WO2013185552A1 - Dual-signal independent chimeric antigen receptor and use thereof - Google Patents

Description

 Dual-signal independent chimeric antigen receptor and use thereof

 The present invention relates to a chimeric antigen receptor, in particular a dual signal independent chimeric antigen receptor, the present invention also relates to an immunoreactive cell expressing said chimeric antigen receptor, and said immune response cell for use in the preparation of a therapeutic Use in drugs for malignant tumors and viral infectious diseases. Background technique

 Adoptive cell therapy (ACT) is a method of returning treated autologous or allogeneic immune cells (mainly autologous cells) to a tumor patient to enhance the patient's immune function and achieve therapeutic goals. Current tumor ACT progresses rapidly, using tumor infiltrating lymphocytes

 The adoptive immunotherapy program (tumor-infiltrating lymphocyte, TIL) has achieved very good clinical results for melanoma (Science 2002; 298: 850-4). However, T cell activation requires stimulation of two signals, two signals related to T cell activation. Among them, T cell surface

The TCR-CD3 complex binds to the antigen peptide-MHC molecule and provides the first signal of T cell activation, which determines the kill specificity of T cells; the costimulatory molecule on the surface of T cells (such as CD28) binds to the corresponding ligand (such as B7). Provides a second signal of T cell activation that promotes T cell activation, proliferation and survival. However, the lack of or the decreased expression of the first signal stimulus source (such as MHC molecule) and the second signal ligand (such as B7) in tumor cells cannot effectively provide T cell activation-related signals, thereby activating T cell immune response. Therefore, it is necessary to genetically engineer T cells. At present, genetic modification of this T cell is mainly achieved by transgenic TCR (T cell receptor) and chimeric antigen receptors (CAR) (Blood 2010; 116: 1035-44; Nat Rev Clin Oncol 2011; 8: 577-85).

 The TCR modification has a relatively large restriction. 1 The transgenic TCR chain may be mismatched with the patient's endogenous TCR chain, resulting in a decrease in the TCR density of tumor-reactive T cells on the surface of the T cell; 2 TCR recognizes the antigen presented by the MHC molecule, However, different patients have different MHC molecules, so it is necessary to isolate TCR specific for all MHC haplotypes, and the operability is low; 3 most TCRs cannot recognize carbohydrates or glycolipid antigens, and the antigen selection range is narrow; 4 The first signal related to T cell activation is provided, and the second signal is not provided, and the therapeutic effect is insufficient. Thus, chimeric antigen receptor CAR receptors are more favored.

Chimeric antigen receptor CAR consists of a scFv single-chain antibody (from the amino acid sequence of the VL region of the antibody and The amino acid sequence of the VH region is linked by Linker, and is constructed by a hinge structure linked to a transmembrane and intracellular signal structure derived from a TCR complex or an IgE high affinity receptor. T cells expressing CAR can react with antigens via a non-MHC restricted pathway. In addition, CAR is not limited to protein antigens compared to protein antigens that can only be targeted by conventional TCRs, but also includes sugars and glycolipids, TAA, which are not as susceptible to mutations as protein antigens (Curr Opin Immunol 2009; 21 : 215-23; Blood 2010; 116: 1035-44; Cancer Res 2011; 71: 3175-81; J Cancer 2011; 2: 378-82). Since 1989, Eshhar and his colleagues first proposed the concept of CAR, which has undergone three different stages of development. The first generation of CAR receptors, including scFv fragments that specifically recognize tumor antigens, and intracellular activation signals by IT3 of CD3 or FcsRIy

 (The immunoreceptor tyrosine-based activation motifs) are transmitted by signal chains. However, the first-generation CAR receptor lacks the costimulatory signal of T cells, which leads to T cells only exerting transient effects, short time in the body and less secretion of cytokines. The second generation of CAR receptors adds an intracellular domain of a costimulatory signaling molecule to the first generation of CAR, providing two signals for T cell activation, including CD28, CD134/OX40, CD137/4-1BB, Lymphocyte-specific protein tyrosine kinase (LCK), inducible T-cell co-stimulator (ICOS) and

The DNAX-activation protein 10 (DAP10) and other domains enhance the proliferation of T cells and the secretion of cytokines, and increase IL-2, IFN-γ and GM-CSF, thereby breaking the immunosuppression of the tumor microenvironment and prolonging AICD. (activate induced cell death, AICD). The third-generation CAR receptor, based on the second-generation CAR, adds an intracellular domain of another co-stimulatory signaling molecule, such as recombining a second-level co-stimulation between the co-stimulatory structure CD28 and the ITAM signal chain. Stimulating molecules such as 4-1BB produce a triple-signal CAR receptor, and third-generation CAR receptor-modified T cells have better effector function and survival time in vivo.

 At present, there are 29 CAR-modified T cell adoptive treatment programs in the United States.

(Blood 2010; 116: 1035-44; Nat Rev Clin Oncol 2011; 8: 577-85). It can be seen that the first generation of CAR only provides the first signal of T cell activation, and the second and third generation of CAR combines the two signals required for T cell activation, and the second signal CD28 or / and The 4-1BB intracellular signal region is directly linked to the CD3z molecule, thereby bypassing the tumor cell. The second signal, such as B7, lacks the barrier that T cells cannot activate. The combination of the first signal and the second signal greatly improves the pair. T cell activation, proliferation and killing ability, so that its efficacy is greatly increased. However, it brings certain risks to clinical treatment, because the second-generation and third-generation CAR-modified T cells will produce a strong response to certain small tissues expressing tumor-associated antigens in a small amount, resulting in a "fall" overdose increase. , causing an overactive immune response to normal tissues. There are currently two injections of CAR modified T Case reports of cells leading to death. In one case, a third-generation CAR (for Her2) was used, and patients died of acute pulmonary edema because CAR+ T cells mistakenly attack Herp-expressing lung epithelial cells (Mol Ther 2010; 18: 843-51); another case Apply second generation CAR (for

