CN112480264A - Chimeric antigen receptor taking TIGIT and PD-1 as targets, CAR-T cell and preparation method thereof - Google Patents

Abstract

The invention provides a chimeric antigen receptor taking TIGIT and PD-1 as targets, which comprises a TIGIT extracellular region and a PD-1 extracellular region; the invention also provides CAR-T cells comprising the chimeric antigen receptor and methods of making the same. The CAR-T cell prepared by the chimeric antigen receptor taking the immune check points TIGIT and PD-1 as targets can promote the proliferation of the CAR-T cell, improve the killing capacity of the CAR-T cell, avoid the tumor cell from immune escape and improve the effectiveness of CAR-T cell treatment. The Anti-TIGIT-PD-1-CAR-T cell prepared by the invention has the strongest proliferation capacity, the effect of killing target cells is most obvious, and the killing capacity is about 3 times of that of single-target Anti-TIGIT-CAR or Anti-PD-1-CAR.

Description

Chimeric antigen receptor taking TIGIT and PD-1 as targets, CAR-T cell and preparation method thereof

Technical Field

The invention relates to the technical field of genes, in particular to a novel CAR construction method and application aiming at solid tumors and taking immune check points TIGIT and PD-1 as targets.

Background

Tumors seriously endanger human health all the time, and in recent years, tumor immunotherapy is rapidly developed and becomes a hot spot and a breakthrough in the field of tumor treatment. Tumor immunotherapy includes immune cell therapy, immune checkpoint inhibitors, cytokines, cell vaccines, and the like. The Chimeric Antigen Receptor T-Cell (CAR-T Cell) therapy is a novel Cell therapy, the T Cell of a tumor patient is mainly artificially modified through a genetic engineering technology, a large amount of in vitro culture is carried out to generate a tumor specific CAR-T Cell, and the tumor specific CAR-T Cell is returned to the body of the patient to attack cancer cells, so that the targeting is more accurate, the killing power is stronger, and the effect is more durable.

Researchers are always searching for a new generation of immune checkpoint inhibitor and PD-1/PD-L1 combination to improve the effect of tumor immunotherapy. T cell immunoglobulins and ITIM domain protein (TIGIT), as newly discovered immunosuppressive molecules, have become one of the hotspots in the development of cancer immunotherapy. TIGIT is an inhibitory receptor that inhibits T cell activation by interacting with PVR (D155) expressed on antigen presenting cells or tumor cells. TIGIT is highly expressed in tumor infiltrating T cells of multiple cancer types. Monoclonal antibodies blocking human TIGIT have been developed for use as monotherapy or in combination with anti-PD-1/PD-L1 monoclonal antibodies in treating patients with advanced solid malignancies. Recent research shows that blocking the immunosuppressive point can promote T cell activation, regulate tumor microenvironment, increase the generation number of memory T cells and generate more effector T cells. Therefore, the TIGIT and PD-1 immune checkpoint signaling pathways are blocked simultaneously, T cells can be synergistically activated, the anti-tumor activity is enhanced, and a better tumor killing effect is achieved.

The prior art of the immune cell therapeutic index of solid tumors combines checkpoint inhibitors (PD-1, CTLA-4 and LAG-3) and CAR-T cells to improve the tumor microenvironment and enhance the killing effect of CAR-T cells. In addition, the immune cell therapy is combined with operation and radiotherapy and chemotherapy to treat solid tumors, and the research shows that the immune cell therapy is safe and feasible. The novel replicative oncolytic adenovirus capable of simultaneously blocking immune checkpoints PD-L1 and TIGIT and the application (CN 201910462985.2) are disclosed, the regulation and control mechanism of the oncolytic adenovirus is single, and the problems of marker loss, tumor microenvironment influence, easy off-target effect and the like cannot be effectively solved. CAR-T (CN 201610226230.9) taking the PD-1 extracellular region as a single target point has a certain killing effect, but the tumor treatment effect is poor by simply blocking the PD-1 pathway of effector lymphocytes. No reports have been found relating to the application of TIGIT to CAR.

The existing tumor immunity technology mostly adopts an anti-PD-1 antibody, an anti-PD-l 1 antibody and a CTLA-4 antibody to treat tumors, but the drugs also exist in other inhibiting proteins including TIGIT while being treated by anti-checkpoint drugs, so that the side effect is large after long-term administration of the antibodies, the drug resistance is easy to occur, and the anti-tumor effect is not obvious.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a chimeric antigen receptor taking TIGIT and PD-1 as targets, a CAR-T cell and a preparation method thereof, the activation of the T cell is enhanced through synergistic action, the combination efficiency of the CAR-T and tumor cells is improved, the problems of recurrence and metastasis caused by tumor immune escape are solved to a greater extent, and the killing activity to target cells is improved.

In order to solve the technical problems, the invention adopts the following technical scheme:

a chimeric antigen receptor taking TIGIT and PD-1 as targets comprises a TIGIT extracellular region and a PD-1 extracellular region.

The following is a further improvement of the above technical solution:

the nucleic acid artificial sequence of the TIGIT extracellular region is shown as SEQ ID NO. 2; the nucleic acid artificial sequence of the PD-1 extracellular region is shown in SEQ ID NO. 4.

The chimeric antigen receptor comprises a CD8 leader, a TIGIT extracellular region, a PD-1 extracellular region, a CD8 Hinge region, a chimeric antigen receptor and a monoclonal antibody,

The CD8 transmembrane region, the 41BB costimulatory region, and the CD3 zeta signaling region.

A CAR-T cell comprising the chimeric antigen receptor of claim 1.

