CN111560075B

CN111560075B - Carrier containing double-target chimeric antigen receptor gene, CAR-T cell and application thereof

Info

Publication number: CN111560075B
Application number: CN202010332161.6A
Authority: CN
Inventors: 廖兴华; 戴周彤; 王易澳; 项园; 王君; 张慧敏; 李佳蓬; 李慧; 张同存
Original assignee: Wuhan University of Science and Engineering WUSE
Current assignee: Wuhan University of Science and Engineering WUSE
Priority date: 2020-04-24
Filing date: 2020-04-24
Publication date: 2023-06-23
Anticipated expiration: 2040-04-24
Also published as: CN111560075A

Abstract

The invention discloses a vector containing a double-target chimeric antigen receptor gene, a CAR-T cell and application thereof. The double-target chimeric antigen receptor is obtained by splicing a signal peptide, a CD44 single-chain antibody ScFv, an EpCAM single-chain antibody ScFv, a strep II tag, a CD8 molecule hinge region, a CD28 molecule transmembrane region, an intracellular domain thereof and intracellular co-stimulatory domains 4-1BB and a CD3 molecule zeta chain in sequence from the N end to the C end. Meanwhile, the invention constructs a lentiviral expression recombinant plasmid pTK-881-CD44-EpCAM, carries out lentiviral packaging, and infects T cells to obtain double-target CAR-T cells capable of simultaneously removing CD44 and EpCAM. The invention is based on CAR-T technology, and only utilizes the immunity of the patient to kill the gastric cancer stem cells, thereby assisting the doctor in treating gastric cancer.

Description

Carrier containing double-target chimeric antigen receptor gene, CAR-T cell and application thereof

Technical Field

The invention relates to the field of tumor cell immunotherapy, in particular to a carrier containing a double-target chimeric antigen receptor gene, a CAR-T cell and application thereof.

Background

Gastric cancer is one of the most common malignant tumors worldwide and is also the third leading cause of death in malignant patients. At present, multidisciplinary combined treatment is used for improving prognosis of patients with advanced gastric cancer, and although the treatment aspect is greatly advanced, the prognosis effect of patients with gastric cancer is still poor, mainly because the diagnosis is relatively late, and difficulties are brought to diagnosis and treatment. Cancer stem cell theory is a recently proposed hypothesis that cancer stem cells and normal stem cells have similar characteristics, have self-renewal ability, can produce different offspring, are different from normal stem cells in that they have tumorigenic activity and have higher resistance to chemotherapeutic drugs for treating malignant tumors, and are also a culprit for causing tumor metastasis. Therefore, the elimination of cancer stem cells in vivo provides a new direction for early diagnosis of gastric cancer.

Chimeric antigen receptor (chimeric antigen receptor, CAR) T cell therapies are methods in which a single-chain antibody variable region gene fragment capable of recognizing a specific tumor surface antigen is artificially overexpressed on the surface of T cells by genetic engineering techniques, thereby allowing T cells to recognize the specific antigen and kill target cells expressing the antigen. The CAR-T cell therapy is a very promising cancer treatment method, is widely applied to blood tumors at present, has remarkable curative effect in acute lymphoblastic leukemia and non-Hodgkin lymphoma by taking CD19 as a target, and has almost 100% curative effect in multiple myeloma by taking BCMA as a target. The safety and effectiveness of solid tumors (e.g., gastric cancer, liver cancer, pancreatic cancer, etc.) have also been demonstrated.

CD44 is a class i transmembrane glycoprotein that is involved in a variety of biological processes of cells, such as survival, proliferation, differentiation, as a cell membrane receptor. The research shows that the gastric adenocarcinoma highly expresses CD44, and the CD44+ cells in the gastric cancer cell lines are further separated and cultured in a serum-free and non-adhesive way, so that the tumor stem cell balls can be obtained, and tumors can be formed after the stomach and the subcutaneous tissue of the immunodeficient mice are transplanted. Epithelial cell adhesion molecules (Epithelial cell adhesion molecule, epCAM) belong to the class i transmembrane glycoproteins, and are also considered as markers for tumor stem cells, with increased expression in a variety of tumor cells of epithelial origin. Recent studies have found that EpCAM is not only involved in cell adhesion, but also is closely related to cell signaling, metastasis, proliferation and differentiation.

However, there is currently little research on CAR-T cell therapies for gastric cancer, and there are fewer reports on dual-target CAR-T treatment of gastric cancer. Constructing a dual-target CAR that can simultaneously clear CD44 and EpCAM is an effective way to treat gastric cancer recurrence, metastasis and drug resistance caused by gastric cancer stem cells.

Disclosure of Invention

In view of the above, the invention provides a vector containing a double-target chimeric antigen receptor gene and CAR-T cells, which can accurately and effectively kill cells co-expressed by CD44 and EpCAM in a patient, thereby providing possibility for curing gastric cancer.

The technical scheme of the invention is realized as follows:

in a first aspect, the invention provides a double-target chimeric antigen receptor, which is obtained by splicing a signal peptide, a CD44 single-chain antibody ScFv, an EpCAM single-chain antibody ScFv, a strep II tag, a CD8 molecule hinge region, a CD28 molecule transmembrane region and an intracellular domain thereof, an intracellular co-stimulatory domain 4-1BB and a CD3 molecule zeta chain in sequence from N end to C end, wherein the CD44 single-chain antibody ScFv can specifically recognize a CD44 protein on the surface of a gastric cancer stem cell, and is obtained by connecting in series an antibody light chain and a heavy chain variable region aiming at the CD44 protein on the surface of the gastric cancer stem cell, and the EpCAM single-chain antibody ScFv can specifically recognize an EpCAM protein on the surface of the gastric cancer stem cell, and is obtained by connecting in series an antibody light chain and a heavy chain variable region aiming at the EpCAM protein on the surface of the gastric cancer stem cell.