CD19), the cause of death is complex, but accompanied by an increase in cytokine levels in the blood (Hum Gene Ther 2010; 21: 1039-42; Mol Ther 2010; 18: 666-8). In order to solve this safety hazard, the researchers introduced HSV-TK, AFas. iCasp9, CD20-Rituximab and other suicide systems into the T-cell CAR modification to exert a braking effect and avoid excessive proliferation of T cells (J Cancer 2011; 2: 378- 82; N Engl J Med 2011; 365: 1673-83). However, the "waterfall" effect of CAR+'s T-cell off-target is very fast, and these suicide systems may not work in time. Another approach is to reduce the number of CAR+ sputum cells, which in turn reduces the therapeutic efficacy. Therefore, it is very necessary to modify CAR's own response system to improve the clinical safety of CAR-mediated tumor ACT. Summary of the invention

 The present invention provides a dual signal independent chimeric antigen receptor, an immunoreactive cell expressing the chimeric antigen receptor, and the use of the immune response cell. specifically,

 A first aspect of the invention relates to a dual-signal chimeric antigen receptor (dsCAR) consisting of two independent chimeric antigen receptors, each of which transmits two signals, Antigen receptor 1 is comprised of one or several (eg, two) chimeric antigens comprising a ligand capable of binding a tumor-specific antigen or a tumor-associated antigen, a transmembrane region, and an intracellular immunoreceptor tyrosine activation motif. In vivo, chimeric antigen receptor 2 consists of one or several (eg, two) ligands that bind to membrane receptors that are widely expressed by tumor cells, transmembrane regions, and intracellular domains of intracellular costimulatory signaling molecules. Combined with antigen receptor composition.

 In the present invention, the two signals refer to a first signal and a second signal of T cell activation. The two independent chimeric antigen receptors are those in which the chimeric antigen receptors that transmit the T cell activation first signal and the second signal are independent of each other, and transmit the first signal and the second signal of T cell activation, respectively.

According to the chimeric antigen receptor of the first aspect of the invention, the tumor-specific antigen or tumor-associated antigen is selected from the group consisting of CD19, CD20, CEA, GD ₂ (also known as B4GALNT1, beta-l, 4-N-acetyl-galactosaminyl Transferase 1 ) , FR ( Flavin reductase ) , PSMA ( Prostate-specific membrane antigen ) , gpl00 ( PMEL premelanosome protein ) , CA9 ( carbonic anhydrase IX ) , CD171/L1-CAM , IL-13Ra2 , MART-1 ( also known as melan -A ), ERBB2, NY-ESO-K, also known as CTAG1B, cancer/testis Antigen IB ) , MAGE ( Melanoma-associated antigen El ) family protein, BAGE ( B melanoma antigen family ) family protein, GAGE ( growth hormone releasing factor ) family protein, AFP ( alpha-fetoprotein ) , MUCl ( mucin 1, cell surface associated ), CD22, CD23, CD30, CD33, CD44v7/8, CD70, VEGFR1. VEGFR2. IL-llRou EGP-2, EGP-40, FBP, GD ₃ (also known as ST8SIA1, ST8 alpha-N-acetyl-neuraminide alpha- 2,8-sialyltransferase 1 ), PSCA (promoting stem cell antigen ), FSA (also known as KIAA1109 ) , PSA (also known as KLK3 , kallikrein-related peptidase 3 ), HMGA2 , fetal acetylcholine receptor , LeY ( also known as FUT3 ) , EpCAM , MSLN ( mesothelin ), IGFR1, EGFR, EGFRvIII. ERBB3, ERBB4, CA125 (also known as MUC16, mucin 16, cell surface associated ), CA15-3, CA19-9, CA72-4, CA242, CA50, CYFRA21-1, SCC (also known as SERPINB3), AFU (also known as FUCA1), EBV-VCA, POA (also known as VDR, vitamin D (1,25- dihydroxyvitamin D3) receptor), P2-MG (beta-2-microglobulin) and PROGRP ( GRP ga One or several of strin-releasing peptides. In one embodiment of the invention, the tumor-specific antigen or tumor-associated antigen is MUC1.

 According to the chimeric antigen receptor of the first aspect of the invention, the membrane receptor widely expressed by the tumor cell is selected from the group consisting of CD19, CD20, MUCl. EGFR, EGFRvIII. ERBB2, ERBB3, ERBB4, VEGFR1. VEGFR2, EpCAM, CD44 and One or several of IGFR. Preferably, the tumor-specific antigen or tumor-associated antigen is different from a membrane receptor widely expressed by tumor cells.

 According to the chimeric antigen receptor of the first aspect of the present invention, preferably, the ligand capable of binding to a membrane receptor widely expressed by a tumor cell is capable of simultaneously binding to two or more of the above membrane receptors, so that the present invention The chimeric antigen receptor is capable of undergoing signal stimulation of a heterogeneous tumor cell population to prolong the effect time of the immune response cell.

 In one embodiment of the invention, the ligand capable of binding to a tumor cell to express a membrane receptor broadly refers to a ligand capable of binding to an EGFR family protein (including EGFR, ERBB2, and/or ERBB4) and the EGFR mutant EGFRvIII; Preferably, the ligand is HERIN.

A chimeric antigen receptor according to the first aspect of the invention, wherein said intracellular immunoreceptor tyrosine activating motif comprises an immunoreceptor tyrosine activating motif signal chain selected from the group consisting of CD3 and FCSRIY; The intracellular domain of the intracellular costimulatory signal molecule comprises an intracellular domain selected from the group consisting of CD28, CD134/OX40, CD137/4-lBB, LCK, ICOS, DAP10, preferably, the above.

 In one embodiment of the invention, the intracellular immunoreceptor tyrosine activation motif comprises

The immunoreceptor tyrosine activation motif signal chain of CD3.

 In one embodiment of the invention, the intracellular domain of the intracellular costimulatory signaling molecule comprises the intracellular domain of CD28 and CD137/4-1BB.