The CAR-T cell is prepared by adopting the following method: the lentiviral expression plasmid containing the gene fragment of the chimeric antigen receptor was packaged into a lentivirus, and the recombinant lentivirus was used to infect T cells.

The T cell is a TIGIT and PD-1 double-gene knockout T cell.

The gene knockout rate of TIGIT of the T cell is more than 75%, and the gene knockout rate of PD-1 is more than 75%.

The preparation of the TIGIT and PD-1 double-gene knockout T cell comprises the construction of a TIGIT and PD-1 expression vector knocked out by CRISPR/CAS9 and the electrotransfection of the T cell;

the construction of the TIGIT and PD-1 knockout expression vector of the CRISPR/CAS9 is characterized in that a TIGIT sgRNA nucleic acid sequence and a PD-1 sgRNA nucleic acid sequence are respectively inserted into a standard vector and connected to a CRISPR/CAS9 expression vector to respectively obtain a CRISPR/CAS9 knockout TIGIT vector and a CRISPR/CAS9 knockout PD-1 vector.

The TIGIT sgRNA nucleic acid sequence is ccagtcgctgaccgtgaacgata; the PD-1 sgRNA nucleic acid sequence is acatgagcgtggtcagggcc.

The invention provides a novel anti-tumor immunotherapy strategy by combining TIGIT and PD-1 extracellular domains together as the extracellular domain of CAR and CD3 Zeta as a stimulation signal domain based on the latest tumor immunotherapy method-CAR-T technology. The activation of T cells is enhanced through synergistic effect, the combination efficiency of CAR-T and tumor cells is improved, and the problems of recurrence and metastasis caused by tumor immune escape are solved to a greater extent. In addition, the invention also knocks out corresponding immune suppressive factors (TIGIT and PD-1) on the surface of the T cell by a gene editing technology (CRISPR/CAS 9) to inhibit the expression of the T cell, cuts off an immune checkpoint signaling pathway, improves the activity of the CAR-T cell and enables the CAR-T cell to be more effective in treating solid tumors.

Compared with the prior art, the invention has the following beneficial effects:

(1) the CAR-T cell prepared by the chimeric antigen receptor taking the immune check points TIGIT and PD-1 as targets,

can promote the proliferation of CAR-T cells, improve the killing capacity of the CAR-T cells, avoid the immune escape of tumor cells and improve the treatment effectiveness of the CAR-T cells.

When the CAR-T cell prepared by the chimeric antigen receptor taking the immune check points TIGIT and PD-1 as targets and a positive cell (namely a target cell) expressing PVR and PD-L1 genes are co-cultured for 48 hours, the secretion amounts of IL-2, TNF-alpha and IFN-gamma of effector cells are respectively detected by an ELISA method, the secretion amount of IL-2 is 4500 pg/ml, the secretion amount of TNF-alpha is 3900 pg/ml, and the secretion amount of IFN-gamma is 4320 pg/ml.

The IL-2 secretion amount of the experimental group A (Anti-TIGIT-PD-1-CAR-T cell and target cell coculture) is 3 times that of the experimental group B (Anti-TIGIT-CAR-T cell and target cell coculture), the TNF-alpha secretion amount is 3.25 times that of the experimental group B, and the IFN-r secretion amount is 3.9 times that of the experimental group B; the IL-2 secretion of the experimental group A was 2.5 times that of the experimental group C (Anti-PD-1-CAR-T cells and target cells co-cultured), the TNF- α secretion was 2.9 times that of the experimental group C, and the IFN-r secretion was 3 times that of the experimental group C.

(2) The Anti-TIGIT-PD-1-CAR-T cell prepared by the invention has the strongest proliferation capacity, the effect of killing target cells is most obvious, and the killing capacity is about 3 times that of single target Anti-TIGIT-CAR or Anti-PD-1-CAR and about 4 times that of a control group.

The CAR-T cell prepared by the chimeric antigen receptor taking the immune check points TIGIT and PD-1 as targets has the killing activity of 98.5 percent on positive cells (target cells) expressing PVR and PD-L1 genes.

Drawings

FIG. 1 schematic diagram of Anti-TIGIT-PD-1-CAR expression vector structure.

FIG. 2 pX330A-TIGIT expression vector structure diagram.

FIG. 3 pX330A- -schematic diagram of PD-1 expression vector structure.

FIG. 4 is a graph of the expression efficiency of Anti-TIGIT-PD-1-CAR vector on the surface of T cells detected by flow cytometry.

FIG. 5 is a flow cytometry image for detecting the expression efficiency of Anti-TIGIT-CAR vector on the surface of T cells.

FIG. 6 is a flow cytometry graph for detecting the expression efficiency of Anti-PD-1-CAR vector on the surface of T cells.

FIG. 7 is a graph comparing the secretion of IL-2, TNF-alpha and IFN-gamma factors by different CAR-T cells.

FIG. 8 is a graph comparing different CAR-T cell killing activities.

Detailed Description

Example 1 construction of TIGIT and PD-1 Dual-target CAR expression vector

Schematic diagram 1 of Anti-TIGIT-PD-1-CAR module. The nucleic acid sequences of each module of the Anti-TIGIT-PD-1-CAR vector are as follows:

(1) CD8 Leader nucleic acid artificial sequence (SEQ ID NO.1)

(2) Nucleic acid artificial sequence of TIGIT extracellular region (SEQ ID NO.2)

(3) Linker nucleic acid artificial sequence (SEQ ID NO.3)

(4) Nucleic acid artificial sequence of PD-1 extracellular region (SEQ ID NO.4)

(5) CD8 Hinge region (CD8 alpha) nucleic acid artificial sequence (SEQ ID NO.5)

(6) CD8 transmembrane region (CD8 TM) nucleic acid artificial sequence (SEQ ID NO.6)

(7) 41BB costimulatory region nucleic acid artificial sequence (SEQ ID NO.7)

(8) CD3 zeta signaling region nucleic acid artificial sequence (SEQ ID NO.8)

(9) pLent-C-GFP vector (SEQ ID NO. 9).

