CN108699163B - Polygene recombinant chimeric antigen receptor molecule and application thereof - Google Patents

Abstract

A multi-gene recombinant chimeric antigen receptor molecule comprises an extracellular region peptide segment, a transmembrane region peptide segment and an intracellular domain peptide segment which are sequentially connected in series, wherein the extracellular region peptide segment comprises a LIGHT protein peptide segment and an immune binding protein peptide segment which are connected, wherein the LIGHT protein peptide segment comprises at least one extracellular region peptide segment of human LIGHT protein or a fragment thereof, and the immune binding protein peptide segment comprises an extracellular region peptide segment of a protein or a fragment thereof which is combined with an immunosuppressive molecule on the surface of a tumor cell and/or the surface of a tumor stromal cell; also provides the application of the polygene recombinant chimeric antigen receptor molecule, which comprises coding nucleotide, recombinant vector and recombinant cell; the polygene recombinant chimeric antigen receptor molecule has higher specificity and killing property.

Description

Polygene recombinant chimeric antigen receptor molecule and application thereof

The present application claims priority from the chinese patent application entitled "a multigene recombinant chimeric antigen receptor molecule and uses thereof", filed 2016, 28/9, 201610855799.1, the entire contents of which are incorporated herein by reference.

Technical Field

The invention relates to the field of molecular biology, in particular to a polygene recombinant chimeric antigen receptor molecule and application thereof.

Background

Malignant tumors are a serious life-threatening disease for humans. The pathogenesis of malignant tumor or cancer is various, and the common expression of the malignant tumor or cancer is that the variant tumor cells are not cleared by the immune system of the body, can be propagated and spread without limit, and damage the normal cells and functions of the surrounding tissues. The medical community has long made a great deal of effort in attempting to cure and control the course of neoplastic disease, but with little success. At present, the main clinical auxiliary treatment means in the medical field are radiotherapy, chemotherapy and antibody therapy except surgery in the treatment of malignant tumors.

In 1985, american scientists attempted to isolate mononuclear cells from patients and induced, activated, and generated killer T Cells (CIK) in vitro using various cytokines, and then found that these cells had a killing effect on tumor growth after they were infused back into the patients by intravenous infusion. After the clinical application and the technical development of the last thirty years, the treatment of tumors by using the immune killer cells has become a fourth accepted adjuvant therapy means for malignant tumors. Common immune killer cells for clinical treatment: natural killer cells (NK), γ δ T cells, Cytotoxic T Lymphocytes (CTL), NK-like T lymphocytes (NKT), Th1 Effector cells (Effector cells), and the like. Dendritic Cells (DCs), one of the antigen presenting cells, are also commonly used in immune cell therapy applications, where DCs can enhance the specificity of and the viability of immune killer cells.

The recognition and killing effect of immunocompetent cells on tumor cells depends on the expression of receptor molecules on the surface of tumor cell membranes, and at least two factors cause that T lymphocytes in vivo cannot well recognize cancer cells: (1) cancer cells down-regulate the expression of antigen presenting molecules, (2) presented antigens have poor affinity for T cell receptors. Although T lymphocytes highly specific to cancer cells are present in cancer patients, too small a number does not play a role in treating cancer. In order to overcome the defect of low specificity of the immune killer cells, the American scientist invents a transgenic method: the hypervariable region sequence of a monoclonal antibody recognizing a tumor surface specific antigen is recombined and subcloned into a Single-chain antibody fragment (scFv) in vitro, then fused with transmembrane protein fragments of other genes and an intracellular signal peptide to form an artificial Chimeric Antigen Receptor (CAR), and transfected into a T cell to form a Chimeric antigen receptor T cell (CAR-T). The chimeric antigen receptor is mainly composed of two parts, one end is positioned outside cells and can specifically recognize a certain antigen on the surface of a cancer cell, and the other end is positioned in cells and contains a signal activation element (such as a Zeta chain of a T cell receptor) which plays a role in transmitting signals to activate T cells.

The current CAR-T cell therapy taking CD19 as a target achieves obvious effects on clinical treatment of primary and secondary blood system tumors, but the CAR-T cell therapy is slow in progress in the research of solid tumors, and no significant breakthrough exists at present. One of the reasons is that 95% of the lymphoid leukemia cancer cells express the B cell antigen CD19, while the specific antigens of other solid tumor cells are expressed between 40-70%; therefore, in the treatment of solid tumors, it is unlikely that a single monoclonal anti-CAR-T cell will kill cancer cells expressing other tumor-specific antigens. The second reason is the rate and number of infiltration of the immune killer cells into the solid tumor through blood/lymph circulation. The third reason is the negative regulatory effect of tumor tissue cells on immunocompetent cells.