Further, the nucleotide sequence of the ScFv gene encoding the CD44 single-chain antibody is shown as SEQ ID NO.1 in a sequence table, the nucleotide sequence of the ScFv gene encoding the EpCAM single-chain antibody is shown as SEQ ID NO.2 in the sequence table, the nucleotide sequence of the ScFv gene encoding the signal peptide is shown as SEQ ID NO.3 in the sequence table, the nucleotide sequence of the ScFv gene encoding the strep II tag gene is shown as SEQ ID NO.4 in the sequence table, the nucleotide sequence of the ScFv gene encoding the CD8 molecular hinge region is shown as SEQ ID NO.5 in the sequence table, the nucleotide sequences of the gene encoding the CD28 molecular transmembrane region and the intracellular domain thereof are shown as SEQ ID NO.6 and SEQ ID NO.7 in the sequence table, the nucleotide sequence of the gene encoding the intracellular co-stimulatory domain 4-1BB is shown as SEQ ID NO.8 in the sequence table, and the nucleotide sequence encoding the CD3 molecular zeta chain gene is shown as SEQ ID NO.9 in the sequence table.

On the basis of the technical scheme, preferably, the C end of the zeta chain of the CD3 molecule is further spliced with F2A peptide, IL-7, F2A peptide and CCL19 in sequence.

In a second aspect, the invention provides a gene for encoding a double-target chimeric antigen receptor, the nucleotide sequence of which is shown as SEQ ID NO.10 in the sequence Listing.

In a third aspect, the invention provides a vector comprising a double-target chimeric antigen receptor gene, which vector is a nucleic acid construct, plasmid or lentivirus incorporating the double-target chimeric antigen receptor gene of the second aspect.

Based on the above technical scheme, preferably, the plasmid is a recombinant plasmid taking pTK-881 plasmid as a framework.

In a fourth aspect, the invention provides a CAR-T cell that is a genetically engineered T cell capable of expressing the above-described dual-target chimeric antigen receptor.

In a fifth aspect, the present invention further provides a construction method of a CAR-T cell, specifically, a three-plasmid packaging system is adopted to perform lentiviral packaging, the three plasmids include a lentiviral expression plasmid containing the double-target chimeric antigen receptor encoding gene, a lentiviral packaging plasmid VSVG and a vector plasmid GAG, 293T cells are used as lentiviral packaging cells, culturing is performed, virus liquid is collected, and after the virus liquid is concentrated, T cells are infected, so as to obtain the CAR-T cell.

In a sixth aspect, the invention also provides the use of the CAR-T cell of the fourth aspect for the preparation of a live cell medicament or kit for the treatment of gastric cancer.

The vector containing the double-target chimeric antigen receptor gene, the CAR-T cell and the application thereof have the following beneficial effects compared with the prior art:

(1) The CAR-T cells constructed by the invention can simultaneously express tumor markers CD44 and EpCAM aiming at the cell surface of gastric cancer stem cells, so that the recognition range of the CAR-T cells is greatly increased, and the killing range of gastric cancer cells is wider;

(2) The CAR-T cell provided by the invention is applied to gastric cancer treatment, and can avoid multiple infusions of the CAR-T cell embedded with a single antigen receptor, so that the injury to a patient is reduced, and the economic pressure of the patient is also reduced;

(3) The CAR structure provided by the invention endows T cells with stronger proliferation and lasting vitality, and infiltrates tumor tissues through IL-7T cells to inhibit release of immune factors in tumors, so that the T cells can overcome the local tumor immunosuppression microenvironment and break the host immune tolerance state, quickly establish tumor immunity and eliminate small or large tumor burden.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a partial sequence of a double-target chimeric antigen receptor gene of the invention, wherein the first gray is a signal peptide and the second gray is a single-chain inter-antibody hinge Inner-Linker.

FIG. 2 is a map of lentiviral expression plasmid pTK-881.

FIG. 3 is a graph of the results of flow cytometry measurement of CAR-T cells for oncolysis.

Detailed Description

The following description of the embodiments of the present invention will clearly and fully describe the technical aspects of the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, are intended to fall within the scope of the present invention.

Example 1 double target chimeric antigen receptor for treating gastric cancer and encoding genes thereof