Sequentially synthesizing the whole expression frame of the sequence of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 by committee bioengineering (Shanghai) limited company and inserting the whole expression frame into a vector pUC57 to obtain pUC-TIGIT-PD-1-CAR. After the pUC-TIGIT-PD-1-CAR and pLent-C-GFP (SEQ ID NO.9) vectors were subjected to Fast Digest AsiSI (purchased from Thermo Fisher) and Fast Digest NotI (purchased from Thermo Fisher), the vectors were subjected to double digestion at 16 ℃ overnight to form a pLent-TIGIT-PD-1-CAR dual-target expression vector. Coli (DH5 α) was transformed with the above-described pLent-TIGIT-PD-1-CAR. Extracting a plasmid, sequencing by committee of Biotechnology engineering (Shanghai) Co., Ltd, and naming the plasmid as Anti-TIGIT-PD-1-CAR after sequencing is correct.

The Anti-TIGIT-CAR and Anti-PD-1-CAR single-target vectors are constructed in the same steps as the Anti-TIGIT-PD-1-CAR, and the Anti-TIGIT-CAR and Anti-PD-1-CAR expression vectors with correct sequencing are finally obtained.

Example 2 preparation of TIGIT and PD-1 double Gene knockout T cells

1. Construction of TIGIT and PD-1 knockout expression vector for CRISPR/CAS9

sgRNA (see Table 1 below) is designed according to website http:// chopchopchopchop. cbu. uib. No./and sgRNA fragments (TIGIT sgRNA) with few off-target sites and strong specificity are screened from TIGIT gene (SEQ ID NO.10), and sgRNA fragments (PD-1 sgRNA) are screened from PD-1 gene (SEQ ID NO. 11).

TABLE 1

The screened TIGIT sgRNA nucleic acid sequence and PD-1 sgRNA nucleic acid sequence are synthesized by the committee biotechnology (Shanghai) GmbH, and are respectively inserted into a standard vector to be connected to the BbsI enzyme cutting site of a CRISPR/CAS9 expression vector pX330A (purchased from the Shanghai daying medicine science and technology Co., Ltd., SEQ ID NO. 12) to respectively obtain a pX330A-TIGIT vector and a pX 330A-PD-1 vector (see figures 2 and 3).

Preparation of T cells

50ml of patient autologous peripheral blood was isolated using TBD sample density separation medium (purchased from Tianjin tertiary ocean organism) to obtain PMBC. After 24-hour induction culture in 100ml DMEM medium (purchased from CORNING corporation, 88-551-CM) containing 1000IU/ml recombinant interferon alpha 2a (purchased from Shenyang Sansheng), 1000IU/ml recombinant IL-2 (purchased from Shenyang Sansheng), 50ng/ml OKT-3 and 5% autologous plasma of patients were added for further 24-hour induction. Adding liquid at intervals of two days in a doubling manner (adding 100ml of DMEM medium to the 4 th day, 200ml of DMEM medium to the 7 th day, 400ml of DMEM medium to the 10 th day and 800ml of DMEM medium to the 13 th day), culturing to the 16 th day, and detecting the positive expression rate of CD3+ and CD56+ in T cells by flow cytometry (CD3-FITC, CD16/CD56-PE antibodies are purchased from BECKMAN company, A07735). CD3+ positivity >80%, CD3+ CD56+ double positivity >20%, considered successful T cell induction.

Co-electrotransfection of T cells with pX330A-TIGIT vector and pX 330A-PD-1 vector

(1) Cleaning the electric rotating cup, soaking the electric rotating cup in 75% alcohol for 2h, and irradiating under an ultraviolet lamp for 15 min.

(2) Take 1X 10⁷For each induced T cell, 500. mu.L of an electrotransfer buffer was added to resuspend the cells and blow them up and down uniformly.

(3) Respectively adding pX330A-TIGIT vector (5 ug) and pX 330A-PD-1 vector plasmid (5 ug) into the cell suspension, blowing and beating uniformly up and down, transferring into an electric rotor (0.2 cm) with corresponding specification, and setting parameters according to preset conditions: 300V, 10ms, 2 electric shock operations are carried out.

(4) After the electric shock was completed, the electric rotor was incubated on ice for 10min to allow the nucleic acid to sufficiently enter the cells.

(5) Taking out the electric shock cup from ice, transferring cells from the electric shock cup, filtering, counting, and determining cell density (3 × 10)⁶/ml) was inoculated in fresh DMEM medium and cultured in a 5% CO2 incubator at 37 ℃.

(6) After culturing for 48-72 h, the cell electric conversion efficiency can be detected.

The expression rates of TIGIT and PD-1 of the T cells before electrotransfection are respectively 38% and 42% through flow cytometry, the expression rates after electrotransfection are 8% and 9.2%, and the electrotransfection efficiency is calculated according to the following formula: (gene expression rate before electrotransfection-gene expression rate after electrotransfection)/gene expression rate before electrotransfection 100%, TIGIT electrotransfection efficiency = (38% -8%)/38% + 100% =78.9%, PD-1 electrotransfection efficiency = (42% -9.2%)/42% + 100% =78.1%, namely the TIGIT and PD-1 double gene knockout efficiency constructed by the invention is about 78%, the subsequent experiment requirements can be met, and the T cell with TIGIT and PD-1 double gene knockout can be successfully obtained.

The electric transfer efficiency is the gene knockout rate.