Normally, small amounts of active killer cells, such as NK, γ δ T cells, etc., are present in the human blood circulation and function to clear aging, mutated histiocytes, or resist viral invasion. The activity of immune cells is mainly controlled by T Regulatory cells (tregs), which when weakened, may cause immune hyperfunction or autoimmune disease; when the control is enhanced, the immune function is lowered, and tumors or other viral skin diseases are propagated. The protein factors currently identified as being involved in down-regulation are Cytotoxic T lymphocyte-associated antigen 4 (CTLA 4) and Programmed cell death protein 1/Programmed cell death protein1 ligand 1(Programmed cell death protein 1/Programmed cell death protein1 ligand 1, PD-1/PD-L1) molecules. The inhibitory T regulatory cells express CTLA4, and can interact with B7 protein subunits on the surfaces of immune cells DC and T cells to inhibit the functions of the immune cells. The B7 protein family members include B7-H1(PD-L1), B7-H2(PD-1L2) and the like. B7-H1(PD-L1) can also further inhibit the active function of target cells by binding to lymphocyte PD-1 molecules. Inhibition of T cell function by the interaction of PD-1 and PD-L1 is a major obstacle to immune cell therapy of tumors. In most solid tumor tissues, the cancer cells express PD-L1/PD-1 at an increased level, and direct inhibition is generated on activated lymphocytes infiltrating into the cancer tissues, which is also one of the immune regulation mechanisms of a tumor escape organism; the expression of PD-1 on the surface of the activated T cell is increased, and the activated T cell is more easily inhibited by negative regulation. The most recent antibody drugs, keytruda and opdivo, have been approved by the FDA in the united states for clinical treatment of malignant tumors, and their action principle is to block the mutual binding of CTLA4/B7 and PD-1/PD-L1, thereby releasing the inhibition of active T-killer cells by T-regulatory cells or cancer cells in vivo, and allowing the tumor cells to be killed and eliminated by immunocompetent cells in vivo.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a polygene recombinant chimeric antigen receptor molecule with higher specificity and higher killing property and application thereof.

The invention provides a multi-gene recombinant chimeric antigen receptor molecule, which comprises an extracellular region peptide segment, a transmembrane region peptide segment and an intracellular domain peptide segment which are sequentially connected in series, wherein the extracellular region peptide segment comprises a LIGHT protein peptide segment and an immune binding protein peptide segment which are connected, wherein the LIGHT protein peptide segment comprises at least one extracellular region peptide segment of human LIGHT protein or a fragment thereof, and the immune binding protein peptide segment comprises an extracellular region peptide segment of a protein or a fragment thereof which is combined with an immunosuppressive molecule on the surface of a tumor cell and/or the surface of a tumor stromal cell.

LIGHT, also known as TNFSF14 or CD258, is a member of the ligand TNF superfamily (TNFSF). Its name is derived from a lymphoid toxin, exhibits inducible expression, and competes with HSV glycoprotein D for the herpes virus invasion mediator (HVEM), a receptor expressed by T lymphocytes. LIGHT is expressed on the surface of T cells in a tightly regulated manner by activation (Castellano et al (2002) J.biol.chem.27742841-51). LIGHT modulates its biological effects by binding to one of 3 TNF superfamily receptors, including lymphotoxin beta receptor (LT. beta.R) (Crowe et al (1994) Science 264707-10, Browning et al (1997) JImmunol 159: 3288-98), herpes virus invasion medium (HVEM) (Montgomery et al (1996) Cell 87 (3): 427-36) and anti-trap receptor 3(DeR3) (Yu et al (1999) J.biol.Chem.27413733-6). LIGHT exhibits a variety of immunostimulatory activities upon interaction with its receptors, including modulation of chemokine expression and cell adhesion molecules (Wang, J.et al (2002) Eur.J.Immunol.32: 1969-1979). For example, LIGHT and LT α 1 β 2 act synergistically in lymphoid organogenesis and lymphoid structure development (Scheu, S.et al. (2002) J.Exp.Med.195: 1613-. The signaling of LT β R by the LIGHT transgene was shown to be sufficient to induce up-regulation of chemokine and adhesion molecule expression (Wang, J.et al (2004) J.Clin.invest.113: 826-. In addition, LIGHT has also been shown to modulate the CD 28-independent co-stimulatory activity of T-cell priming and expansion, which may result in enhanced T-cell immunity and/or enhanced autoimmunity against tumors (Tamada, K.et al (2000) Nat.Med.6: 283-.

Further, the LIGHT protein peptide fragment has one or more LIGHT-related activities, including but not limited to: (i) binding to one or more LIGHT receptors, such as lymphotoxin beta receptor (LT β R), herpes virus invasion mediator (HVEM), and/or anti-trap receptor 3(DcR 3); (ii) inducing expression of one or more of a chemokine or cytokine (e.g., CXCL10(IP-10), CCL21, CXCL9, IL-5, IL-8 and/or TNF), a chemokine or cytokine receptor (e.g., IL-10RA), an adhesion molecule and/or a co-stimulatory molecule; (iii) activated T cells, such as lymphocytes (e.g., cytotoxic T lymphocytes), T cells expressing CD4 or CD8, and/or regulatory T cells; (iv) recruiting T cells into a hyperproliferative (e.g., tumor) cell or tissue; (v) activating and/or enhancing proliferation of tumor-reactive T cells; (vi) for example, under hyperproliferative conditions (e.g., tumor cells or tissues), a lymphoid-like microenvironment is created; (vii) inducing apoptosis of hyperproliferative (e.g., tumor) cells or tissues; and/or (viii) stimulating an immune response in a subject, e.g., stimulating the subject's immune system against a hyperproliferative (e.g., tumor or cancer) cell or tissue.

Further, the sequence of the LIGHT protein peptide fragment is shown as SEQ ID NO: 1 is shown.

Further, the LIGHT protein peptide fragment and the immunological binding protein peptide fragment may be connected with or without a connecting peptide. Specifically, the sequence of the connecting peptide is shown as SEQ ID NO: 2, respectively.

Further, the peptide fragment of the immunological binding protein includes an extracellular region peptide fragment of PD-1 or HAC. Specifically, the extracellular region peptide fragment sequence of the PD-1 is shown as SEQ ID NO: 3, respectively. According to the article "Engineering high-affinity PD-1 variants for optimized immunization and immunization-PET imaging", the mutant HAC of PD-1 has higher affinity with PDL-1 molecules on the surface of tumor; the sequence of the extracellular region peptide fragment of the HAC is shown as SEQ ID NO: shown at 13.