The embodiment provides a double-target chimeric antigen receptor for treating gastric cancer, which comprises the following specific splicing method: the signal peptide, the CD44 and EpCAM single-chain antibody ScFv, strepII, CD range which can respectively identify the surface of gastric cancer stem cells, the zeta chain of the leukocyte antigen differentiation group molecule transmembrane region CD28-TM+ICD, 4-1BB, CD3 (leukocyte antigen differentiation group molecule 3), the F2A peptide, the IL-7, the F2A peptide and the CCL19 are spliced in sequence from the N end to the C end, and finally the complete Chimeric Antigen Receptor (CAR) molecule which can treat gastric cancer is obtained. The above structural fragments can perform the following functions: the signal peptide can secrete the CAR extracellular; the CD44 single-chain antibody ScFv specifically recognizes CD44 protein on the surface of gastric cancer stem cells, and the EpCAM single-chain antibody ScFv specifically recognizes EpCAM protein on the surface of gastric cancer stem cells; the strep II tag protein facilitates the purification of the fusion protein; the CAR of this example is anchored to the cell membrane by CD28tm+icd, connecting ScFv and intracellular signaling molecules via the transmembrane region and intracellular domain of the CD28 molecule; cd3ζ is an intracellular signaling activating molecule, which activates a signal after ScFv binds antigen, initiating the killing activity of lymphocytes; the F2A peptide is Linker, two genes can be connected into an ORF through the F2A peptide, mRNA is translated into a fusion protein, and the fusion protein can be cut into two proteins by the protease for recognizing F2A; chemokines IL-7 and CCL19 can chemotactic T cells to infiltrate tumor cells, mediate tumor immunity, inhibit release of immunosuppressive factors in tumors, indirectly promote antigen presentation of immune cells, inhibit angiogenesis, and finally inhibit growth of tumors.

The embodiment also provides a gene for encoding the double-target chimeric antigen receptor for treating gastric cancer, and the nucleotide sequence of the gene is shown as SEQ ID NO. 10. The CD44 single-chain antibody ScFv is obtained by connecting variable regions of light chain and heavy chain of an antibody aiming at CD44 protein on the surface of gastric cancer stem cells in series, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 1. The EpCAM single-chain antibody ScFv is obtained by connecting variable regions of a light chain and a heavy chain of an antibody aiming at the EpCAM protein on the surface of gastric cancer stem cells in series, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 2. The positions of the monoclonal antibodies and the signal peptide are shown in the color distinction in FIG. 1, wherein the first gray is the signal peptide, and the second gray is the hinge Inner-Linker between single chain antibodies. The nucleotide sequence of the encoding signal peptide gene is shown as SEQ ID NO.3 in a sequence table, the nucleotide sequence of the encoding strep II tag gene is shown as SEQ ID NO.4 in the sequence table, the nucleotide sequence of the encoding CD8 molecule hinge region gene is shown as SEQ ID NO.5 in the sequence table, the nucleotide sequences of the encoding CD28 molecule transmembrane region and intracellular domain genes thereof are respectively shown as SEQ ID NO.6 and SEQ ID NO.7 in the sequence table, the nucleotide sequence of the encoding intracellular co-stimulatory domain 4-1BB gene is shown as SEQ ID NO.8 in the sequence table, and the nucleotide sequence of the encoding CD3 molecule zeta chain gene is shown as SEQ ID NO.9 in the sequence table.

Example 2 construction of double-target chimeric antigen receptor recombinant expression vector for treatment of gastric cancer

The embodiment provides a double-target chimeric antigen receptor recombinant expression vector and a construction method thereof, wherein the construction method of the vector specifically comprises the following steps:

s1 construction of recombinant expression plasmid pTK-881-CD44-EpCAM

(1) Preparation of experimental materials

(1) The preparation method of the LB liquid medium comprises the following steps: weighing 5g of liquid culture medium dry powder by an electronic balance, putting the liquid culture medium dry powder into a 500mL conical flask, adding 100mL of ultrapure water, sealing, sterilizing in a high-pressure steam sterilizing pot, cooling to 40-50 ℃, adding ampicillin in a ratio of 1000:1, carefully mixing, transferring into a clean 500mL reagent bottle for later use, and storing at a temperature of 4 ℃.

The preparation method of the LB solid medium comprises the following steps: weighing 5g of solid culture medium dry powder by an electronic balance, putting the solid culture medium dry powder into a 500mL conical flask, adding 100mL of ultrapure water, sealing, sterilizing in a high-pressure steam sterilizing pot, cooling to 40-50 ℃, adding ampicillin at a ratio of 1000:1, carefully mixing, pouring into a plate, solidifying, and storing the sealing film at 4 ℃.

(2) 3 glycerins respectively containing lentiviral expression plasmid pTK-881 (plasmid map is shown in figure 2), VSVG and GAG are taken out from a refrigerator at-80 ℃,5 mu L of each glycerol strain is inoculated into 5mL of ampicillin-resistant LB liquid medium, 8 tubes are respectively taken, marks are respectively made, and shaking culture is carried out for 12-16 h at the constant temperature of 37 ℃ and 250 rmp.

(3) Plasmid extraction was performed according to the specification using plasmid miniextract kit from Tiangen Biochemical technology (Beijing) limited to obtain lentiviral expression plasmid pTK-881, lentiviral packaging plasmid VSVG and vector plasmid GAG, respectively. Measuring the concentration of the product by using an enzyme-labeled instrument, making a label, and storing the label at the temperature of-20 ℃ for standby.

(2) Enzyme digestion, ligation and transformation

(1) And (3) enzyme cutting: the lentiviral expression plasmid pTK-881 was digested simultaneously with restriction enzymes ScaI and ApeI in a water bath at 37℃for 1-2h.

(2) The pTK-881 plasmid enzyme was subjected to 1.0% agarose gel electrophoresis: weighing 0.2g of agarose, putting into a 100mL conical flask, adding 20mL of 1 xTAE, heating and dissolving in a microwave oven, cooling to 50-60 ℃, adding 2 mu L of nucleic acid dye, shaking uniformly, pouring into a comb gel plate, and solidifying for later use; and after the gel is cooled and solidified, adding 6×loading buffer according to the final volume into the plasmid enzyme digestion product, blowing and mixing uniformly, and sequentially adding into a sample Loading hole. Put into an electrophoresis tank, 90V for 30min, and observed and recorded by a gel imager. After electrophoresis, gel recovery is carried out by using an agarose gel recovery kit of Tiangen company according to the specification, and the concentration of the plasmid enzyme digestion products is detected by using an enzyme-labeled instrument.