 A chimeric antigen receptor according to the first aspect of the invention, wherein said transmembrane region refers to a portion of a membrane protein within a cell membrane, such as CD28, CD8, CD3, CD134, CD137, ICOS, and DAP10 transmembrane regions Preferably, the transmembrane regions of the two chimeric antigen receptors are different to prevent mismatching. In one embodiment of the invention, the transmembrane region of the chimeric antigen receptor 1 is a CD8 transmembrane region, and the transmembrane region of the chimeric antigen receptor 2 is a CD28 transmembrane region.

 According to the chimeric antigen receptor of the first aspect of the present invention, the ligand is a molecule which specifically binds to the tumor-specific antigen or tumor-associated antigen, and a membrane receptor widely expressed by a tumor cell, and may be, for example, a protein. a polypeptide or an antibody; the antibody may be, for example, a monoclonal antibody, a single chain antibody, a Fab antibody, or the like, and in one embodiment of the invention, the antibody is a single chain antibody (ScFv); in another embodiment of the present invention In the embodiment, the ligand is a polypeptide, and in a specific embodiment, the polypeptide is a HERIN molecule.

 According to the chimeric antigen receptor of the first aspect of the invention, the ligand is single copy or multiple copies, and the multiple copies are, for example, double copies.

 A chimeric antigen receptor according to the first aspect of the invention, which is co-expressed by a vector or separately expressed by two identical or different vectors. In one embodiment of the invention, it is co-expressed by a vector. Preferably, a protein precursor processing enzyme recognition sequence, such as Furin-2A, is ligated between the two chimeric antigen receptors, and when the two chimeric antigen receptors are expressed, they are cleaved into two independent proteins. Transported to the cell membrane separately.

 The vector of the present invention is a vector known in the art and can be used for protein cloning and expression, for example, a eukaryotic expression plasmid, a recombinant virus; and the eukaryotic expression plasmid can be, for example, pSV2, pRSV, pcDNA3.1, pCI and pVAXl. The transposon plasmid; the recombinant virus may be, for example, a recombinant retrovirus, a recombinant lentivirus, or a recombinant adenovirus. In one embodiment of the invention, the vector is pcDNA3.1(+).

 In the present invention, cells are genetically modified by introducing a vector expressing a chimeric antigen receptor into an immunoreactive cell to express a chimeric antigen receptor.

The method for introducing a vector into an immunoreactive cell can be a method commonly used in the art, for example, a gene gun method, a transfection method, an electrotransfer method, and a virus transduction method. In one embodiment of the invention, the chimeric antigen receptor 1 is a ScFv, a CD8 transmembrane region, a CD3 signal chain of MUC1. The chimeric antigen receptor 2 is the amino acid sequence HERIN, the CD28 transmembrane region, the CD28 intracellular region, and the 4-1BB costimulatory peptide encoded by the eighth intron of the human Her2 gene.

 According to the chimeric antigen receptor of the first aspect of the invention, the amino acid sequence of the chimeric antigen receptor 1 comprises the amino acid sequence of SEQ ID NO: 31, and the amino acid sequence of the chimeric antigen receptor 2 comprises SEQ ID NO: The amino acid sequence shown by 33.

 In a particular embodiment of the invention, the amino acid sequence of the chimeric antigen receptor 1 is the amino acid sequence set forth in SEQ ID NO:31.

 In a particular embodiment of the invention, the amino acid sequence of the chimeric antigen receptor 2 is the amino acid sequence set forth in SEQ ID NO:33. A second aspect of the invention relates to an engineered immunoreactive cell expressing the chimeric antigen receptor of any one of the first aspects of the invention.

 According to the immunoreactive cell of the second aspect of the present invention, the immunoreactive cell may be selected, for example, from a T cell, a monocyte, a natural killer cell (NK cell), a neutrophil cell; wherein the T cell can be, for example, For cytotoxic T lymphocytes, KT cells, helper T cells, or inhibitory/regulatory T cells. A third aspect of the invention relates to a kit comprising the immunoreactive cell of any of the second aspects of the invention, and optionally instructions for use. A fourth aspect of the invention relates to the chimeric antigen receptor of any one of the first aspect of the invention, or the immunoreactive cell of any one of the second aspect, for use in the preparation of a prophylactic and/or therapeutic malignancy and viral infectivity Use in medicines for diseases.

 The use according to the fourth aspect of the present invention, wherein the malignant tumor can be any malignant tumor, such as lung cancer, hepatocellular carcinoma, lymphoma, colon cancer, rectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, bile duct Cancer, biliary cancer, esophageal cancer, kidney cancer, glioma, melanoma, pancreatic cancer or prostate cancer.

The use according to the fourth aspect of the present invention, wherein the virus may be any virus that infects cells, such as HIV (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus ( Epstein-Barr virus ), Papillomavirus, Herpesvirus or cytomegalovirus. A fifth aspect of the invention relates to a method of modifying an immunoreactive cell, comprising the step of expressing the chimeric antigen receptor of any one of the first aspects of the invention on an immunoreactive cell.

 According to the method of the fifth aspect of the present invention, the immune response cell is selected from the group consisting of a T cell, a monocyte, a natural killer cell, and a neutrophil; wherein the T cell can be, for example, a cytotoxic T lymphocyte or an NKT. Cells, helper T cells, or inhibitory/regulatory T cells. A sixth aspect of the invention relates to a method of preventing and/or treating a malignant tumor and a viral infectious disease, the method comprising preventing or treating a subject in need thereof an effective amount of any one of the first aspects of the invention A step of a chimeric antigen receptor or an immune response cell according to any one of the preceding aspects.

 The method according to the sixth aspect of the present invention, wherein the malignant tumor can be any malignant tumor, such as lung cancer, hepatocellular carcinoma, lymphoma, colon cancer, rectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, bile duct Cancer, biliary cancer, esophageal cancer, kidney cancer, glioma, melanoma, pancreatic cancer or prostate cancer.