Example 3 Lentiviral packaging and Titer assay

The lentivirus packaging cell line 293T is inoculated in a 10cm culture dish containing DMEM and 10% FBS, and cultured under the conditions of 37 ℃ and 5% CO2, and transfection is prepared when the anchorage rate is 70% -80%. A sterile 1.5ml EP tube was used to prepare the reaction system by following the components: serum-free DMEM: 3 ml; anti-TIGIT-PD-1-CAR plasmid: 10 mu g of the mixture; GM easy (TM) Lentiviral Mix: 10 μ L (10 μ g); HG Transgene TM Reagent: 60 μ L. After mixing, the mixture was left at room temperature for 20min, and then dropped into a 293T cell culture dish and cultured in a CO2 incubator. After 24h of transfection, the cell culture solution was carefully aspirated off and discarded in a waste liquid cup containing a disinfectant solution, and then 15ml of fresh culture medium containing 10% serum was added to continue the culture. After 48h of liquid change, the cell supernatant was aspirated into a 50ml centrifuge tube, centrifuged at 500g for 5min at 4 ℃, filtered through a 0.45 μm filter and transferred to a new centrifuge tube. And (4) after the viruses are harvested, concentrating to obtain concentrated TIGIT-PD-1-CAR virus solution, and directly detecting the titer or storing in a low-temperature refrigerator at-70 ℃ for later use. The above viruses were titrated with TCID 50, and 293T cells in logarithmic growth phase were grown at 1X 10⁴ The amount of Cells/well was seeded in 96-well cell culture plates, and samples were diluted 10-fold in serial concentrations in 5% FBS DMEM into 96-well plates, 10 wells for each concentration, and 2-well blanks were set. Culturing at 37 deg.C and 5% CO2, observing the cell for 5-7 days, and calculating TCID 50 result according to the concentration and number of wells. The results show that the Anti-TIGIT-PD-1-CAR recombinant lentivirusTiter 4.36X 10⁸pfu/mL。

The Anti-TIGIT-CAR and Anti-PD-1-CAR vector recombinant lentivirus have the virus titer of 5.22 x 10 respectively⁸pfu/mL and 5.16X 10⁸pfu/mL。

Example 4 preparation of TIGIT and PD-1 Dual-target CAR-T cells

After activation of T cells (T cells obtained by TIGIT and PD-1 double knockout in example 2), 1X 10 cells were taken out⁶The cells were treated with concentrated Anti-TIGIT-PD-1-CAR virus (titer 4.36X 10 as prepared in example 3 above) and added⁸pfu/mL virus solution), mixed well, MOI 8, and cultured in 5% CO2 incubator at 37 ℃. The transfected T cells were subjected to a relevant assay one week later. And detecting the expression of the chimeric antigen receptor by a fluorescence microscope, wherein the positive cells for detecting GFP are positive cells for expressing the TIGIT-PD-1 chimeric antigen receptor due to the co-expression of GFP and CAR.

FIG. 4 shows the expression of Anti-TIGIT-PD-1-CAR vector constructed according to the present invention on the surface of T cells as detected by flow cytometry. The result shows that 38.6% of the cells are positive to GFP (GFP protein is expressed by the lentivirus vector), which indicates that Anti-TIGIT-PD-1-CAR constructed by the invention can be expressed on the surface of T cells, and the transfection efficiency is 38.6%.

The transfection efficiencies of Anti-TIGIT-CAR-T and Anti-PD-1-CAR-T were 37.2% and 38.1%, respectively.

Example 5 preparation of target cells expressing PVR and PD-L1 genes

PVR (SEQ ID NO. 13), SEQ ID NO.3 and PD-L1 (SEQ ID NO. 14) sequences of Venezo Bioengineering (Shanghai) Limited are sequentially synthesized into a whole gene fragment and inserted into a standard vector pUC57 to obtain pUC-PVR-PD-L1. After subjecting the pUC-PVR-PD-L1 and pLent-C-GFP (SEQ ID NO.9) vectors to double digestion with Fast Digest AsiSI (available from Thermo Fisher) and Fast Digest NotI (available from Thermo Fisher), the vectors were ligated overnight at 16 ℃ to form pLent-PVR-PD-L1 vector and transformed into E.coli (DH 5. alpha.). Extracting plasmids, sequencing by committee of Biotechnology engineering (Shanghai) Co., Ltd, and reserving after sequencing is correct.

The above plasmids were lentivirally packaged and transfected into 293T cells, and the expression of GFP (the lentivirus vector itself expresses GFP protein, and GFP and genes are co-expressed) on the surface of 293T cells was examined by flow cytometry. The results showed that 98% of the cells were positive for GFP, indicating that the present invention successfully constructed positive cells (i.e., target cells) expressing the PVR and PD-L1 genes.

Example 6 CAR-T cells Co-culture with target cells cytokine secretion assay

Positive cells expressing PVR and PD-L1 genes constructed in example 5 were used as target cells, Anti-TIGIT-PD-1-CAR-T cells, Anti-TIGIT-CAR-T cells, Anti-PD-1-CAR-T cells, T cells (TIGIT and PD-1 double gene knockout T cells prepared in example 2) were used as effector cells, and the expression of the receptor genes was determined as follows: target cell =5:1, when the cells are cultured for 48 hours in a mixed way, the secretion amounts of IL-2, TNF-alpha and IFN-gamma of effector cells are respectively detected by an ELISA method.