Further, the multigene recombinant chimeric antigen receptor molecule further comprises a signal peptide. The signal peptide can improve the secretion effect of the fusion protein of the multi-gene recombinant chimeric antigen receptor molecule, and is finally cut by protease after the signal peptide and other amino acid sequences of the fusion protein are expressed together. The protease has a certain recognition sequence, and the signal peptide is fused with the peptide segment behind the signal peptide to form a new amino acid sequence, so that if the selected signal peptide is improper, the protease can be cut by mistake, and the protein is inactivated. The signal peptide can be selected from signal peptides of immunoglobulin light chains or signal peptides secreted by PD-1 protein, and the sequences of the signal peptides are respectively shown in SEQ ID NO: 4 and 5.

It should be noted that the transmembrane domain peptide segment is a region spanning cell membranes in a protein sequence, and is generally in an alpha-helical structure, and has about 20 to 25 amino acid residues, and most of the amino acids are hydrophobic amino acids; wherein, the transmembrane peptide segment includes but is not limited to CD8 transmembrane peptide segment or PD-1 transmembrane peptide segment, and when the transmembrane peptide segment is CD8 transmembrane peptide segment, the extracellular domain peptide segment and the CD8 transmembrane peptide segment are further connected by a hinger domain peptide segment of CD 8. The sequence of the CD8 transmembrane peptide segment is shown as SEQ ID NO: 6, the sequence of the hinge region peptide fragment of the CD8 is shown as SEQ ID NO: 7, the sequence of the PD-1 transmembrane domain peptide segment is shown as SEQ ID NO: shown in fig. 8.

Further, the intracellular domain peptide segment is a costimulatory signal molecule, and is selected from one or more of 4-1BB (also named as CD137), CD28 and CD3 delta. Preferably, the intracellular domain peptide segment is selected from the group consisting of 4-1BB and CD3 δ intracellular domain peptide segments linked to each other, the sequences of which are set forth in SEQ ID NO: 9 and 10.

Further, the multi-gene recombinant chimeric antigen receptor molecule comprises the amino acid sequence shown as SEQ ID NO: 3, and (b) is the sequence shown in the specification.

Preferably, the multi-gene recombinant chimeric antigen receptor molecule of the invention is formed by sequentially connecting a signal peptide of an immunoglobulin LIGHT chain, a LIGHT protein peptide segment, a connecting peptide, an extracellular region peptide segment of PD-1, a hinge region peptide segment of CD8, a transmembrane region peptide segment of CD8, a 4-1BB intracellular domain peptide segment and a CD3 δ intracellular domain peptide segment, and the sequence of the multi-gene recombinant chimeric antigen receptor molecule is as shown in SEQ ID NO: shown at 11.

In a second aspect, the present invention provides a nucleotide encoding the chimeric antigen receptor molecule provided in the first aspect of the present invention.

Further, comprising or being as set forth in SEQ ID NO: 12.

In a third aspect, the invention provides a recombinant vector comprising the nucleotide provided in the second aspect of the invention.

Furthermore, the vector is a lentiviral vector, and can effectively integrate an exogenous gene or exogenous shRNA (short hairpin ribonucleic acid) onto a host chromosome, thereby achieving the effect of persistently expressing a target sequence. Can effectively infect various cells such as neuron cells, liver cells, cardiac muscle cells, tumor cells, endothelial cells, stem cells and the like in the aspect of infection capacity, thereby achieving good gene therapy effect. For some cells which are difficult to transfect, such as primary cells, stem cells, undifferentiated cells and the like, the lentiviral vector is used, so that the transduction efficiency of the target gene or the target shRNA can be greatly improved, the probability of integrating the target gene or the target shRNA into the host cell genome is greatly increased, and the long-term and stable expression of the target gene or the target shRNA can be conveniently and quickly realized.

It should be noted that the lentiviral vector used in the present invention should include, but is not limited to, pRRSLIN lentiviral expression vector and pLVX vector, preferably pRRSLIN lentiviral expression vector.

In a fourth aspect, the present invention provides a recombinant cell comprising the recombinant vector provided in the third aspect of the present invention. The recombinant cell is preferably a T cell or NK cell. The encoding gene of the chimeric antigen receptor can be transferred into a T cell or an NK cell through the carrier, and is used for modifying the T cell or the NK cell into a CAR-T or CAR-NK cell; the T cell or NK cell modified by the chimeric antigen receptor can kill tumor cells by recognizing tissue factors on the surface of the tumor cells, and can be used for treating tumors.

The term "tumor stromal cells" refers to some cells in the tumor microenvironment that support the malignant proliferation, anti-apoptosis, invasion, metastasis, escape from immune monitoring and other vital activities of tumor cells, and mainly includes fibroblasts, tumor-associated macrophages (TAMs), regulatory T cells (tregs), undifferentiated bone marrow cells, endothelial cells, pericytes and platelets, endothelial cells and the like.

The term "immunosuppressive molecule" refers to a molecule produced by tumor cells or tumor stromal cells that acts to suppress immunity, allow tumor cells to escape from immune surveillance of the body, and induce immune tolerance.

The term "Co-stimulatory signaling molecules" (Co-stimulatory molecules) refers to some adhesion molecules on the surface of immune cells, such as CD28, CD134/OX40, CD137/4-1BB, CD40, etc., which activate the secondary signal of immune cells by binding to their ligands, enhance the proliferation capacity of immune cells and the secretion function of cytokines, and prolong the survival time of activated immune cells.