(3) The recombinant CAR gene complete sequence is synthesized by the Shanghai Co., ltd, and the gene fragment and gel are recovered to obtain pTK-881 plasmid enzyme digestion product, which is connected with T4 DNA ligase in a PCR instrument for 4h at 16 ℃.

(4) Conversion: taking out competent cells from a refrigerator at-80 ℃ and dissolving the competent cells on ice, taking out LB solid culture from 4 ℃ and preheating the solid culture based on a incubator at 37 ℃; transferring the connection product into a 1.5mL centrifuge tube, slowly adding 50 mu L of competent cells into the centrifuge tube, stirring while adding, and uniformly mixing the two; then placing the centrifuge tube on ice for 30min, and then carrying out heat shock for 90s in a water bath kettle at 37 ℃; immediately taking out and placing on ice for 2min after heat shock; then 950 mu L of LB liquid medium is added into the mixture, and the mixture is subjected to shaking culture for 40 to 60 minutes at the constant temperature of 37 ℃ and 200 rmp; centrifuging at 6000rpm for 2min, removing most of supernatant, and keeping 40-60 mu L; the re-suspension bacteria liquid is blown by a gun head, and the bacteria liquid is dripped on a preheated and cooled solid culture medium, marks are made, and the incubator is kept at 37 ℃ overnight.

(3) Plasmid extraction, enzyme digestion verification and sequencing

(1) And (3) picking part of bacterial colonies into 5mL of LB liquid medium, and culturing at the constant temperature of a shaking table for 12-16 h at the temperature of 37 ℃ and the pressure of 250 rmp.

(2) Plasmid extraction was performed using a plasmid miniprep kit purchased from Tiangen biotechnology limited, and the concentration was determined; 500. Mu.L of each was taken out in 1.5mL Ep tubes prior to plasmid extraction and used as strain stock for storage at-80 ℃.

(3) The obtained plasmid is subjected to sci and AgeI double digestion, and water bath is carried out for 1-2h at 37 ℃.

(4) The plasmid enzyme cleavage products were verified by agarose gel electrophoresis. 1. Mu.g of the plasmid with the correct band was taken and sent to the engineering (Shanghai) Limited for sequencing, and the plasmid with the correct sequencing result was the recombinant expression plasmid pTK-881-CD44-EpCAM.

S2, virus package

(1) Virus liquid collection

Lentivirus packaging was performed using a three plasmid packaging system. The three plasmids are respectively lentiviral expression plasmid pTK-881-CD44-EpCAM containing a CAR structure, lentiviral packaging plasmid VSVG and vector plasmid GAG. The cells were 293T cells.

The specific implementation steps are as follows:

plating was performed within 24h prior to transfection: selecting cells with passage times within 3 generations, regulating the cell density according to the cell growth density and state, growing 293T cells with good growth state and 80% growth density in a 10cm dish, and sucking waste liquid;

to this was slowly attached 3mL PBS to wash the cells;

slowly attaching 1mL of pancreatin to the waste liquid after waste liquid is sucked and removed, and digesting for 2min in a constant temperature incubator;

3mL of DMEM complete medium (10% FBS) was added to the digested cells to terminate digestion, and transferred to a 15mL centrifuge tube, and centrifuged at 800rpm for 3min;

discarding the supernatant, adding 5mL of DMEM complete medium (10% FBS) to the cell sediment to resuspend the cells, and slowly blowing and uniformly mixing;

6cm dishes were taken and 5mL of DMEM complete medium (10% FBS) was added;

adding 720 mu L of cell suspension into each 6cm dish, and carefully and uniformly mixing to ensure that the cells are completely adhered and uniformly adhered;

when the growth density reaches 60% -90%, the cell state is good, and virus packaging can be performed;

according to the number of transfected cells, the three plasmid mixtures required for each 6cm dish were prepared as follows:

recombinant plasmid 5. Mu.g

VSVG 2μg

GAG 1μg；

Adding 2mL of Opti-MEM into the plasmid mixed solution, and standing at room temperature for 5min;

in addition, a lipofectamine3000 (stored at 4 ℃) mixed solution is required to be prepared, each 6cm dish of lipofectamine2000 is required to be 2 mu L/mu g of three-plasmid mixed solution, 2mL of Opti-MEM is slowly added dropwise into the three-plasmid mixed solution, and the three-plasmid mixed solution is kept stand for 5min at room temperature;

mixing the above materials in a tube, standing at room temperature for 20min;

taking out the paved 293T cells, absorbing and discarding the waste liquid, and carefully attaching to 1mL of DMEM culture medium to prevent the cells from floating;

culturing for 72h;

the supernatant was collected for 72 hours and cultured to give a virus starting solution, which was filtered through a 0.45 μm filter membrane for use.

