably, the extracellular domain capable of binding an antigen refers to a receptor extracellular domain of the antigen, or a single-chain variable fragment of a specific antibody of the antigen; Preferably, the antigen is a tumor antigen and/or a tumor-associated helper cell antigen; Preferably, the tumor antigen and/or tumor-associated helper cell antigen is selected from the group consisting of 5T4, α5β1-integrin, 707-AP, AFP, ART-4, BAGE, β-catenin/m, Bcr-abl, MN/ C IX antibody, CA125, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22, CD25, CDC27/m, CD30, CD33, CD52, CD56, CD80, CDK4/m, CEA, CT, Cyp- B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2/new, HLA-A*0201-R170I, HPV-E7, HSP70- 2M, HST-2, hTERT, hTRT, iCE, IGF-1R, IL-2R, IL-5, KIAA0205, LAGE, LDLR/FUT, MAGE, MART-1/melan-A, MART-2/Ski, MC1R, Mesothelin, myosin/m, PSCA, GPC3, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, PAP, Protease-3, p190minor bcr-abl, Pml/RARα, PRAME, PSA, PSM, PSMA, RAGE, RU1, RU2, SAGE, SART-1, SART-3, survivin, TEL/AML1, TGFβ, TPI/m, TRP-1, TRP-2, TRP-2/INT2, VEGF , WT1, BCMA, CD123, PD-1, PD-L1/2, TIM3, LAG3, A2ARA2AR, B7H3, B7H4, BTLA, IDO, KIR, CD160, 2B4, VISTA, TSHR, CD171, CS-1, CLL-1 , GD3, Tn Ag, FLT3, CD38, CD44v6, B7-H3, CD117, IL-13Ra2, IL-11Ra, PRSS21, VEGFR2, Lewis antigen, CD24, PDGFR-β, SSEA-4, NCAM, CAIX, EphA2, GM1 , sLe, TGS5, HMWMAA, OAcGD2, folate receptor β, CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, Lymphocyte antigen 6 complex, LY6K, OR51E2, TARP, ETS, SPA17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, ERG, androgen receptor, cyclin B1 Any one or a combination of at least two of SART3, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, FCRL5, IGLL1, NY-Eso-1 or NY-Eso-B ; Further preferably, the antigen is any one or a combination of at least two of PCSA, Mesothelin or GPC3 antigens; more preferably, the extracellular domain is a receptor that specifically recognizes the antigen Mesothelin, PSCA or GPC3 Extracellular segments and/or antibodies; Preferably, the transmembrane domain is a transmembrane domain corresponding to a transmembrane domain or an intracellular segment costimulatory signal molecule corresponding to an extracellular segment receptor, preferably a CD28 transmembrane domain. A nucleic acid encoding a chimeric antigen receptor according to claim 1 or 2 or a nucleic acid having at least 80% identity, preferably at least 90% identity thereto. A chimeric antigen receptor-expressing cell into which the nucleic acid according to claim 3 is introduced; preferably, the cell is an immune effector cell, further preferably a T cell, a B cell, an NK cell, an NKT cell, or a dendritic cell Any one or a combination of at least two of the cells or macrophages. A method of producing a chimeric antigen receptor-expressing cell according to claim 4, which comprises the step of introducing the nucleic acid of claim 7 into a cell; preferably, the cell is an immune effector cell, further preferably a T cell Any one or a combination of at least two of B cells, NK cells, NKT cells, dendritic cells or macrophages. A recombinant vector comprising the nucleic acid of claim 3; preferably, the vector is any one or a combination of at least two of a recombinant cloning vector, a recombinant eukaryotic expression plasmid or a recombinant lentiviral vector, preferably For recombinant lentiviral vector; Preferably, the recombinant cloning vector comprises any one or a combination of at least two of pUC18, pUC19, pMD19-T, pGM-T vector, pUC57, pMAX or pDC315; Preferably, the eukaryotic expression plasmid comprises a pCDNA3 series vector, a pCDNA4 series vector, a pCDNA5 series vector, a pCDNA6 series vector, a pRL series vector, a pUC57 vector, pMAX or pDC315, or a combination of at least two; Preferably, the recombinant lentiviral vector comprises any one or a combination of at least two of a recombinant adenovirus vector, a recombinant adeno-associated virus vector, a recombinant retroviral vector, a recombinant herpes simplex virus vector or a recombinant vaccinia virus vector. A recombinant virus comprising the recombinant vector obtained by co-transfecting a mammalian cell with the recombinant vector of claim 6 and a packaging helper plasmid; Preferably, a chimeric antigen receptor T cell which is expressed by transfecting the recombinant virus of claim 7 into a T cell; Preferably, the chimeric antigen receptor according to claim 1 or 2, the nucleic acid according to claim 3, the recombinant vector according to claim 6 or the recombinant virus according to claim 7 is used for amplifying CAR T cells. A pharmaceutical composition comprising the chimeric antigen receptor of claim 1 or 2, the nucleic acid of claim 3, the chimeric antigen receptor expression cell of claim 4, or the method of claim 6. Said expression vector, and optionally a pharmaceutically acceptable excipient. The chimeric antigen receptor according to claim 1 or 2, the nucleic acid according to claim 3, the chimeric antigen receptor expression cell according to claim 4 or the expression vector according to claim 6 in preparation Use in a medicament for treating and/or preventing an autoimmune disease or tumor; Preferably, the tumor is a solid tumor and/or a hematoma, preferably leukemia, liver cancer or lung cancer. A method of treating a subject having an autoimmune disease and/or a disease associated with expression of a tumor antigen, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 8 drug; Preferably, the tumor is a solid tumor and/or a hematoma, preferably leukemia, liver cancer or lung cancer.

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