The term "extracellular region" refers to a segment of a membrane protein that is located outside of a cell.

The term "structural domain" refers to a region with specific structure and independent function in protein biomacromolecule, the number of amino acid residues of common structural domain is 100-400, the smallest structural domain is only 40-50 amino acid residues, and the large structural domain can exceed 400 amino acid residues.

The term "single-chain antibody" (scFv) refers to an antibody fragment having the ability to bind to an antigen, which is obtained by linking an amino acid sequence of a VL region and an amino acid sequence of a VH region of an antibody with a Linker.

The term "PD-1" refers to HUMAN Programmed cell death factor 1(Programmed cell death protein1), the gene name PDCD1_ HUMAN, the corresponding protein sequence number UniProtKB-Q15116, is a T cell immunosuppressive molecule, the extracellular domain of which is similar to the variable region (V-section) of immunoglobulin and has the characteristic of specifically binding to the ligands PD-L1 and PD-L2(Programmed cell death protein1 ligand 1/2). PD-1 is commonly expressed in activated T lymphocytes, as well as in a variety of malignant cells.

The terms "PD-L1", "PD-L2" refer to the currently found ligands 1 and 2 of human programmed cell death factor 1(programmed cell death protein1 ligand 1/2). The extracellular domain has immunoglobulin-like V and C1 regions, and is bound to the V region of PD-1 via the V region (4zqk Structure 232341 2348, 2015). It is usually expressed in dendritic cells DC, T regulatory cells and Th cells, macrophages, Mast cells and bone marrow in small amounts, and also in a variety of malignant cells.

By the scheme, the invention at least has the following advantages: immune checkpoint therapies such as PD-1 antibodies are not effective for a wide range of tumor patients, and increasing the objective remission rate becomes an urgent challenge. Studies have demonstrated that sufficient T cell infiltration in tumor tissue is a prerequisite for the occlusion of PD-L1 and the onset of action of PD-1 antibody. Targeting the tumor necrosis factor superfamily member LIGHT molecules is a tumor-activating lymphotoxin B-receptor signal that results in the production of enormous amounts of chemokines to recruit T cells. The LIGHT-PD1CAR-T cell with higher specificity and higher killing property is prepared by combining the characteristics of LIGHT molecules and the killing property of CAR-T cells and introducing the LIGHT and PD-1 molecules into extracellular segments simultaneously so as to treat solid tumors.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.

Drawings

FIG. 1 is a schematic representation of the construction of a lentiviral expression vector;

FIG. 2 is a graph showing the flow results of LIGHT-PD-1 CAT-T cell infection for 7 days in the present invention;

FIG. 3 is a graph showing the results of killing of different target cells by LIGHT-PD-1 CAT-T cells in the present invention;

FIG. 4 is a graph showing the results of in vitro proliferation of LIGHT-PD-1 CAT-T cells in the present invention against various kinds of target cells;

FIG. 5 is a graph showing the results of measuring cytokines of LIGHT-PD-1 CAT-T cells versus different kinds of target cells in the present invention;

FIG. 6 is a graph showing the results of detection of MCF7/MCF7-PDL1/HeLa/SMC 77214 cell line CD3/CD8 by CAR-LIGHT CAT-T cells in the present invention;

FIG. 7 is a graph comparing the results of the killing effect of PD-1 CAR-T and LIGHT-PD1CAR-T in the present invention.

Detailed Description

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

EXAMPLE 1 Lentiviral expression vector preparation

The invention provides a preparation method of a lentivirus expression vector for expressing a chimeric antigen receptor molecule, which comprises the following steps:

s1, synthesizing a LIGHT gene segment according to the sequence information of the LIGHT molecule reported in the article of facility T Cell Infiltration in Tumor microorganisms Resistance experiments. Known human PD-1 sequence, CD8 transmembrane gene sequence, human 4-1BB intracellular region gene sequence and CD 3. delta. intracellular region gene sequence were searched from GenBank databases.

S2, connecting the gene sequences in sequence according to a LIGHT gene, a PD-1 gene, a CD8 membrane gene, a 4-1BB intracellular region gene and a CD3 delta intracellular region gene, introducing different enzyme cutting sites at the connection positions of the sequences to form complete LIGHT-PD1-CD8-4-1BB-CD3 delta (LIGHT-PD 1-CAR for short) gene sequence information,

s3, connecting the gene sequence of LIGHT-PD1-CD8-4-1BB-CD3 delta into a pRRSLIN vector through enzyme digestion and transformation, wherein the upstream of the gene is an EP-1 alpha promoter. Transforming the vector into a Stbl3 escherichia coli strain, transferring the strain into a solid culture medium containing ampicillin for propagation and screening to obtain positive clones, extracting plasmids, carrying out enzyme digestion to identify the clones, confirming the success of vector construction through sequencing to obtain a pRRSLIN-LIGHT-PD1 lentiviral expression vector, wherein the schematic diagram of the construction of the lentiviral expression vector is shown in figure 1.

Example 2 Lentiviral preparation

The invention provides a method for preparing lentivirus by expressing a lentivirus expression vector in example 1, which comprises the following steps:

s1, 24 hours before transfection, at about 8X 10 per dish⁶293T cells were seeded into 15cm dishes. Ensure that the cells are confluent at around 80% and evenly distributed in the culture dish during transfection.

S2, preparation solution A and solution B

Solution A: 6.25mL of 2 XHEPES buffer.