(2) Concentration of virus liquid

5 XPEG 8000NaCl preparation: 8.766g of NaCl and 8000 50g of PEG are respectively weighed and dissolved in 200mL of Milli-Q pure water, and the mixture is subjected to wet heat sterilization at 121 ℃ for 30min and stored at 4 ℃;

every 30mL of filtered virus initial solution is added with 7.5mL of 5 XPEG-8000 NaCl mother solution;

mixing once every 20-30 min for 3-5 times;

standing at 4 ℃ overnight;

centrifuging at 4 ℃ for 20min at 4000 g;

removing the supernatant, standing the tube for 1-2 min, and removing residual liquid;

every 30mL of filtered virus initial solution is added with 500 mu L of PBS to dissolve slow virus sediment;

the collected virus suspension is split into 50 mu L of each part, and the 50 mu L of each part is stored in a finished product tube to obtain concentrated virus liquid, namely the slow virus vector containing the targeting CD44 and EpCAM double-target chimeric antigen receptor, and the slow virus vector is frozen by broken dry ice and then stored at the temperature of-80 ℃ for standby (the repeated freezing and thawing of the concentrated virus liquid are avoided).

(3) Virus titer assay

293T cells with good growth state were taken, counted after digestion, and the number was 2X 10 ⁵ Uniformly spreading the cells to a 24-hole cell culture plate by the cells/holes, and culturing for 6-10 hours until the cells adhere to the wall;

adding concentrated virus liquid with different concentrations into cell culture supernatant in a 24-hole plate, gently beating the edge of the 24-hole plate, uniformly mixing the virus liquid with the culture solution, and carrying out infection for 48 hours in an incubator;

after 48h of infection, cells were digested and collected, positive cell percentages were detected by flow cytometry and viral titers were calculated according to the following formula in TU/mL: t= (number of cells at plating x percentage of positive cells x 1000)/volume of virus liquid added (μl).

Example 3 preparation of double-target chimeric antigen receptor T cells (CAR-T) for the treatment of gastric cancer

S1, PBMC cell separation

About 6mL of human peripheral fresh blood was collected with a heparin-containing vacuum blood collection tube;

dilution: adding PBS with the same volume at room temperature, lightly blowing and uniformly mixing;

sample adding: taking a 50mL centrifuge tube, sucking 6mL of Ficoll (lymphocyte separation liquid) into the centrifuge tube (the volume ratio of the Ficoll to the blood before dilution is 1:1), inclining the tube by 45 degrees, and slowly adding the diluted blood to the Ficoll along the tube wall at a position about 1cm above the liquid level of the Ficoll;

and (3) centrifuging: centrifuging at 2000rpm for 30min at 18-20deg.C, and separating four layers from bottom to surface, which are red blood cell and granulocyte layer, stratified liquid layer, mononuclear cell layer, and plasma layer;

and (3) recycling: directly inserting a pipette into the mononuclear cell layer (or sucking the upper plasma layer firstly), gently sucking out the mononuclear cell layer, and placing the mononuclear cell layer into a new centrifuge tube;

washing: adding PBS which is 3 times the volume of the PBMC (peripheral blood mononuclear cells), and centrifuging at 2000rpm for 10min at 18-20 ℃ for two times;

cell count: the supernatant was discarded, 1mL of RPMI-1640 medium (containing 10% fetal calf serum) was added, and the mixture was blown and mixed to prepare a PBMC cell suspension. Counting using a hemocytometer: one drop of PBMC suspension was mixed with one drop of 2% trypan blue dye solution and added to a hemocytometer, and the total number of cells in 4 grids was counted under a microscope. Cell number/mL = total number of 4 large square cells/4 x 10 ⁴ X 2 (dilution fold).

S2, activation of T cells and lentiviral infection

Adjusting cell density to 1×10 ⁶ cell/mL, cytokine and antibody complex (final concentration of 100U/mL IL-2, 100ng/mL Anti-CD3 (OKT 3), 250ng/mL Anti-CD 28) were added, and culture was continued for 48h;

the required viral load was calculated at moi=20. The calculation formula is as follows: required viral load (mL) = (MOI x cell number)/viral titer.

After the viruses were removed from the-80 ℃ refrigerator, they were rapidly thawed in a 37 ℃ water bath. Adding the calculated virus amount into a six-hole plate, adding polybrene with the final concentration of 8 mug/mL, fully and uniformly mixing, sealing the hole plate by using a sealing film, and placing the hole plate into a centrifuge, and centrifuging for 60min at 800 g;

taking out the pore plate, placing the pore plate at 37deg.C and 5% CO ₂ In the incubator of (2) and continuing to culture for 24 hours;

250g was centrifuged for 10min, the virus-containing medium supernatant was removed, the cell pellet was resuspended in fresh medium, and the cells were transferred to a new six-well plate and cultured for 3-6 days.

S3, verifying whether the CAR structure is expressed or not by using Westernblot

The expression of the CAR in the cell is detected by detecting the CD3 zeta chain in the CAR structure.

Example 4 double target chimeric antigen receptor T cell killing Activity study for treatment of gastric cancer

The specific operation flow of cytotoxicity analysis and detection is as follows:

(1) setting detection plates, and simultaneously setting 4 contrast: the maximum release group, the volume correction control group, the background control group and the natural release group of target cells, and the experimental group selects BGC803 cell lines according to the target cells of 20:1, 10:1, 5:1 and 1:1 (effector cells: target cells);

(2) cell lysis and harvest of supernatant: mu.L of lysis solution (10X), 37℃and 5% CO were added per 100. Mu.L of medium ₂ Incubating for 45min under the condition, and centrifuging for 4min at 250 g;

(3) transfer 50 μl of supernatant to another well plate, take 12mL of thawed assay buffer in the dark, and freeze the remainder quickly (thawed in a 37 ℃ water bath, but not allowed to stand for too long). Adding 12mL of detection buffer solution into a bottle bottom mixture (which can be used for two 96-well plates), and mixing the mixture in an inverted manner;