Cell co-culture

The concentrations of effector cells and target cells were adjusted with 10% FBS (fetal bovine serum) in DMEM and seeded in a 96-well plate at 100. mu.L/well and 6X 10 cells/well⁴Perwell (E: T =5:1, i.e. 5X 10 cells for effector and target cells, respectively⁴Hole and 1X 10⁴Hole/bore). The method is divided into four groups:

experimental group a: co-culturing Anti-TIGIT-PD-1-CAR-T cells and target cells;

experimental group B: co-culturing Anti-TIGIT-CAR-T cells and target cells;

experimental group C: co-culturing Anti-PD-1-CAR-T cells and target cells;

control group: co-culturing T cells (TIGIT and PD-1 double gene knockout) and target cells;

each set of three replicates was CO-cultured at 37 ℃ in a 5% CO2 incubator for 72 hours, and culture supernatants were collected for cytokine detection.

Detection of cell culture supernatants IL-2, TNF-alpha and IFN-gamma

1) The TNF-alpha and IFN-gamma kit was removed and allowed to stand at room temperature, and 5 standard concentration gradients of TNF-alpha and IFN-gamma were prepared as described. And adding 100 mu L of standard substance into the antibody pre-coated hole.

2) Sample preparation, 100 μ L of cell culture supernatant was added directly to the well plate, while a blank control was set. .

3) Adding 50 mu L of biotin-labeled primary antibody diluted according to the proportion into each hole, and incubating for 90min at 37 ℃; after the incubation is finished, the supernatant is discarded, and the washing solution is used for washing for 4-5 times, 1min each time; adding 100 mu L of enzyme-conjugated secondary antibody diluted according to the proportion, and incubating for 30min at 37 ℃; after incubation, the supernatant was discarded, and the solution was washed 4-5 times for 1min each time.

4) Adding 100 mu L of substrate TMB (3, 3',5,5' -tetramethyl benzidine), incubating at 37 ℃ for developing for 5-15min, adding 100 mu L of stop solution after developing obviously, and placing in an enzyme-linked immunosorbent assay (OD 450 nm) for reading.

The results show that:

the CAR-T cells of experiment group A released IL-2, TNF- α and IFN-r factors in greatly increased amounts compared to control, experiment group B and experiment group C (FIG. 7). Wherein, the secretion of each factor of the experiment group A (Anti-TIGIT-PD-1-CAR-T cell) is the highest, compared with the unmodified T cell of the control group, the secretion of IL-2 is 12.7 times of that of the control group, the secretion of TNF-alpha is 12.5 times of that of the control group, and the secretion of IFN-r is 10.5 times of that of the control group; the IL-2 secretion amount of the experimental group A is 3 times that of the experimental group B, the TNF-alpha secretion amount is 3.25 times that of the experimental group B, and the IFN-r secretion amount is 3.9 times that of the experimental group B; the IL-2 secretion amount of the experimental group A was 2.5 times that of the experimental group C, the TNF-. alpha.secretion amount was 2.9 times that of the experimental group C, and the IFN-. r secretion amount was 3 times that of the experimental group C.

TABLE 2 cytokine secretion amounts (pg/ml) of CAR-T cells prepared according to the present invention

Example 7 CAR-T cell in vitro killing experiment

Cell killing activity was determined using LDH release: positive cells expressing the PVR and PD-L1 genes constructed in example 5 were used as target cells, Anti-TIGIT-PD-1-CAR-T cells, Anti-TIGIT-CAR-T cells, Anti-PD-1-CAR-T cells, T cells (TIGIT and PD-1 double gene knock-out) were used as effector cells, and the expression of the receptor genes in the effector cells: targetCell (E: T) =5:1, inoculating into 96-well plate, adding 200 μ L cell suspension per well, the cell number is 6 × 10⁴The cells were cultured in 3 wells each containing 100ul of natural target cell release pore and 100ul of culture medium, 100ul of maximum target cell release pore and 2.5% Triton for 4 hours at 37 ℃ in a 5% CO2 incubator. The specific grouping is as follows:

experimental group a: co-culturing Anti-TIGIT-PD-1-CAR-T cells and target cells;

experimental group B: co-culturing Anti-TIGIT-CAR-T cells and target cells;

experimental group C: co-culturing Anti-PD-1-CAR-T cells and target cells;

control group: co-culturing T cells (TIGIT and PD-1 double gene knockout) and target cells;

blank group 1: a target cell natural release hole and a culture solution;

blank group 2: maximum release pore of target cells and 2.5% Triton.

And (3) centrifuging the 96-well plate for 5min at 1500rpm of a multi-plate centrifuge, taking 100ul of supernatant from each well, adding the supernatant into corresponding wells of the flat-bottom 96-well plate, simultaneously adding 100ul of Lactate Dehydrogenase (LDH) matrix solution into each well, reacting for 10min, adding 30ul of HCl (1 mol/L) into each well, and measuring the absorbance (A) at 490nm of an enzyme labeling instrument. Cell killing activity was calculated according to the following formula: killing activity (%) = [ (reaction pore a value-natural release pore a value)/(maximum release group a value-natural release pore a value) ] × 100%.

As shown in FIG. 8, the killing activity of A, B, C on the target cells was 98.5%, 32.3% and 36.4% in the experimental group, while the killing activity of the cells in the control group was only 23.3%. The result shows that the Anti-TIGIT-PD-1-CAR-T cell prepared by the invention has the strongest proliferation capacity and the most obvious effect of killing target cells, and the killing capacity is about 3 times that of single target Anti-TIGIT-CAR or Anti-PD-1-CAR and about 4 times that of a control group.