Solution B: the following plasmid mixture was added: 112.5 μ g pRRSLIN-LIGHT-PD1(target plasmid); 39.5 μ G pMD2.G (VSV-G envelop); 73. mu.g pCMVR8.74(gag, pol, tat, rev); 625 μ L of 2M calcium ion solution. Total volume of solution B: 6.25 mL.

S3, fully mixing the solution B, adding the solution B dropwise while slightly swirling the solution A, and standing for 2-3 minutes. The mixed solution of A and B was vortexed gently, added dropwise to a culture dish containing 293T cells, and the dish was shaken gently back and forth to uniformly distribute the mixture of DNA and calcium ions. Placing the mixture in an incubator to be cultured for 16-18 hours. Replacing fresh culture medium, culturing, centrifuging at 25 deg.C at 500g for 10min, and filtering with PES membrane (0.45 μm); sterilizing centrifuge tube (Beckmann Coultra-clearSW 28centrifugetubes) with 70% ethanol, and sterilizing under ultraviolet lamp for 30 min; the filtered lentivirus-containing supernatant was transferred to a centrifuge tube, carefully layered with a 20% sucrose (1 mL sucrose per 8mL supernatant) on the bottom of the tube, equilibrated with PBS and centrifuged at 25,000rpm (82,700g) at 4 ℃ for 2 h; carefully taking out the centrifugal tube, pouring out the supernatant, and inverting the centrifugal tube to remove residual liquid; adding 100 μ LPBS, sealing the centrifuge tube, standing at 4 deg.C for 2h, gently vortexing once every 20min, centrifuging at 500g for 1min (25 deg.C), and collecting virus supernatant; cooling on ice, and storing at-80 deg.C.

Example 3 CAR-T cell preparation

The invention provides a method of making CAR-T cells from lentivirus-infected cells of example 2, comprising the steps of:

s1, taking 0.5mL of blood to carry out rapid pathogenic microorganism detection, and eliminating the infection of microorganisms such as HBV, HCV, HDV and HEV, HIV-1/2, treponema pallidum, parasites and the like; under the aseptic condition, 50mL (heparin anticoagulation) of blood is collected by a heparin bottle and immediately (at 4 ℃ within 24 hours) is sent to a cell preparation laboratory, so that the process is ensured to be free from the pollution of pathogenic microorganisms. After obtaining the blood of the patient, the surface of the heparin bottle is wiped by an alcohol cotton ball in a GMP preparation room for disinfection and then is put into a biological safety cabinet.

S2, opening 2 50mL centrifuge tubes in advance, transferring blood into two 50mL centrifuge tubes, and screwing; placing the two 50mL centrifuge tubes filled with the blood into a centrifuge for centrifugation, centrifuging for 10min at 400g (2000rpm), centrifuging at room temperature, collecting upper plasma, and leaving a precipitate layer; the collected autologous plasma is inactivated at 56 deg.C for 30min, placed at 4 deg.C for 15min, then 900g, centrifuged for 30min (4 deg.C), and the supernatant is taken for use.

S3, diluting the enriched blood cells to 30 mL/tube with normal saline, opening 2 new 50mL centrifuge tubes, adding 15mL of human lymphocyte separation solution into each centrifuge tube, slowly adding the diluted blood cell solution into the centrifuge tube containing the human lymphocyte separation solution with a pipette, and screwing. Note that blood is added to the upper layer of the lymph separation fluid without breaking the interface with the human lymph separation fluid. The added blood cell fluid is put into a centrifuge, the lifting speed is adjusted to the minimum, 400g (2000rpm) is centrifuged for 20min (normal temperature). The middle leukocyte layer of the two tubes was collected in a 15mL sterile centrifuge tube, and 5mL physiological saline was added thereto, and washed twice (400g, centrifugation for 10min) to obtain Peripheral Blood Mononuclear Cells (PBMC).

S4, preparing a complete growth medium, adding autologous AB (FBS) into V-VIVO15 at a concentration of 5%, and adding interleukin-2 (IL-2) at a concentration of 40ng/mL, and diluting the PBMC obtained by separation into 2X 10 with the culture medium⁶Per mL, 50. mu.L of the flow assay PBMC were used for purity of T cells.

S5 and Day 0, preparing a buffer solution (1% Fetal Bovine Serum (FBS) is added into a PBS buffer solution), selecting microbeads as cell culture carriers, shaking the microbeads for 30S or shaking up and down manually for 5min, and mixing the microbeads with T cells according to the dosage ratio of 3: 1 placing CD3/CD28 microbeads into a 1.5mL EP tube, adding 1mL buffer solution to clean the microbeads, then sucking the microbeads outwards from the EP tube for 1min by using a magnet, abandoning the washing solution, repeating the steps twice, then re-suspending the microbeads to the original volume by using a culture medium, and finally re-suspending the cells and the microbeadsMixing the microbeads according to a ratio of 2X 10⁶PBMC/mL were added to the appropriate flask.

S6, Day 2 adjusting the cell density to 3-5X 10⁶PermL, the PRRSLIN-LIGHT-PD1 lentiviral vector prepared in example 2 was added at a ratio of viral vector to cells of 1:5, and polybrene (polybrene) was added at 4. mu.g/mL and IL-2 at 40 ng/mL. After 4h, the cell density was adjusted to 1X 10 by adding fresh complete medium⁶The culture was continued at/mL. All cells were centrifuged, fresh medium was added and the culture was continued.

S7, half-amount liquid change is carried out every 2-3 days, and the cell density is maintained at 0.5-1 × 10⁶/mL。

S8, Day 10-12, the cell number reaches 10⁹Grade, immune cells were centrifuged at 400g for 5min and washed twice with pre-chilled PBS (400g, 5 min).