(4) 50. Mu.L/well of diluted substrate mixture was added and incubated at room temperature for 30min in the dark (unused diluted substrate mixture was placed at-20℃for 6-8 weeks), and 50. Mu.L of stop solution was added;

(5) removing bubbles contained in the holes, detecting an absorption value (490 or 492 nm) within 1h, and detecting an absorption value at least twice;

and (3) result statistics:

all experimental groups, effector cell spontaneous release groups and target cell spontaneous release groups should have their absorbance subtracted by the background average absorbance; the absorbance of the maximum release group of target cells should be subtracted from the average absorbance of the volume corrected control group. The corrected values were used for killing statistics:

cell killing (%) = (experimental group release-effector cell spontaneous release-target cell spontaneous release)/(target cell maximum release-target cell spontaneous release) ]x100%.

The results are expressed in two ways: visual observation with naked eyes and measurement of LDH reflect the cell killing rate. Visual inspection shows that the number of target cells in the experimental group is reduced compared with that of the target cells in the spontaneous release group (the number of target cells in each group is the same during plating, and the plating is even), and the reduction of the number of target cells is more obvious along with the increase of the ratio of the effector cells to the target cells in the experimental group, which indicates that the effector cells have killing property on the target cells.

S1, experiment one: in vitro tumor killing function detection of pTK-CD44-EpCAM-CAR-T cells by adopting calcein detection method

Taking appropriate amount of MGC-803 gastric cancer cells as target cells, and performing cell proliferation at 1×10 ⁶ Cell suspension/mL (PBS, 5% fetal bovine serum) was added to Calcein-acetyl hydroxymethyl ester (Calcein-AM) to a final concentration of 25. Mu.M and incubated in an incubator for 30min. After washing twice at normal temperature, the cells were resuspended to 1.5X10 ⁵ /mL. pTK-881-CD44-EpCAM-CAR-T cells were added at different target ratios, centrifuged for 30 seconds and incubated at 37℃for 2-3 h. The supernatant was taken after the incubation was completed, the fluorescence intensity of calcein therein was measured, and the percent target cell lysis was calculated from the spontaneous release control and the maximum release control. The results are shown in Table 1 below.

TABLE 1 killing effect of CAR-T cells and Normal T cells on gastric cancer cells

The results of the above table show that pTK-CD44-EpCAM-CAR-T cells have specific killing activity against gastric cancer cells with T cells not modified by CAR molecules as a reference.

S2, experiment II: determination of tumor killing efficiency using flow cytometry

After the gastric cancer cells MGC-803, the normal gastric cancer tissue cells GS1 and the cells treated for 24 hours by adding the CAR-T cells are digested by pancreas, the cells are directly subjected to immunofluorescence staining: cell concentration was determined by adding 1X 10 to a 1.5ml EP tube ⁶ The method comprises the steps of (1) performing cell separation, 1000 rpm, centrifuging for 5min, flushing with PBS for 2 times, then respectively adding different combinations of CD44, epCAM and CD4 monoclonal antibodies and nonspecific homotype monoclonal antibodies into different EP tubes, marking, standing in a dark room for 15-30 min, flushing with PBS for 2 times again, and configuring cell resuspension to be detected.

The results are shown in FIG. 2, wherein pTK-CD44-EpCAM-CAR-T cells have specific killing activity against gastric cancer cells.

The results show that the CAR-T cells obtained by lentiviral infection can specifically kill tumor cells by expressing and recognizing antibodies against specific antigens on the surface of the tumor cells.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Sequence listing

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aaacctggcc aggctcccag gctcctcatc tatggtgcat ccagcagggc cactggcatc 600

ccagacaggt tcagtggcag tgggtctggg acagacttca ctctcaccat cagcagactg 660

gagcctgaag attttgcagt atattactgt cagcagtatg gtcattcacc t 711

<210> 2

<211> 740

<212> DNA

<213> (Artificial sequence)

<400> 2

ctgcatgtag gctgtgctgg aggattcgtc tacagtcaat gtgaccttgt ccttgaactc 60

ttgattgtag ttagtataaa tataagaagg ataaatattt ccgatccact caaggccttg 120

tccaggcctc tgcttcaccc agtttatcca gtagttggtg aaggtgtagc cagaagcctt 180

gcaggacagc ttcactgaag ccccaggcct caccagttca gcccctgact gctgcagctt 240

gacctgctca gcagcccgac atctgaggac tctgcggtct attactgtac aagatcccct 300

tatggttacg acgagtatgg tctggactac tggggccaag gcaccacggt caccgtctcc 360

tcaggtggag gcggttcagg cggaggtggc tctggcggtg gcggatcgga catcgagctc 420

actcagtctc catcctccct gactgtgaca gcaggagaga aggtcactat gaactgcaag 480

tccagtcaga gtctgttaaa cagtagaaat acttgacctg gtaccagcag aaaccagggc 540

agcctcctaa actgttgata tactgggcat ccactaggga atctggggtc cctgatcgtc 600

tcacaggcag tggatctgga acagatttca ctctcaccat cagcagtgtg caggctgaag 660

acctggcagt ttattactgt cagaatgatt atgtttatcc gctcacgttc ggtgctggga 720

ccaagctgga aataaaacgg 740

<210> 3

<211> 60

<212> DNA

<213> (Artificial sequence)