Sequence listing

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tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 9

<211> 7387

<212> DNA

<213> ethnic species (Homo sapiens)

<400> 9

gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg 60

atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt 120

gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc 180

tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat cgcgttgaca 240

ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata 300

tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 360

cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt 420

ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt 480

gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca 540

ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt 600

catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt 660

tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca 720

ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg 780

cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt actgggtctc 840

tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 900

agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 960

ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg 1020

cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg 1080

gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat 1140

tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg 1200

gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa 1260

attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct 1320

gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac 1380

aggatcagaa gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca 1440

aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa 1500

aagtaagacc accgcacagc aagcggccgg ccgctgatct tcagacctgg aggaggagat 1560

atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta 1620

ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga 1680

ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca 1740

atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat 1800

ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag 1860

cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg 1920

atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg 1980

agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa 2040

attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa 2100

aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac 2160

ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt 2220

ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca 2280

ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa 2340

gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg atcggcactg 2400

cgtgcgccaa ttctgcagac aaatggcagt attcatccac aattttaaaa gaaaaggggg 2460

gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac 2520

taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag 2580

cagagatcca gtttggttag taccgggccc gctctagaca tgtccaatat gaccgccatg 2640

ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag 2700

cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc 2760

caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg 2820

gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca 2880

tcaagtgtat catatgccaa gtccgccccc tattgacgtc aatgacggta aatggcccgc 2940

ctggcattat gcccagtaca tgaccttacg ggactttcct acttggcagt acatctacgt 3000

attagtcatc gctattacca tggtgatgcg gttttggcag tacaccaatg ggcgtggata 3060

gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt 3120

ttggcaccaa aatcaacggg actttccaaa atgtcgtaat aaccccgccc cgttgacgca 3180

aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt tagtgaaccg 3240

tcagaatttt gtaatacgac tcactatagg gcggccggga ttcgtcgact ggatccggta 3300

ccgaggagat ctgccgccgc gatcgccggc gcgccagatc tcaagcttaa ctagctagcg 3360

gaccgacgcg tacgcggccg ctcgagatgg tgagcaaggg cgaggagctg ttcaccgggg 3420

tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg 3480

gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg 3540

gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct 3600

tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag 3660

gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg 3720

aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca 3780

aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct 3840

atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca 3900

tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg 3960

gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc 4020

ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc 4080

tcggcatgga cgagctgtac aagaataagg tttatccgat ccaccggatc tagataagat 4140

aaacggccgg ccgcggtctg tacaagtagg attcgtcgag ggacctaata acttcgtata 4200

gcatacatta tacgaagtta tacatgttta agggttccgg ttccactagg tacaattcga 4260

tatcaagctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat 4320

tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca 4380

tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc 4440

tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc 4500

tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt 4560

cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg 4620

gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc 4680

ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta 4740

cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg 4800

gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc 4860

cccgcatcga taccgtcgac ctcgatcgag acctagaaaa acatggagca atcacaagta 4920

gcaatacagc agctaccaat gctgattgtg cctggctaga agcacaagag gaggaggagg 4980

tgggttttcc agtcacacct caggtacctt taagaccaat gacttacaag gcagctgtag 5040

atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa 5100

gacaagatat ccttgatctg tggatctacc acacacaagg ctacttccct gattggcaga 5160

actacacacc agggccaggg atcagatatc cactgacctt tggatggtgc tacaagctag 5220

taccagttga gcaagagaag gtagaagaag ccaatgaagg agagaacacc cgcttgttac 5280

accctgtgag cctgcatggg atggatgacc cggagagaga agtattagag tggaggtttg 5340

acagccgcct agcatttcat cacatggccc gagagctgca tccggactgt actgggtctc 5400

tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 5460

agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 5520

ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagcatgt 5580

gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 5640

ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 5700

acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 5760

ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 5820

cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 5880

tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 5940

gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 6000

ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 6060

acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 6120

gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 6180

ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 6240

tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 6300

gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 6360

tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac 6420

ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga 6480

taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc 6540

cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca 6600

gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta 6660

gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg 6720

tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc 6780

gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg 6840

ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt 6900

ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt 6960

cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata 7020

ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc 7080

gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac 7140

ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa 7200

ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct 7260

tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat 7320

ttgaatgtat ttagaaaaat aaacaaatag gggtcccgcg cacatttccc cgaaaagtgc 7380

cacctga 7387

<210> 10

<211> 831

<212> DNA

<213> ethnic species (Homo sapiens)

<400> 10

cgtcctatct gcagtcggct actttcagtg gcagaagagg ccacatctgc ttcctgtagg 60

ccctctgggc agaagcatgc gctggtgtct cctcctgatc tgggcccagg ggctgaggca 120

ggctcccctc gcctcaggaa tgatgacagg cacaatagaa acaacgggga acatttctgc 180

agagaaaggt ggctctatca tcttacaatg tcacctctcc tccaccacgg cacaagtgac 240

ccaggtcaac tgggagcagc aggaccagct tctggccatt tgtaatgctg acttggggtg 300

gcacatctcc ccatccttca aggatcgagt ggccccaggt cccggcctgg gcctcaccct 360

ccagtcgctg accgtgaacg atacagggga gtacttctgc atctatcaca cctaccctga 420

tgggacgtac actgggagaa tcttcctgga ggtcctagaa agctcagtgg ctgagcacgg 480

tgccaggttc cagattccat tgcttggagc catggccgcg acgctggtgg tcatctgcac 540

agcagtcatc gtggtggtcg cgttgactag aaagaagaaa gccctcagaa tccattctgt 600

ggaaggtgac ctcaggagaa aatcagctgg acaggaggaa tggagcccca gtgctccctc 660

acccccagga agctgtgtcc aggcagaagc tgcacctgct gggctctgtg gagagcagcg 720

gggagaggac tgtgccgagc tgcatgacta cttcaatgtc ctgagttaca gaagcctggg 780

taactgcagc ttcttcacag agactggtta gcaaccagag gcatcttctg g 831

<210> 11

<211> 864

<212> DNA

<213> ethnic species (Homo sapiens)