 The method according to the sixth aspect of the present invention, wherein the virus may be any virus that infects cells, such as HIV (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), and Epstein-Barr virus ( Epstein-Barr virus), Papillomavirus, Herpesvirus or cytomegalovirus. Detailed description of the invention

 The term "chimeric antigen receptor" in the present invention is an artificially engineered receptor capable of anchoring a specific molecule (such as an antibody) that recognizes a tumor antigen to an immune cell (such as a T cell), so that the immune cell recognizes a tumor antigen or virus. Antigen and cells that kill tumor cells or virus infection.

 The term "T cell activation-associated signal" in the present invention means that two signals required for activation of T cells, that is, a TCR-CD3 complex on the surface of a T cell, bind to an antigen peptide-MHC molecule, and provide a first signal for T cell activation, which is determined. Killing specificity of T cells; co-stimulatory molecules on the surface of T cells (such as CD28) bind to the corresponding ligand (such as B7), providing a second signal of T cell activation, promoting T cell activation, proliferation and survival.

The term "immunoreceptor tyrosine activation motif" ( immunoreceptor) in the present invention Tyrosine-based activation motifs (ITAM) are tyrosine residues (tyrosine, Y) shared by the cytoplasmic regions of immune cell activation-associated receptors (eg, BCR/Iga/Igp, TCR/CD3. FcaR and FcRy, etc.) A basic amino acid sequence motif characterized by two tyrosine residues separated by an amino acid residue of about 13 (...YXX [L/V] X 7-11 YXX [L/V] ...), where tyrosine is a protein kinase phosphorylation site that is phosphorylated to bind to a signaling molecule downstream of the signal transduction pathway, resulting in activation of the cell.

 The term "co-stimulating molecule" in the present invention means some adhesion molecules on the surface of an immune cell, such as CD28, CD134/OX40. CD137/4-1BB. CD40, etc., activated by binding to its ligand. The second signal of immune cells enhances the proliferative capacity of immune cells and the secretory function of cytokines, prolonging the survival time of activated immune cells.

 In the invention, the term "tumor specific antigen" (TSA) is a new antigen unique to tumor cells or present only in certain tumor cells but not in normal cells.

 In the invention, the term "tumor-associated antigen" (TAA) refers to an antigen which is unique to non-tumor cells and which is also present on normal cells and other tissues, but an antigen whose content is markedly increased when the cells are cancerous.

 In the invention, the term "single-chain antibody variable region fragment (scFv)" refers to an antibody fragment having the ability to bind antigen by linking the amino acid sequence of the VL region of the antibody and the amino acid sequence of the VH region via Linker.

 In the present invention, the term "EGFR" refers to the human epidermal growth factor receptor, also referred to as ERBB1 or HER1, and its family members include EGFR, ERBB2 (HER2), ERBB3 (HER3), and ERBB4 (HER4).

 The term "Herin" in the present invention refers to a DNA sequence encoding the C-terminal 79 amino acids of Herstatin in the 8th intron of human Her2, which encodes the C-terminus of Herstatin in the 8th intron of human Her2. A 79 amino acid sequence that binds to members of the EGFR family (including EGFR, ERBB2, ERBB4) and the EGFR mutant EGFRvIII.

 In the present invention, the dual signal independent means that the chimeric antigen receptor that transmits the first signal of T cell activation and the chimeric antigen receptor of the second signal are independent of each other, respectively bind to the respective ligands, and respectively generate signals after binding. Transfer into the cells.

In the present invention, the Linker is a polypeptide fragment that links between different proteins or polypeptides, the purpose of which is to maintain the spatial conformation of the linked protein or polypeptide to maintain the function or activity of the protein or polypeptide. In the present invention, the polypeptide generally refers to a peptide chain molecule having a length of from 1 to 100 amino acids; a protein generally refers to a peptide chain molecule having a length of more than 100 amino acids.

 The term "selected from" as used in the present invention means one or several selected from the listed items. Advantageous effects of the invention

 In the embodiment of the present invention, the T cell is taken as an example, and the second generation and the third generation CAR which are the most popular in the world are reconstructed, and the first signal and the second signal are separated from the single CAR to construct a double Signal-independent chimeric antigen receptors (dsCAR). The two CARs identify antigens from two different families of tumor cells, respectively, and transmit two signals related to T cell activation. Among them, a CAR transmits a first signal related to T cell activation by binding a single-chain antibody or peptide of a tumor-specific antigen or a tumor-associated antigen to determine T cell killing specificity; another CAR is widely expressed by binding to tumor cells. Single-chain antibodies or peptides of membrane receptors (such as EGFR family member proteins) transmit a second signal associated with T cell activation, promoting T cell activation, proliferation, and survival. It is ensured that the T cells modified by dsCAR proliferate in a large amount in the tumor environment in which the first signal and the second signal source coexist, thereby specifically killing the tumor cells; and the normal cells do not simultaneously express the receptors of the two stimulation signals, even the normal cells. Low expression of the first source, only mild accidental injury, will not cause a "fall" over-expression of CAR+ T cells, leading to serious consequences; and in some normal tissue environments with only the second source, T The cells do not exert a killing effect, so the safety is greatly improved. The present invention can avoid potential safety problems while maintaining the efficacy of second and third generation CAR.

 In addition to T cells, the invention is equally applicable to other immune response cells, such as monocytes, NK cells, neutrophils.

 Meanwhile, the chimeric antigen receptor 2 of the present invention comprises a ligand capable of binding to a membrane receptor widely expressed by tumor cells, and thus is suitable for various malignant tumors, viral infectious diseases, and the same type of malignant cells or The virus-infected cells express different membrane receptors due to heterogeneity, thereby achieving the effect of prolonging the effect time of the immune response cells. DRAWINGS

Figure 1: Schematic diagram of CAR1 (CARIMUCI) binding to MUC1 and CAR2 (CAR2 _EGFR ) binding to EGFR family proteins, and third generation CAR (G3-CAR _MU ci) binding to MUC1.

SP: signal peptide; LI - L5: Linker 1 - Linker 5;

Figure 2: Structure of the expression vector for CAR1 _C1 CAR2 _EGFR (abbreviation) pcDNA3.1-CARl: 2).

