Preparation and construction method of CAR-T vector for use in the treatment of colon cancer

Abstract

The present invention provides a preparation method and construction method of a CAR-T vector for use in the treatment of colon cancer. The therapeutic vector of the present invention consists of two parts: a lentiviral expression vector pCDH-CMV-MCS-EF1-Puro and a CAR expression structure. The CAR structure of the present invention is specifically CD8leader-EpCAMscfv-CD8α-CD28-CD137-CD3ζ-IRES-PD-1mAb. The therapeutic vector of the present invention and the packaging plasmids PSPAX2 and pMD2G adopt a three-plasmid packaging system according to (PSPAX2: pMD2G = 8:1): vector plasmid = 3:2 to infect 293T cells to obtain CAR-T cells. The CAR-T cells of the present invention can target and kill colon cancer cells that positively express EpCAM.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a preparation method and a construction method of a CAR-T vector for use in the treatment of colon cancer. Background Art

[0002] Colon cancer is a malignant lesion of the colon mucosal epithelium caused by a variety of carcinogenic factors such as the environment or genetics. It is one of the common malignant tumors. In recent years, the incidence and mortality of colon cancer in my country have been on the rise. At present, the main treatment methods include surgery, chemotherapy, radiotherapy, and traditional Chinese medicine. However, the treatment effects of the above methods are limited, and the toxic side effects are large. Although the multidisciplinary team (MDT) diagnosis and treatment model of colon cancer based on surgery and immunotherapy have gradually played an increasingly important role, they cannot achieve satisfactory results. For example, immunotherapy lacks the ability to specifically recognize tumor associated antigens (TAA) and kill specific tumor target cells, and the expression of the major histocompatibility complex (MHC) on the surface of tumor cells decreases, resulting in immune escape.

[0003] However, in recent years, chimeric antigen receptor (CAR)-T cell therapy has made important progress in the treatment of malignant tumors. CAR-T technology extracts T lymphocytes from patients and transforms them using genetic engineering methods, so that the transformed T lymphocytes can secrete CARs and have the ability to specifically recognize and bind to tumor antigens. The transformed T lymphocytes are amplified in vitro and then returned to the patient's body to achieve specific killing of tumor cells. Compared with traditional immunotherapy, CAR-T therapy has shown better targeting and killing in vitro and in clinical trials. There are still differences in the effects of CAR-T therapy in many tumor treatment studies. The effect of CAR-T in the treatment of malignant tumors depends on the targeting of its CAR to the tumor and the survival time and activity of CAR-T cells in the human body. Therefore, the preparation of CAR-T vectors has an important impact on the treatment of malignant tumors using CAR-T technology.

[0004] When T lymphocytes play an anti-tumor role, they recognize and kill tumor cells through the PD-1 (Programmed cell death protein 1) programmed cell death receptor 1 on their surface. However, studies have found that PD-L1 is upregulated in a variety of tumor cells. Tumor cells secrete PD-L1 (programmed death ligand 1) programmed cell death receptor ligand 1 and bind to the PD-1 receptor of T cells, thereby causing T cells to be inactivated, but it does not kill tumor cells. After binding to PD-1 on T cells, it can inhibit T cell proliferation and activation, making T cells in an inactivated state, thus causing tumor immune escape.

[0005] In the process of using CAR-T for anti-tumor treatment, the most critical thing is the construction of the vector. Although there have been many reports on the construction technology of CAR-T preparation vectors, there are currently no CAR-T treatment vectors and related research that can effectively counteract the interference of PD-L1 of colon cancer cells on T cells and have good recognition ability for colon cancer cells. Summary of the invention

[0006] The purpose of the present invention is to provide a CAR-T vector for use in the treatment of colon cancer, a construction method thereof, and an application thereof in the treatment of colon cancer.

[0007] One of the purposes of the present invention is to provide a CAR-T vector for use in the treatment of colon cancer, which includes a lentiviral expression vector and a CAR gene sequence connected to the lentiviral expression vector, wherein the CAR gene sequence includes an EpCAM single-chain antibody gene sequence and a PD-1 monoclonal antibody gene sequence, wherein the EpCAM single-chain antibody nucleotide sequence is shown in SEQ ID NO.2, and the PD-1 monoclonal antibody nucleotide sequence is shown in SEQ ID NO.8. By inserting the gene sequence of the PD-1 monoclonal antibody into the CAR gene sequence, the T cells express the PD-1 monoclonal antibody and specifically bind to the PD-1 on the surface of the T cells to reduce the binding of the PD-L1 of the cancer cells to PD-1, thereby improving the killing power of the CAR-T cells to the tumor cells. In addition, by inserting the EpCAM single-chain antibody gene sequence into the CAR gene sequence, the recognition power of the CAR-T cells to the cancer cells can be improved.