<400> 11

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggtcagccg gccagttcca aaccctggtg gttggtgtcg tgggcggcct gctgggcagc 540

ctggtgctgc tagtctgggt cctggccgtc atctgctccc gggccgcacg agggacaata 600

ggagccaggc gcaccggcca gcccctgaag gaggacccct cagccgtgcc tgtgttctct 660

gtggactatg gggagctgga tttccagtgg cgagagaaga ccccggagcc ccccgtgccc 720

tgtgtccctg agcagacgga gtatgccacc attgtctttc ctagcggaat gggcacctca 780

tcccccgccc gcaggggctc agctgacggc cctcggagtg cccagccact gaggcctgag 840

gatggacact gctcttggcc cctc 864

<210> 12

<211> 8964

<212> DNA

<213> ethnic species (Homo sapiens)

<400> 12

aggtacccgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc 60

gcccattgac gtcaatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 120

atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc 180

ctattgacgt caatgacggt aaatggcccg cctggcattg tgcccagtac atgaccttat 240

gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg 300

tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt 360

atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg 420

cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg 480

cagccaatca gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc 540

ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc tgcgacgctg ccttcgcccc 600

gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc 660

ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg 720

taagggttta agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg 780

cctgaaatca ctttttttca ggttggaccg gtgccaccat ggactataag gaccacgacg 840

gagactacaa ggatcatgat attgattaca aagacgatga cgataagatg gccccaaaga 900

agaagcggaa ggtcggtatc cacggagtcc cagcagccga caagaagtac agcatcggcc 960

tggacatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca 1020

gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg 1080

gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca 1140

gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg 1200

agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag 1260

aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc 1320

acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg 1380

ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc 1440

tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg 1500

tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca 1560

aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc 1620

tgcccggcga gaagaagaat ggcctgttcg gaaacctgat tgccctgagc ctgggcctga 1680

cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg 1740

acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc 1800

tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga 1860

acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc 1920

accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag 1980

agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc 2040

aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac 2100

tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca 2160

gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt 2220

tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc 2280

cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga 2340

gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc 2400

agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc 2460

ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat 2520

acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg 2580

tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact 2640

tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg 2700

cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca 2760

atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca 2820

gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga 2880

agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg 2940

gcatccggga caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg 3000

ccaacagaaa cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc 3060

agaaagccca ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg 3120

gcagccccgc cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga 3180

aagtgatggg ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga 3240

ccacccagaa gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca 3300

aagagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg 3360

agaagctgta cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg 3420

acatcaaccg gctgtccgac tacgatgtgg accatatcgt gcctcagagc tttctgaagg 3480

acgactccat cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca 3540

acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg 3600

ccaagctgat tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga 3660

gcgaactgga taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa 3720

agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc 3780

tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg 3840

atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc 3900

tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg 3960

tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa 4020

tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg 4080

agattaccct ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa 4140

ccggggagat cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca 4200

tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt 4260

ctatcctgcc caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta 4320

agaagtacgg cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag 4380

tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca 4440

tggaaagaag cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag 4500

aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg 4560

gccggaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc 4620

cctccaaata tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc 4680

ccgaggataa tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga 4740

tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca 4800

aagtgctgtc cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata 4860

tcatccacct gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca 4920

ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc 4980

accagagcat caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca 5040

aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa gaaaaagtaa gaattcctag 5100

agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc 5160

ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga 5220

ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca 5280

ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggagc ggccgcagga 5340

acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg 5400

gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc 5460

gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat 5520

ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg 5580

gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct 5640

cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta 5700

aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa 5760

cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct 5820

ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc 5880

aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg 5940

ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt 6000

acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc 6060

cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct 6120

tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca 6180

ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg 6240

ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct 6300

atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga 6360

taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 6420

cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 6480

aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 6540

aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact 6600

tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc 6660

ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 6720

catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 6780

aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 6840

ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 6900

gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 6960

aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg 7020

gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 7080

gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc actggggcca 7140

gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 7200

gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 7260

gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg 7320

atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 7380

ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 7440

ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 7500

ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 7560

ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 7620

ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 7680

tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 7740

tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 7800

tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 7860

tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 7920

gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 7980

tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 8040

ttcctggcct tttgctggcc ttttgctcac atgtgagggc ctatttccca tgattccttc 8100

atatttgcat atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa 8160

cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc 8220

agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt 8280

cgatttcttg gctttatata tcttgtggaa aggacgaaac accgggtctt cgagaagacc 8340

tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag 8400

tggcaccgag tcggtgcttt tttgttttag agctagaaat agcaagttaa aataaggcta 8460

gtccgttttt agcgcgtgcg ccaattctgc agacaaatgg ctctagagac cggcgccgct 8520

acagggcgcg tcccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg 8580

ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg 8640

ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta 8700

atacgactca ctatagggcg aattgggtac cgggcccccc ctcgaggtcc tccagctttt 8760

gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg 8820

tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta 8880

aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg 8940

ctttccaccg gtggtctctt ctag 8964

<210> 13

<211> 1038

<212> DNA

<213> ethnic species (Homo sapiens)