And S9, counting by using a blood counting chamber, and detecting the cell group and the CAR-T cell proportion by using a flow cytometer. The color change, cell density, cell morphology of the culture medium were observed daily and recorded accordingly. In the process of gradually expanding culture, interleukin-2 required by the total volume is added.

Example 4 CAR-T cell flow assay

The CAR-T cells prepared in example 3 were subjected to flow analysis, which specifically comprises the following steps:

s1, 5X 10⁴Cells (including T cells, CAR-T cells) are used for staining;

s2, incubating the cells and the antibody (the antibody can be identified and combined with PD-1 molecules and coupled with FITC fluorescent molecules) for 45min, and placing the cells and the antibody on ice in 50 mu l;

s3, eluting twice with PBS;

s4, resuspending the cells with 120. mu.l FACS reagent;

s5, measuring the FITC fluorescent signal by a flow cytometry instrument, and if the FITC fluorescent signal of the CAR cell is compared with that of the control T cell, the FITC fluorescent signal of the CAR cell is enhanced, so that the construction of the surface CAR cell is successful.

The CAR-T cell flow-through staining effect is shown in fig. 2, wherein the ordinate is the flow SSC-H side scatter signal and the abscissa is the FITC fluorescence signal, the stronger the signal value, indicating that the more PD-1 molecules are expressed on the membrane, the higher the successful rate of CAR-T cell transfection. Panels A and B are control groups, which are T cells that are not infected with virus; FITC-conjugated CAR molecule-detecting antibodies do not detect CAR molecule expression; panels C and D, which are flow-based assays of T cells transfected with PRRSLIN-LIGHT-PD1 lentivirus, compare panel A and B, with successful transfection of cells; after the virus infects the T cell, the infection efficiency of flow detection can reach 47 percent, which indicates that the LIGHT-PD1CAR-T cell is successfully prepared.

Example 5 LIGHT-PD1CAR-T cell in vitro Activity assay

The method for detecting the killing effect of LIGHT-PD1CAR-T cells on engineering cell strains MCF-7/PDL1 and tumor cells highly expressing PDL1 by adopting an LDH release method and detecting the LDH release by an ELISA method comprises the following steps:

s1, adjusting the target cells to 5X 10 with 5% calf serum-containing RPMI-1640 culture solution⁴/mL。

S2, adding target cells into a 96-well cell culture plate, and adding 100 mu L of target cells into each well. 3 wells were used as control wells for the natural release of effector cells (LIGHT-PD 1CAR-T cells) with no target cells added, and only 100. mu.L of culture medium was added.

S3, adding 100 mu L of effector cells into each hole, wherein the ratio of the effector cells to the target cells is 10: 1; 5: 1; 1:1. The natural release hole is not added with effector cells, only 100 mu L of culture solution is added, the effector cells and target cells are incubated for 6 hours, and three multiple holes are arranged in each experiment.

S4, Add 10. mu.L of lysine Solution (10X) to the maximum release well (positive control), incubate for 45min-60min, place three replicates per experiment.

S5, taking 50 mu L of each sample to be detected and the control sample in the steps 3 and 4, adding the samples into a fresh 96-hole enzyme label plate, adding the reaction solution and the substrate, and keeping out of the sun for 30 min.

S6, adding 50 mu L of stop solution.

S7, the optical density (OD value) of each well was measured on an enzyme-linked immunosorbent assay device, and the measurement was completed within 1 hour at a measurement wavelength of 490nm or 492 nm.

S8 calculation of specific killing efficiency

Kill rate ═ experimental set LDH (OD)/maximum LDH release set (OD).

Calculating the formula: the killing efficiency ═ 100% (experimental group-effect natural release-target natural release)/(target maximum release-target natural release).

9. Cytokine secretion was measured by CBA kit, and proliferation in each group of CAR-T cells was calculated, and staining with CD3 and CD8 antibodies confirmed the proportion of CD 8-positive T cells in the proliferated T cells.

As shown in fig. 3, the abscissa of the graph indicates different effective target ratios of CAR-T cells to tumor cells, the ordinate indicates killing efficiency, the different types of histograms indicate different tumor cells, and LIGHT-PD1CAR-T can significantly kill tumor cells under different effective target ratios against four tumor cells of MCF-7, MCF-1/PDL-1, SMCC7721 and Hela, and is most obvious for SMCC7721 and Hela cells; accordingly, as shown in fig. 4, the abscissa represents the effective target ratio of T cells to tumor or CAR-T cells to tumor, the ordinate represents the number of cells, T represents T cells, and LIG represents LIGHT-PD1CAR-T cells, it can be seen that CAR-T cells can be specifically activated and proliferated after being exposed to tumor cell stimulation, and the effective target ratio is more significant than that of ordinary T cells.

As shown in the killing experiment of FIG. 5, the abscissa represents the different effect-target ratio of CAR-T cells and tumor cells, the ordinate represents the content of cytokines, and the cytokines in the culture supernatant are detected, so that the secretion of IL-2 (FIG. 5A) and TNF-alpha (FIG. 5B) in the CAR-T killing experiment group is remarkably increased. As shown in FIG. 6, for the CD3/CD8 flow antibody detection of the proportion of CART to MCF7/MCF7-PDL1/HeLa/SMC 77214 cell strains killed by CD 8T cells, the PE signal of the ordinate indicates the specific detection of CD3 molecule expression, the FITC signal of the abscissa indicates the detection of CD8 molecule expression, and the LIGHT-PD1CAR-T cells are activated by flow cytometry, so that the specific proliferation of CD 8T cells is mainly generated.