<400> 3

atggccctgc ctgtgacagc tctgctcctc cctctggccc tgctgctcca tgccgccaga 60

<210> 4

<211> 42

<212> DNA

<213> (Artificial sequence)

<400> 4

aactggagcc acccccagtt cgagaagggc ggtggcggaa gc 42

<210> 5

<211> 135

<212> DNA

<213> (Artificial sequence)

<400> 5

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 6

<211> 81

<212> DNA

<213> (Artificial sequence)

<400> 6

ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60

gcctttatta ttttctgggt g 81

<210> 7

<211> 120

<212> DNA

<213> (Artificial sequence)

<400> 7

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120

<210> 8

<211> 126

<212> DNA

<213> (Artificial sequence)

<400> 8

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210> 9

<211> 336

<212> DNA

<213> (Artificial sequence)

<400> 9

agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60

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> 10

<211> 4084

<212> DNA

<213> (Artificial sequence)

<400> 10

atgctgctgc tggtgaccag cctgctgctg tgcgagctgc cccaccccgc ccccctgctg 60

atcccccgag cgcacctggt acaatcaggg actgcgatga agaaaccggg ggcctcagta 120

agagtctcct gccagacctc tggatacacc tttaccgccc acatattatt ttggttccga 180

caggcccccg ggcgaggact tgagtgggtg gggtggatca agccacaata tggggccgtg 240

aattttggtg gtggttttcg ggacagggtc acattgactc gagacgtata tagagagatt 300

gcgtacatgg acatcagagg ccttaaacct gacgacacgg ccgtctatta ctgtgcgaga 360

gaccgttcct atggcgactc ctcttgggcc ttagatgcct ggggacaggg aacgacggtc 420

gtcgtctccg cgggcggagg gggttcaggt ggaggaggct ctggcggtgg cggaagctac 480

atccacgtga cccagtctcc gtcctccctg tctgtgtcta ttggagacag agtcaccatc 540

aattgccaga cgagtcaggg tgttggcagt gacctacatt ggtatcaaca caaaccgggg 600

agagccccta aactcttgat ccaccatacc tcttctgtgg aagacggtgt cccctcaaga 660

ttcagcggct ctggatttca cacatctttt aatctgacca tcagcgacct acaggctgac 720

gacattgcca catattactg tcaagtttta caatttttcg gccgagggag tcgactccat 780

attaaaacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 840

cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 900

gggctggact tcgcctgtga tttttgggtg ctggtggtgg ttggtggagt cctggcttgc 960

tatagcttgc tagtaacagt ggcctttatt attttctggg tgaggagtaa gaggagcagg 1020

ctcctgcaca gtgactacat gaacatgact ccccgccgcc ccgggcccac ccgcaagcat 1080

taccagccct atgccccacc acgcgacttc gcagcctatc gctccaaacg gggcagaaag 1140

aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 1200

gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact gagagtgaag 1260

ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc agaaccagct ctataacgag 1320