Figure 3: Proliferation of Jurkat cells (Jurkat ^CAR1 , Jurkat ^CAR2 , Jurkat ^CAR1CAR2, and Jurkat ^3G - ^CAR ) treated with different treatments after exposure to A431, MCF7, and U-20S.

Figure 4: Secretion of INFry after co-culture with A431, MCF7, and U-20S cells by Jurkat cells (Jurkat ^CAR1 , Jurkat ^CAR2 , Jurkat ^CAR1CAR2, and Jurkat ^3G - ^CAR ) treated with different treatments.

Figure 5: Killing effect of differently treated Jurkat cells (Jurkat ^CAR1 , Jurkat ^CAR2 , Jurkat ^CAR1CAR2 and Jurkat ^3G - ^CAR ) on A431, MCF7, U-20S. detailed description

 The embodiments of the present invention are described in detail below with reference to the accompanying drawings. If no specific conditions are specified in the examples, they are carried out according to the general conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that are commercially available. Example 1: Synthesis of CAR expression cassette and construction of expression vector

 According to the amino acid sequence and coding sequence constituting each component of CAR, the entire fused amino acid sequence and the coding DNA expression frame are spliced, wherein:

 The amino acid residue sequence of HERI is:

 GTHSLPPRPAAVPVPLRMQPGPAHPVLSFLRPSWDLVSAFYSLP LAPLSPTSVPISPVSVGRGPDPDAHVAVDLSRYEG (SEQ ID NO: 1) The coding sequence of HERI is:

 GGTACCCACTCACTGCCCCCGAGGCCAGCTGCAGTTCCTGTCC

 GGTATGAAGGC (SEQ ID NO: 2)

 The amino acid residue sequences of the CD28 transmembrane and intracellular regions (CD28) are:

 PFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNM

TPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 3) The coding sequence of CD28 transmembrane and intracellular regions (CD28) is:

TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGC

 CCCCACCACGCGACTTCGCAGCCTATCGCTCC (SEQ ID NO: 4) The amino acid residue sequence of the 4-1BB costimulatory signal domain peptide (41BB) is:

 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

 (SEQ ID NO: 5)

 The coding sequence of the 4-1BB costimulatory signal domain peptide (41BB) is:

 AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCA

TGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO: 6)

 The amino acid residue sequence of CD3 is:

 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 7)

 The coding sequence of CD3 is:

 A AGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCA

 AGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGA

CGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 8)

 The amino acid residue sequence of MUC1 scFv-VH ( VHMUCI ) is:

EVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLE WVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDT GIYYCTFGNSFAYWGQGTTVTVSS (SEQ ID NO: 9)

The coding sequence of MUC1 scFv-VH ( VHMUCI ) is: GAGGTCCAGCTGCAGCAGTCAGGAGGAGGCTTGGTGCAACC

 ID NO: 10)

 The amino acid residue sequence of MUC1 scFv-VL (VLMUCI) is:

 DIVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDH

LFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCAL WYSNHWVFGGGTKLTVLGSE (SEQ ID NO: 11)

 The coding sequence of MUC1 scFv-VL ( VLMUCI ) is:

 GATATCGTTGTGACTCAGGAATCTGCACTCACCACATCACCT

 CAAACTGACTGTCCTAGGATCCGAG (SEQ ID NO: 12)

 The amino acid residue sequence of the CD8 transmembrane region peptide (CD8TM) is:

 YIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 13)

 The coding sequence of the CD8 transmembrane region peptide (CD8TM) is:

 TACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTC CTGTCACTGGTTATCACCCTTTACTGC (SEQ ID NO: 14)

 The amino acid residue sequence of signal peptide 1 is:

 MEFWLSWVFLVAILKGVQC (SEQ ID NO: 15)

 The signal peptide 1 coding sequence is:

ATGGAGTTTTGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAA AAGGTGTCCAGTGT (SEQ ID NO: 16) The amino acid residue sequence of signal peptide 2 is:

 MEAPAQLLFLLLLWLPDTTG (SEQ ID NO: 17)

 The signal peptide 2 coding sequence is:

 ATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGGC TCCCAGATACCACCGGA (SEQ ID NO: 18)

 The amino acid residue sequence of Linkerl is:

 GGSGSGGSGSGGSGS (SEQ ID NO: 19)

 The coding sequence of Linkerl is:

 GGTGGTTCTGGTTCTGGCGGCTCCGGTTCCGGTGGATCCGG CTCT (SEQ ID NO: 20)

 The amino acid residue sequence of Linker2 is:

 EPKSCDKTHTCPPCPAPE (SEQ ID NO: 21)

 The coding sequence of Linker2 is:

CCAGCACCTGAA (SEQ ID NO: 22)

 The amino acid residue sequence of Linker3 is:

 PKLEEGEFSEARVDIVLTQSP (SEQ ID NO: 23)

 The coding sequence of Linker3 is:

 CCCAAGCTTGAAGAAGGTGAATTTTCAGAAGCACGCGTAGAT ATCGTTCTCACTCAATCTCCA (SEQ ID NO: 24)

 The amino acid residue sequence of Linker 4 is:

 GGGGSGGGGSGGGGS (SEQ ID NO: 25)

 The Linker 4 coding sequence is:

 GGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCG GGTCG (SEQ ID NO: 26)

 The amino acid residue sequence of Linker 5 is:

 GGGGGGGGG (SEQ ID NO: 27)

 The coding sequence of Linker 5 is:

 GGTGGAGGTGGAGGTGGAGGTGGAGGT (SEQ ID NO: 28) The amino acid residue sequence of Furin-2A is:

RAKRAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 29) The coding sequence of Furin-2A is: CGTGCTAAACGAGCTCCTGTTAAACAGACTTTGAATTTTGAC CTTCTCAAGTTGGCGGGAGACGTCGAGTCCAACCCTGGGCCC

(SEQ ID NO: 30)

CARIMUCI is composed of signal-like l-VH _M uci-Linkerl-VL _M uci-Linker2-CD8TM-Linker3-CD3 fusion (see Figure 1), and its amino acid sequence is:

MEAPAQLLFLLLLWLPDTTGEVQLQQSGGGLVQPGGSMKLSC VASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVK GRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVT VSSGGSGSGGSGSGGSGSDIVVTQESALTTSPGETVTLTCRSSTGAVT TSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTI TGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSEEPKSCDKTHTC PPCPAPEYIWAPLAGTCGVLLLSLVITLYCPKLEEGEFSEARVDIVLT QSPRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 31)

 ! The coding sequence of ^^^ is:

ATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGG

CAGGCCCTGCCCCCTCGC (SEQ ID NO: 32)

CAR2 _EGFR is composed of the signal peptide 2-HERIN-Linker4-CD28-Linker5-41BB fusion (see Figure 1), and its amino acid sequence is:

 MEFWLSWVFLVAILKGVQCGTHSLPPRPAAVPVPLRMQPGPAH PVLSFLRPSWDLVSAFYSLPLAPLSPTSVPISPVSVGRGPDPDAHVAVD LSRYEGGGGGSGGGGSGGGGSPFWVLVVVGGVLACYSLLVTVAFII FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSGG GGGGGGGKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG GCEL (SEQ ID NO: 33)

The coding sequence of CAR2 _EGFR is:

 ATGGAGTTTTGGCTGAGCTGGGTTTTCCTTGTTGCTATTTTAA

GTGGCAGCGGCGGTGGCGGGTCGCCCTTTTGGGTGCTGGTGGTGG TATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAG

TCCGGTGGAGGTGGAGGTGGAGGTGGAGGTAAACGGGGCAGAAA

 GAAGAAGGAGGATGTGAACTG (SEQ ID NO: 34)

CAR1MUCICAR2 _E GFR consists of CAR1MU _C1 and CAR2 _EGFR linked by Furin-2A. The amino acid sequence is:

 MEAPAQLLFLLLLWLPDTTGEVQLQQSGGGLVQPGGSMKLSC VASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVK GRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVT VSSGGSGSGGSGSGGSGSDIVVTQESALTTSPGETVTLTCRSSTGAVT TSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTI TGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSEEPKSCDKTHTC PPCPAPEYIWAPLAGTCGVLLLSLVITLYCPKLEEGEFSEARVDIVLT QSPRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPRRAKRAPVKQTLNFDLLKLAG DVESNPGPEFWLSWVFLVAILKGVQCGTHSLPPRPAAVPVPLRMQP GPAHPVLSFLRPSWDLVSAFYSLPLAPLSPTSVPISPVSVGRGPDPDAH VAVDLSRYEGGGGGSGGGGSGGGGSPFWVLVVVGGVLACYSLLVT VAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAA YRSGGGGGGGGGKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP EEEEGGCEL (SEQ ID NO: 35)

The coding sequence of CARl _MUC1 CAR2 _EeFR is:

GAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGCC ATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGG

GAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGCC

 CTG (SEQ ID NO: 36)

The control G3-CAR _MUC1 was composed of the signal peptide l-VH _M uci-Linkerl-VL _M uci-Linker2-CD28-Linker4-41BB-Linker3-CD3 fusion (see Figure 2), and its amino acid sequence was:

 MEAPAQLLFLLLLWLPDTTGEVQLQQSGGGLVQPGGSMKLSC VASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVK GRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVT VSSGGSGSGGSGSGGSGSDIVVTQESALTTSPGETVTLTCRSSTGAVT TSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTI TGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSEEPKSCDKTHTC PPCPAPEPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDY MNMTPRRPGPTRKHYQPYAPPRDFAAYRSGGGGGGGGGKRGRKK LLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELPKLEEGEFSEA RVDIVLTQSPRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 37)

The coding sequence of G3-CARMUCI is:

 ATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGG

分别按 ！^^^的 DNA编码序列（SEQ ID NO: 32)、 CAR2_EGFR的 DNA编码序列（SEQ ID NO: 34)、 CAR1_MUC1CAR2_EGFR的 DNA编码序列 (SEQ ID NO: 36) . G3-CAR_MUC1的 DNA编码序列（SEQ ID NO: 38) , 委托 生工⑧生物工程（上海）有限公司合成其整个表达框， 插入 pCDNA3.1 ( + ) 载体（ Invitrogen ) EcoRl-Xbal位点 （见图 2 ) , 转化到 E. coli ( DH5a ) , 经测序正确后，使用 Qiagen公司的质粒纯化试剂盒提取并纯化质粒， 获得 各重组表达载体的高品质质粒。 实施例 2: T细胞株的遗传修饰

Press separately! DNA coding sequence of ^^^ (SEQ ID NO: 32), DNA coding sequence of CAR2 _EGFR (SEQ ID NO: 34), DNA coding sequence of CAR1 _MUC1 CAR2 _EGFR (SEQ ID NO: 36). G3-CAR _MUC1 The DNA coding sequence (SEQ ID NO: 38) was entrusted to Biotech Engineering (Shanghai) Co., Ltd. to synthesize its entire expression cassette and insert the pCDNA3.1 ( + ) vector ( Invitrogen ) EcoRl-Xbal site (see Figure 2). After transformation to E. coli (DH5a), after sequencing, the plasmid was extracted and purified using Qiagen's plasmid purification kit to obtain a high quality plasmid of each recombinant expression vector. Example 2: Genetic modification of T cell lines

High quality plasmids of each recombinant expression vector constructed and purified in Example 1 were transfected into Jurkat E6.1 (T lymphocyte strain, purchased from American Type Collection, ATCC) using Lipofectamine 2000 (Invitrogen), respectively. Two days later, the transfected Jurkat E6.1 cells were transferred to RPMI 1640 medium with neomycin and the cells were cloned by limiting dilution. After 21 days of screening, Jurkat E6.1 cell line Jurkat ^CAR1 , Jurkat ^CAR2 , Jurkat ^c dish with neomycin resistance and genetic modification by CAR1 _MUC1 , CAR2 _EGFR , CAR1 _MUC1 CAR2 _EGFR and G3-CAR _M uci were ^established . ^AR2 and J _U rkat ^{G3 CAR} . Example 3: Determination of proliferation of T cell strain after genetic modification