As shown in figure 7, CAR-PD-1 shows that the extracellular segment of the expressed CAR molecule is CAR-T cell of a common PD-1 molecule, CAR-LIGHT-PD-1 shows LIGHT-PD1CAR-T cell constructed by the invention, and the result shows that LIGHT-PD1CAR-T cell has better killing effect on SMC7721 tumor cell compared with PD-1 CAR-T cell.

The results prove that the LIGHT-PD1-CART cell can be specifically activated and proliferated, release cytokines and kill tumor cells after being contacted with the tumor cells, wherein the CD8 positive T cell plays a main role. And simultaneously comparing the killing effect of PD-1 CAR-T and LIGHT-PD1CAR-T on the tumor, and the result shows that LIGHT-PD1CAR-T is obviously superior to PD-1 CAR-T.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Lihuashun

<120> polygene recombinant chimeric antigen receptor molecule and application thereof

<130> 2016

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 145

<212> PRT

<213> Artificial sequence ()

<400> 1

Pro Ala Ala His Leu Thr Gly Ala Asn Ser Ser Leu Thr Gly Ser Gly

1 5 10 15

Gly Pro Leu Leu Trp Glu Thr Gln Leu Gly Leu Ala Phe Leu Arg Gly

20 25 30

Leu Ser Tyr His Asp Gly Ala Leu Val Val Thr Lys Thr Gly Tyr Tyr

35 40 45

Tyr Ile Tyr Ser Lys Val Gln Leu Gly Gly Val Gly Cys Pro Leu Gly

50 55 60

Leu Ala Gly Thr Ile Thr His Gly Leu Tyr Lys Arg Thr Pro Arg Tyr

65 70 75 80

Pro Glu Glu Leu Glu Leu Leu Val Ser Gln Gln Ser Pro Cys Gly Arg

85 90 95

Ala Thr Ser Ser Ser Arg Val Trp Trp Asp Ser Ser Phe Leu Gly Gly

100 105 110

Val Val His Leu Glu Ala Gly Glu Lys Val Val Val Arg Val Leu Gly

115 120 125

Lys Arg Leu Val Arg Leu Arg Asp Gly Thr Arg Ser Tyr Phe Gly Ala

130 135 140

Phe

145

<210> 2

<211> 15

<212> PRT

<213> Artificial sequence ()

<400> 2

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 3

<211> 170

<212> PRT

<213> Artificial sequence ()

<400> 3

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu Val

165 170

<210> 4

<211> 19

<212> PRT

<213> Artificial sequence ()

<400> 4

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr

<210> 5

<211> 20

<212> PRT

<213> Artificial sequence ()

<400> 5

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg

20

<210> 6

<211> 24

<212> PRT

<213> Artificial sequence ()

<400> 6

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 7

<211> 45

<212> PRT

<213> Artificial sequence ()

<400> 7

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 8

<211> 19

<212> PRT

<213> Artificial sequence ()

<400> 8

Val Gly Val Val Gly Gly Leu Leu Gly Ser Leu Val Leu Leu Val Trp

1 5 10 15

Val Leu Ala

<210> 9

<211> 42

<212> PRT

<213> Artificial sequence ()

<400> 9

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 10

<211> 112

<212> PRT

<213> Artificial sequence ()

<400> 10

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 11

<211> 554

<212> PRT

<213> Artificial sequence ()

<400> 11

Met Pro Ala Ala His Leu Thr Gly Ala Asn Ser Ser Leu Thr Gly Ser

1 5 10 15

Gly Gly Pro Leu Leu Trp Glu Thr Gln Leu Gly Leu Ala Phe Leu Arg

20 25 30

Gly Leu Ser Tyr His Asp Gly Ala Leu Val Val Thr Lys Thr Gly Tyr

35 40 45

Tyr Tyr Ile Tyr Ser Lys Val Gln Leu Gly Gly Val Gly Cys Pro Leu

50 55 60

Gly Leu Ala Gly Thr Ile Thr His Gly Leu Tyr Lys Arg Thr Pro Arg

65 70 75 80

Tyr Pro Glu Glu Leu Glu Leu Leu Val Ser Gln Gln Ser Pro Cys Gly

85 90 95

Arg Ala Thr Ser Ser Ser Arg Val Trp Trp Asp Ser Ser Phe Leu Gly

100 105 110

Gly Val Val His Leu Glu Ala Gly Glu Lys Val Val Val Arg Val Leu

115 120 125

Gly Lys Arg Leu Val Arg Leu Arg Asp Gly Thr Arg Ser Tyr Phe Gly

130 135 140

Ala Phe Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu

165 170 175

Gln Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro

180 185 190

Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly

195 200 205

Asp Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe

210 215 220

Val Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu

225 230 235 240

Ala Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe

245 250 255

Arg Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val

260 265 270

Arg Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser

275 280 285

Leu Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg

290 295 300

Val Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser

305 310 315 320

Pro Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Thr Thr Thr Pro Ala

325 330 335

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

340 345 350

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

355 360 365

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

370 375 380

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

385 390 395 400

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

405 410 415

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

420 425 430

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

435 440 445

Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn

450 455 460

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

465 470 475 480

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

485 490 495

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

500 505 510

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

515 520 525

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

530 535 540

Ala Leu His Met Gln Ala Leu Pro Pro Arg

545 550

<210> 12

<211> 1665

<212> DNA

<213> Artificial sequence ()