ctcagctcat ggctctcccc gtgaccgctc tgctgctgcc tctggctctg ctcctccacg 1380

ctgctagacc ccaggtacag cagcagcagt caggtccagg actggtgaag ccctcgcaga 1440

ccctctcact cacctgtgcc atctccgggg acagtgtctc tagcaacagt gctgcttgga 1500

actggatcag gcagtcccca tcgagaggcc ttgagtggct gggaaggaca tactacaggt 1560

ccaagtggta taatgagtat gcagaatctg tgaaaagtcg aataatcatc aacgcagaca 1620

catccaagaa ccagttgtcc ctgcagttga actctgtgac tcccgaggac acggctgtgt 1680

attactgtgc aagagggggt tggccctact actactacat ggacgtctgg ggccaaggga 1740

ccctggtcac cgtctcctca gcggccgcaa taacttcgta taatgtgtac tatacgaagt 1800

tattggcgcg ccaggaaatt gtgctgactc aatctccagg caccctgtct ttgtctccag 1860

gggaaagagc caccctctcc tgcagggcca gtcagagtgt tagcagcagc tacttagcct 1920

ggtaccagca gaaacctggc caggctccca ggctcctcat ctatggtgca tccagcaggg 1980

ccactggcat cccagacagg ttcagtggca gtgggtctgg gacagacttc actctcacca 2040

tcagcagact ggagcctgaa gattttgcag tatattactg tcagcagtat ggtcattcac 2100

ctggaggagg cggctccggc ggcggcggct ccggaggagg cggcagcggc ggcggaggct 2160

ccggaggcgg cggcagcctg catgtaggct gtgctggagg attcgtctac agtcaatgtg 2220

accttgtcct tgaactcttg attgtagtta gtataaatat aagaaggata aatatttccg 2280

atccactcaa ggccttgtcc aggcctctgc ttcacccagt ttatccagta gttggtgaag 2340

gtgtagccag aagccttgca ggacagcttc actgaagccc caggcctcac cagttcagcc 2400

cctgactgct gcagcttgac ctgctcagca gcccgacatc tgaggactct gcggtctatt 2460

actgtacaag atccccttat ggttacgacg agtatggtct ggactactgg ggccaaggca 2520

ccacggtcac cgtctcctca ggtggaggcg gttcaggcgg aggtggctct ggcggtggcg 2580

gatcggacat cgagctcact cagtctccat cctccctgac tgtgacagca ggagagaagg 2640

tcactatgaa ctgcaagtcc agtcagagtc tgttaaacag tagaaatact tgacctggta 2700

ccagcagaaa ccagggcagc ctcctaaact gttgatatac tgggcatcca ctagggaatc 2760

tggggtccct gatcgtctca caggcagtgg atctggaaca gatttcactc tcaccatcag 2820

cagtgtgcag gctgaagacc tggcagttta ttactgtcag aatgattatg tttatccgct 2880

cacgttcggt gctgggacca agctggaaat aaaacggatg gccctgcctg tgacagctct 2940

gctcctccct ctggccctgc tgctccatgc cgccagaaac tggagccacc cccagttcga 3000

gaagggcggt ggcggaagca ccacgacgcc agcgccgcga ccaccaacac cggcgcccac 3060

catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc 3120

agtgcacacg agggggctgg acttcgcctg tgatttttgg gtgctggtgg tggttggtgg 3180

agtcctggct tgctatagct tgctagtaac agtggccttt attattttct gggtgaggag 3240

taagaggagc aggctcctgc acagtgacta catgaacatg actccccgcc gccccgggcc 3300

cacccgcaag cattaccagc cctatgcccc accacgcgac ttcgcagcct atcgcaaacg 3360

gggcagaaag aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac 3420

tcaagaggaa gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact 3480

gagagtgaag ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc agaaccagct 3540

ctataacgag ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg 3600

ccgggaccct gagatggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa 3660

tgaactgcag aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg 3720

ccggaggggc aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac 3780

ctacgacgcc cttcacatgc aggccctgcc ccctcgcaat ctaggacgaa gagaggagta 3840

cgatgttttg gacaagagac gtggccggga ccctgagatg gggggaaagc cgagaaggaa 3900

gaaccctcag gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag 3960

tgagattggg atgaaaggcg agcgccggag gggcaagggg cacgatggcc tttaccaggg 4020

tctcagtaca gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg 4080

ctaa 4084

Claims

1. A dual-target chimeric antigen receptor, characterized in that: the double-target chimeric antigen receptor is obtained by splicing a signal peptide, a CD44 single-chain antibody ScFv, an EpCAM single-chain antibody ScFv, a strep II tag, a CD8 molecular hinge region, a CD28 molecular transmembrane region, an intracellular domain thereof, an intracellular co-stimulatory domain 4-1BB and a CD3 molecular zeta chain in sequence from the N end to the C end, wherein the CD44 single-chain antibody ScFv can specifically recognize CD44 protein on the surface of gastric cancer stem cells, is obtained by connecting an antibody light chain variable region and a heavy chain variable region aiming at the CD44 protein on the surface of gastric cancer stem cells in series, and the EpCAM single-chain antibody ScFv can specifically recognize EpCAM protein on the surface of gastric cancer stem cells, is obtained by connecting an antibody light chain variable region and a heavy chain variable region aiming at the surface of gastric cancer EpCAM protein on the stem cells in series;

the nucleotide sequence of the ScFv gene for encoding the CD44 single-chain antibody is shown as SEQ ID NO.1 in a sequence table, the nucleotide sequence of the ScFv gene for encoding the EpCAM single-chain antibody is shown as SEQ ID NO.2 in the sequence table, the nucleotide sequence of the ScFv gene for encoding the signal peptide is shown as SEQ ID NO.3 in the sequence table, the nucleotide sequence of the ScFv gene for encoding the strep II tag gene is shown as SEQ ID NO.4 in the sequence table, the nucleotide sequence of the ScFv gene for encoding the CD8 molecule hinge region is shown as SEQ ID NO.5 in the sequence table, the nucleotide sequences of the ScFv gene for encoding the CD28 molecule transmembrane region and the intracellular domain thereof are respectively shown as SEQ ID NO.6 and SEQ ID NO.7 in the sequence table, the nucleotide sequence of the ScFv gene for encoding the intracellular co-stimulatory domain 4-1BB is shown as SEQ ID NO.8 in the sequence table, and the nucleotide sequence of the ScFv 3 molecule zeta chain gene is shown as SEQ ID NO.9 in the sequence table;

and the C end of the zeta chain of the CD3 molecule is spliced with F2A peptide, IL-7, F2A peptide and CCL19 in sequence.

2. A double-target chimeric antigen receptor gene, characterized in that: the nucleotide sequence of the double-target chimeric antigen receptor is shown as SEQ ID NO.10 in a sequence table.

3. A vector comprising a double-target chimeric antigen receptor gene, characterized in that: the vector is a nucleic acid construct, plasmid or lentivirus incorporating the dual-target chimeric antigen receptor gene of claim 1.

4. The vector comprising a dual-target chimeric antigen receptor gene according to claim 3, wherein: the plasmid is a recombinant plasmid taking pTK-881 plasmid as a framework.

5. A CAR-T cell, characterized by: the CAR-T cell is a genetically engineered T cell capable of expressing the dual-target chimeric antigen receptor of claim 1.

6. The method of constructing a CAR-T cell according to claim 5, wherein: and (3) carrying out lentivirus packaging by adopting a three-plasmid packaging system, wherein the three plasmids comprise a lentivirus expression plasmid containing the double-target chimeric antigen receptor coding gene of claim 1, a lentivirus packaging plasmid VSVG and a vector plasmid GAG, 293T cells are used as lentivirus packaging cells, the packaging cells are cultured, virus liquid is collected, and after the virus liquid is concentrated, T cells are infected, so that the CAR-T cells are obtained.

7. The use of the CAR-T cell of claim 5, wherein: the CAR-T cells are applied to preparation of living cell medicines or kits for treating gastric cancer.