Jurkat ^CAR1 , Jurkat ^CAR2 , Jurkat ^CAR1CAR2 and J _U rkat ^{G3 CAR} and unmodified Jurkat E6.1 cells (5xl0 ⁵ /well, RPMI 1640 medium, containing 20U/ml IL-2) were added to pre-plated radiation Treatment (without disrupting the overall structure, the cells lose the activity of dividing proliferation, see Clin Cancer Res. 2011; 17(7): 1664-73) 6-well plates of A431, MCF7, U-20S (cells) Suspended Jurkat cells were counted on day 3 and day 7, respectively, from ATCC, 5xl0 ⁵ /well. The results showed that Jurkat ^{CARlc AR2} could proliferate in a large amount after ^{exposure to MCI1} and EGFR double positive A431 cells, and the proliferation ratio was higher than that of Jurkat ^{G3 CAR} . After exposure to MCF7 cells with high MUC1 positive and EGFR family protein weak positive, J _U rkat ^CAR1CAR2 proliferation The fold is similar to J _U rkat ^CAR1 , slightly lower than J _U rkat ^G3 - ^CAR ; Jurkat ^CAR1CAR2 does not proliferate after exposure to U-20S cells with weak MUC1 positive and EGFR weak positive, and Jurkat ^G3 - ^CAR can still proliferate in a small amount (see image 3 ). The above results indicate that when both antigens are present (such as MUC1 and EGFR double positive, representing general tumor tissue cells), dsCAR-modified T cells can proliferate in a large amount; when the first signal is present, the second signal is weak (such as MUC1 positive) , EGFR weakly positive, representing a small number of tumor cells), modified by dsCAR The cells can also proliferate effectively; when the first signal is weak, the second signal is present (such as MUC1 weak yang, EGFR weak yang, representing normal tissue cells), and dsCAR-modified T cells do not proliferate. Example 4: Determination of IFNy secretion in T cell strain after genetic modification

Jurkat ^CAR1 , Jurkat ^CAR2 , Jurkat ^CAR1CAR2 and J _U rkat ^{G3 CAR} and unmodified Jurkat E6.1 cells (5xl0 ⁵ /well) in 24-well plates with A431, MCF7, U-20S (all purchased from ATCC, lxlO) Co-culture was carried out at ⁵ /well. After 72 hours, the supernatant was collected, and the amount of IFNy secreted was measured by ELISA kit (BD Biosciences) of IFNy. The results showed that Jurkat ^eARieAR2 could secrete a large amount of γ and secrete more than Jurkat ^G3 - ^CAR after co-culture with MUC1 and EGFR double positive A431 cells. After co-culture with MCF7 cells with high positive MUC1 and weak EGFR family protein, The 诵γ secretion of Jurkat ^CAR1CAR2 is similar to that of Jurkat ^CAR1 , which is lower than that of Jurkat ^G3-CAR . After co-culture with U-20S cells with weak MUC1 and weak EGFR positive, Jurkat ^CAR1CAR2 does not secrete IFNy, and Jurkat ^G3 - ^CAR can still Secreted more IFNy (see Figure 4). The above results indicate that when both antigens are present (such as MUC1 and EGFR double positive, representing general tumor tissue cells), dsCAR-modified T cells can secrete IFNy in large amounts; when the first signal is present, the second signal is weak (such as MUC1). Positive, EGFR weakly positive, representing a small number of tumor cells), dsCAR-modified T cells can also effectively secrete IFNy; when the first signal is weak, the second signal exists (such as MUC1 weak yang, EGFR weak yang, representing normal tissue cells) The T cells modified by dsCAR do not substantially secrete IFNy. Example 5: Determination of in vitro killing effect of T cell strain after genetic modification

Jurkat ^CAR1 , Jurkat ^CAR2 , Jurkat ^CAR1CAR2 and Jurkat ^G3-CAR and unmodified Jurkat E6.1 cells were ^plated with A431 at different target ratios (50:1, 25:1, 5:1, 1:1). Co-culture of MCF7 and U-20S, using LDH-Cytotoxicity Assay Kit (Biovision) to detect the in vitro killing of different types of tumor cells by genetically modified Jurkat E6.1 cells. ability. The method is as follows: The target cells are plated in 96-well plates (5×10 ³ /well), medium background, volume correction, spontaneous LDH release from target cells, maximum LDH release from target cells, control cells spontaneous LDH release control wells, treatment group wells (groups) Prepare according to the kit instructions), repeat 3 wells per group, the final volume of each well is the same and not less than ΙΟΟμ 250g for 4 min, and incubated at 37 ° C, 5% C02 for at least 4 h. 45 min before centrifugation, lOx lysate was added to the largest release well of the target cells, and an equal amount of lysate was added to the volume-corrected well. Centrifuge again, transfer 50 μL of supernatant from each well to a new 96-well plate, add 50 μL of substrate solution, and shield from light at room temperature. Incubate for 30 min. Add 50 μM per well, stop the solution, and measure D490 within 1 h. Cytotoxicity (%) = [( D experimental well - D medium background well) - (D effector cell spontaneous LDH release well - D medium background well) - (D target cell spontaneous LDH release well - D medium background well) 】 / [ ( D target cell maximum LDH release hole - D volume correction hole) - (D target cell spontaneous LDH release hole - D medium background hole)] xl00 results show that Jurkat ^CAR1CAR2 can effectively kill MUC1 and EGFR double positive A431 Tumor cells; the killing effect of MCF7, which is highly positive for MUC1 and weakly positive for EGFR family protein, is similar to Jurkat ^CAR1 , lower than Jurkat ^G3-CAR ; U-20S cells with weak positive MUC1 and EGFR positive are not killed (see Figure 5). . The above results indicate that when both antigens are present (such as MUC1 and EGFR double positive, representing general tumor tissue cells), dsCAR-modified T cells can effectively kill; when the first signal is present, the second signal is weak (such as MUC1 positive) EGFR weakly positive, representing a small number of tumor cells), dsCAR-modified T cells can also effectively play a killing effect; when the first signal is weak, the second signal exists (such as MUC1 weak yang, EGFR weak yang, representing normal tissue cells) The T cells modified by dsCAR are basically not killed. Although specific embodiments of the invention have been described in detail, those skilled in the art will understand. Various modifications and substitutions may be made to those details in light of the teachings of the invention, which are within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