<400> 12

atgccagcag cgcatctcac aggggccaac tccagcttga ccggcagcgg ggggccgctg 60

ttatgggaga ctcagctggg cctggccttc ctgaggggcc tcagctacca cgatggggcc 120

cttgtggtca ccaaaactgg ctactactac atctactcca aggtgcagct gggcggtgtg 180

ggctgcccgc tgggcctggc cggcaccatc acccacggcc tctacaagcg cacaccccgc 240

taccccgagg agctggagct gttggtcagc cagcagtcac cctgcggacg ggccaccagc 300

agctcccggg tctggtggga cagcagcttc ctgggtggtg tggtacacct ggaggctggg 360

gagaaggtgg tcgtccgtgt gctgggtaaa cgcctggttc gactgcgtga tggtacccgg 420

tcttacttcg gggctttcgg tggaggcggt tcaggcggag gtggctctgg cggtggcgga 480

tcgatgcaga tcccacaggc gccctggcca gtcgtctggg cggtgctaca actgggctgg 540

cggccaggat ggttcttaga ctccccagac aggccctgga acccccccac cttctcccca 600

gccctgctcg tggtgaccga aggggacaac gccaccttca cctgcagctt ctccaacaca 660

tcggagagct tcgtgctaaa ctggtaccgc atgagcccca gcaaccagac ggacaagctg 720

gccgccttcc ccgaggaccg cagccagccc ggccaggact gccgcttccg tgtcacacaa 780

ctgcccaacg ggcgtgactt ccacatgagc gtggtcaggg cccggcgcaa tgacagcggc 840

acctacctct gtggggccat ctccctggcc cccaaggcgc agatcaaaga gagcctgcgg 900

gcagagctca gggtgacaga gagaagggca gaagtgccca cagcccaccc cagcccctca 960

cccaggccag ccggccagtt ccaaaccctg gtgaccacga cgccagcgcc gcgaccacca 1020

acaccggcgc ccaccatcgc gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca 1080

gcggcggggg gcgcagtgca cacgaggggg ctggacttcg cctgtgatat ctacatctgg 1140

gcgcccttgg ccgggacttg tggggtcctt ctcctgtcac tggttatcac cctttactgc 1200

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 1260

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 1320

gaactgagag tgaagttcag caggagcgca gacgcccccg cgtaccagca gggccagaac 1380

cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt ggacaagaga 1440

cgtggccggg accctgagat ggggggaaag ccgagaagga agaaccctca ggaaggcctg 1500

tacaatgaac tgcagaaaga taagatggcg gaggcctaca gtgagattgg gatgaaaggc 1560

gagcgccgga ggggcaaggg gcacgatggc ctttaccagg gtctcagtac agccaccaag 1620

gacacctacg acgcccttca catgcaggcc ctgccccctc gctaa 1665

<210> 13

<211> 156

<212> PRT

<213> Artificial sequence ()

<400> 13

Gly Gly Gly Gly Gly Ser Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg

1 5 10 15

Pro Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu

20 25 30

Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser

35 40 45

Phe His Val Val Trp His Arg Glu Ser Pro Ser Gly Gln Thr Asp Thr

50 55 60

Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg

65 70 75 80

Phe Arg Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val

85 90 95

Val Arg Ala Arg Arg Asn Asp Ser Gly Thr Tyr Val Cys Gly Val Ile

100 105 110

Ser Leu Ala Pro Lys Ile Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu

115 120 125

Arg Val Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro

130 135 140

Ser Pro Arg Pro Ala Gly Gln Phe Gln Thr Leu Val

145 150 155

Claims (9)

1. A multigenic recombinant chimeric antigen receptor molecule characterized by: the polypeptide comprises an extracellular domain peptide segment, a transmembrane domain peptide segment and an intracellular domain peptide segment which are sequentially connected in series, wherein the extracellular domain peptide segment comprises a LIGHT protein peptide segment and an immunological binding protein peptide segment which are connected, wherein the sequence of the LIGHT protein peptide segment is shown as SEQ ID NO: 1, the peptide fragment of the immunological binding protein is an extracellular region peptide fragment of PD-1 or HAC, and the sequence of the extracellular region peptide fragment of PD-1 is shown as SEQ ID NO: 3, the sequence of the extracellular region peptide fragment of the HAC is shown as SEQ ID NO: 13 is shown in the figure;

the transmembrane peptide segment is a CD8 transmembrane peptide segment, and the extracellular domain peptide segment is connected with the CD8 transmembrane peptide segment through a hinger domain peptide segment of CD 8;

the intracellular domain peptide segment is a costimulatory signal molecule and is selected from one or more of the intracellular domain peptide segments of 4-1BB, CD28 and CD3 zeta.

2. The multigene recombinant chimeric antigen receptor molecule according to claim 1, characterized in that: the LIGHT protein peptide segment and the immune binding protein peptide segment are connected by using a connecting peptide.

3. The multigene recombinant chimeric antigen receptor molecule according to claim 1, characterized in that: the multigene recombinant chimeric antigen receptor molecule further comprises a signal peptide.

4. A nucleotide sequence characterized in that: encoding a multigene recombinant chimeric antigen receptor molecule according to any one of claims 1 to 3.

5. The nucleotide sequence of claim 4, wherein: comprises or is SEQ ID NO: 12.

6. A recombinant vector characterized by: comprising a nucleotide sequence according to claim 4 or 5.

7. The recombinant vector according to claim 6, wherein: the recombinant vector is a pRRSLIN lentivirus expression vector.

8. A recombinant cell, wherein: comprising the recombinant vector according to claim 6 or 7.

9. The recombinant cell of claim 8, wherein: the recombinant cell is preferably a T cell or NK cell.

Images