<400> 13

ggaaccgggg acgtcgtcgt gcaggcgccc acccaggtgc ccggcttctt gggcgactcc 60

gtgacgctgc cctgctacct acaggtgccc aacatggagg tgacgcatgt gtcacagctg 120

acttgggcgc ggcatggtga atctggcagc atggccgtct tccaccaaac gcagggcccc 180

agctattcgg agtccaaacg gctggaattc gtggcagcca gactgggcgc ggagctgcgg 240

aatgcctcgc tgaggatgtt cgggttgcgc gtagaggatg aaggcaacta cacctgcctg 300

ttcgtcacgt tcccgcaggg cagcaggagc gtggatatct ggctccgagt gcttgccaag 360

ccccagaaca cagctgaggt tcagaaggtc cagctcactg gagagccagt gcccatggcc 420

cgctgcgtct ccacaggggg tcgcccgcca gcccaaatca cctggcactc agacctgggc 480

gggatgccca atacgagcca ggtgccaggg ttcctgtctg gcacagtcac tgtcaccagc 540

ctctggatat tggtgccctc aagccaggtg gacggcaaga atgtgacctg caaggtggag 600

cacgagagct ttgagaagcc tcagctgctg actgtgaacc tcaccgtgta ctacccccca 660

gaggtatcca tctctggcta tgataacaac tggtaccttg gccagaatga ggccaccctg 720

acctgcgatg ctcgcagcaa cccagagccc acaggctata attggagcac gaccatgggt 780

cccctgccac cctttgctgt ggcccagggc gcccagctcc tgatccgtcc tgtggacaaa 840

ccaatcaaca caactttaat ctgcaacgtc accaatgccc taggagctcg ccaggcagaa 900

ctgaccgtcc aggtcaaaga gggacctccc agtgagcact caggcatatc ccgtaacgcc 960

atcatcttcc tggttctggg aatcctggtt tttctgatcc tgctggggat cgggatttat 1020

ttctattggt ccaaatgt 1038

<210> 14

<211> 750

<212> DNA

<213> ethnic species (Homo sapiens)

<400> 14

atgaggatat ttgctgtctt tatattcatg acctactggc atttgctgaa cgcatttact 60

gtcacggttc ccaaggacct atatgtggta gagtatggta gcaatatgac aattgaatgc 120

aaattcccag tagaaaaaca attagacctg gctgcactaa ttgtctattg ggaaatggag 180

gataagaaca ttattcaatt tgtgcatgga gaggaagacc tgaaggttca gcatagtagc 240

tacagacaga gggcccggct gttgaaggac cagctctccc tgggaaatgc tgcacttcag 300

atcacagatg tgaaattgca ggatgcaggg gtgtaccgct gcatgatcag ctatggtggt 360

gccgactaca agcgaattac tgtgaaagtc aatgccccat acaacaaaat caaccaaaga 420

attttggttg tggatccagt cacctctgaa catgaactga catgtcaggc tgagggctac 480

cccaaggccg aagtcatctg gacaagcagt gaccatcaag tcctgagtgg taagaccacc 540

accaccaatt ccaagagaga ggagaagctt ttcaatgtga ccagcacact gagaatcaac 600

acaacaacta atgagatttt ctactgcact tttaggagat tagatcctga ggaaaaccat 660

acagctgaat tggtcatccc agaactacct ctggcacatc ctccaaatga aaggactcac 720

ttggtaattc tgggagccat cttattatgc 750

Claims (9)

1. A chimeric antigen receptor targeting TIGIT and PD-1, characterized by: the chimeric antigen receptor comprises a TIGIT extracellular region and a PD-1 extracellular region.

2. A chimeric antigen receptor targeting TIGIT and PD-1 according to claim 1, wherein: the nucleic acid artificial sequence of the TIGIT extracellular region is shown as SEQ ID NO. 2; the nucleic acid artificial sequence of the PD-1 extracellular region is shown in SEQ ID NO. 4.

3. A chimeric antigen receptor targeting TIGIT and PD-1 according to claim 1, wherein: the chimeric antigen receptor comprises a CD8 leader, a TIGIT extracellular region, a PD-1 extracellular region, a CD8 Hinge region, a chimeric antigen receptor and a monoclonal antibody,

The CD8 transmembrane region, the 41BB costimulatory region, and the CD3 zeta signaling region.

4. A CAR-T cell, characterized in that: the CAR-T cell comprises the chimeric antigen receptor of claim 1.

5. A method of producing a CAR-T cell according to claim 4, wherein: the CAR-T cell is prepared by adopting the following method: the lentiviral expression plasmid containing the gene fragment of the chimeric antigen receptor was packaged into a lentivirus, and the recombinant lentivirus was used to infect T cells.

6. A method of producing a CAR-T cell according to claim 5, wherein: the T cell is a TIGIT and PD-1 double-gene knockout T cell.

7. A method of producing a CAR-T cell according to claim 6, wherein: the gene knockout rate of TIGIT of the T cell is more than 75%, and the gene knockout rate of PD-1 is more than 75%.

8. Method for the preparation of CAR-T cells according to claim 6, characterized in that: the preparation of the TIGIT and PD-1 double-gene knockout T cell comprises the construction of a TIGIT and PD-1 expression vector knocked out by CRISPR/CAS9 and the electrotransfection of the T cell;

the construction of the TIGIT and PD-1 knockout expression vector of the CRISPR/CAS9 is characterized in that a TIGIT sgRNA nucleic acid sequence and a PD-1 sgRNA nucleic acid sequence are respectively inserted into a standard vector and connected to a CRISPR/CAS9 expression vector to respectively obtain a CRISPR/CAS9 knockout TIGIT vector and a CRISPR/CAS9 knockout PD-1 vector.

9. Method for the preparation of CAR-T cells according to claim 8, characterized in that: the TIGIT sgRNA nucleic acid sequence is ccagtcgctgaccgtgaacgata; the PD-1 sgRNA nucleic acid sequence is acatgagcgtggtcagggcc.

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