Rights request A double-signal-independent chimeric antigen receptor consisting of two independent chimeric antigen receptors, each of which transmits two signals, wherein the chimeric antigen receptor 1 consists of one or several (eg, two) Composed of a chimeric antigen receptor capable of binding to a tumor-specific antigen or a tumor-associated antigen, a ligand, a transmembrane region, and an intracellular immunoreceptor tyrosine activation motif, the chimeric antigen receptor 2 consists of one or several ( For example, two) consist of a chimeric antigen receptor comprising a ligand capable of binding to a membrane receptor that is widely expressed by tumor cells, a transmembrane region, and an intracellular domain of an intracellular costimulatory signaling molecule. 2. The chimeric antigen receptor of claim 1, wherein said tumor cell is broadly expressed by a membrane receptor selected from the group consisting of CD19, CD20, MUC1, EGFR, EGFRvIII, ERBB2, ERBB3, ERBB4, VEGFR1, VEGFR2, EpCAM, CD44 and One or several of IGFR. 3. The chimeric antigen receptor of claim 1, wherein said ligand capable of binding to a membrane receptor widely expressed by a tumor cell is capable of binding to a EGFR family protein (including EGFR, ERBB2, and/or ERBB4) and an EGFR mutation. A ligand for EGFRvIII; preferably, the ligand is HERIN. Receptor chimeric antigen of claim 1, wherein said tumor-specific antigen or tumor-associated antigen is selected from _{CD19, CD20, CEA, GD 2} , FR, PSMA, gpl00, CA9, CD171 / L1-CAM, IL -13Ra2, MART-1, ERBB2, NY-ESO-1. MAGE family proteins, BAGE family proteins, GAGE family proteins, AFP, MUC1, CD22, CD23, CD30, CD33, CD44v7/8, CD70, VEGFR1, VEGFR2, IL -llRou EGP-2, EGP-40, FBP, GD ₃ , PSCA, FSA, PSA, HMGA2, fetal acetylcholine receptor, LeY, EpCAM, MSLN, IGFR1, EGFR, EGFRvIII, ERBB3, ERBB4, CA125, CA15-3, CA19 -9, CA72-4, CA242, CA50, CYFRA21-1, SCC, AFU, EBV-VCA, TSGF, POA, One or several of P2-MG and PROGRP. 5. The chimeric antigen receptor of claim 1, wherein said intracellular immunoreceptor tyrosine activating motif comprises an immunoreceptor tyrosine activating motif signal chain selected from the group consisting of CD3 and FcsRIy. 6. The chimeric antigen receptor of claim 1, wherein said intracellular costimulatory signal molecule intracellular domain comprises an intracellular structure selected from the group consisting of CD28, CD134/OX40, CD137/4-1BB, LCK, ICOS and DAP10 area. 7. The chimeric antigen receptor according to any one of claims 1 to 6, wherein the ligand is a protein, a polypeptide or an antibody; and/or the ligand is a single copy or multiple copies, the multiple copies being, for example Double copy. The chimeric antigen receptor according to any one of claims 1 to 7, wherein the amino acid sequence of the chimeric antigen receptor 1 comprises the amino acid sequence of SEQ ID NO: 31, and the chimeric antigen receptor 2 The amino acid sequence comprises the amino acid sequence set forth in SEQ ID NO:33. 9. The chimeric antigen receptor of any of claims 1-8, which is co-expressed by a vector or separately expressed by two identical or different vectors. 10. An engineered immune response cell which expresses the chimeric antigen receptor of any of claims 1-9. 11. The immune response cell of claim 10, wherein said immune response cell is selected from the group consisting of a T cell, a monocyte, a natural killer cell, a neutrophil; wherein said T cell can be, for example, a cytotoxic T lymphocyte, NKT cells, helper T cells, or inhibitory/regulatory T cells. 12. A kit comprising the immune response cell of claim 10 or 11, and optionally instructions for use. Use of the chimeric antigen receptor according to any one of claims 1 to 9 or the immunoreactive cell according to claim 10 or 11 for the preparation of a medicament for preventing and/or treating malignant tumors and viral infectious diseases . 14. The use according to claim 13, wherein the malignant tumor is lung cancer, hepatocellular carcinoma, lymphoma, colon cancer, rectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, biliary cancer, esophageal cancer, Kidney cancer, glioma, melanoma, pancreatic cancer or prostate cancer. 15. The use of claim 13, wherein the virus is HIV (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (Epstein-Barr virus), papillomavirus ( Papillomavirus), Herpesvirus or Cytomegalovirus. A method of modifying an immune response cell, comprising the step of expressing the chimeric antigen receptor of any one of claims 1-9 on an immunoreactive cell. 17. The method of claim 16, wherein the immune response cell is selected from the group consisting of a T cell, a monocyte, a natural killer cell, and a neutrophil; wherein the T cell can be, for example, a cytotoxic lymphocyte, an NKT cell, Helper T cells, or inhibitory/regulatory T cells. 18. A method of preventing and/or treating a malignant tumor and a viral infectious disease, the method comprising preventing or treating a prophylactically effective amount of the chimeric antigen receptor of any one of claims 1-9 to a subject in need thereof Or the step of immunoreactive cells according to claim 10 or 11. 19. The method of claim 18, wherein the malignant tumor is lung cancer, hepatocellular carcinoma, lymphoma, colon cancer, colon cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer, cholangiocarcinoma, Cholecystectomy, esophageal cancer, kidney cancer, glioma, melanoma, pancreatic cancer or prostate cancer. 20. The method of claim 18, wherein the virus is HIV (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus, and porcine tumor virus ( Papillomavirus), Herpesvirus or Cytomegalovirus.