[0008] Furthermore, the structure of the CAR is CD8leader-EpCAMscfv-CD8α-CD28-CD137-CD3ζ-IRES-PD-1mAb. The schematic diagram of the structure of the CAR is as follows: Figure 1As shown in the figure, wherein: CD8leader is the leader peptide part of CD8 protein, which can locate the protein outside the cell, and the nucleotide sequence of CD8leader is shown in SEQ ID NO.1; EpCAMscfv is a specific antigen binding domain designed based on the high expression of EpCAM in colon cancer tissue, and the nucleotide sequence of EpCAMscfv is shown in SEQ ID NO.2; CD8α is a transmembrane region, connecting the extracellular antigen binding domain and the intracellular signaling domain, and can anchor the CAR structure on the T cell membrane, and the nucleotide sequence of CD8α is shown in SEQ ID NO.3; CD28-CD137 is a co-stimulatory domain, which transduces proliferation signals and induces the production of cytokines to stimulate T cell activation, and the nucleotide sequences of CD28-CD137 are shown in SEQ ID NO.4 and SEQ ID NO.5; CD3ζ is a signal transduction domain, and when the extracellular region binds to the target antigen, it will transmit TCR-like signals into the cell, activate T cells, and play a role in targeted killing of colon cancer cells, and the nucleotide sequence of CD3ζ is shown in SEQ ID NO.6; IRES is a ribosome recognition site that can recruit ribosomes to translate mRNA. When IRES is fused with exogenous cDNA, IRES can independently initiate translation. The nucleotide sequence of IRES is shown in SEQ ID NO.7; PD-1mAb can be expressed to bind to PD-1 on the surface of T cells, blocking the binding of PD-1 and PD-L1, thereby upregulating the growth and proliferation of T cells, enhancing T cell recognition of colon cancer cells, and activating their attack and killing functions of PD-1mAb. The nucleotide sequence of PD-1mAb is shown in SEQ ID NO.8.

[0009] Furthermore, the lentiviral expression vector is pCDH-CMV-MCS-EF1-Puro, and its structural diagram is as shown in Figure 2As shown. pCDH-CMV-MCS-EF1-Puro can insert fragments through double digestion with EcoRI and NotI. The expression vector includes: CMV promoter - a mammalian cell-specific promoter with strong driving ability; multiple cloning site (MCS) - contains multiple restriction enzyme sites (restriction site), which is the location for inserting foreign genes; WPRE element - can improve the polyA tailing efficiency of mRNA and improve the expression efficiency of transferred genes; SV40 polyA sequence - can effectively terminate transcription and add PolyA tail to transcribed mRNA; hybrid RSV / 5'LTR - contains regulatory elements such as promoter and enhancer, so that it can express full-length viral transcripts at high levels in 293T cells; genetic elements (cPPT, gag, env, LTRs) - used for packaging, transduction and stable integration of viral expression structures into the host's genomic DNA; SV40origin - allows plasmids to proliferate stably in packaging cells. The lentiviral expression vector can carry large exogenous gene fragments, has high transfection efficiency, and can also achieve satisfactory transfection effects on T cells.

[0010] Furthermore, the lentiviral expression vector pCDH-CMV-MCS-EF1-Puro is double-digested with EcoRI and NotI to insert the CAR gene sequence.

[0011] A second object of the present invention is to provide a method for constructing a CAR-T vector for use in the treatment of colon cancer, which comprises the following steps:

[0012] 1) The CAR gene sequence was synthesized and stored on the PUC19 plasmid;

[0013] 2) After enriching the strain containing the lentiviral expression vector and the glycerol bacteria containing the PUC19 plasmid obtained in step 1), the plasmids were extracted and double-digested with EcoRI and NotI respectively;

[0014] 3) separating the enzyme cleavage products obtained in step 2) by agarose gel electrophoresis, and recovering the target fragments;

[0015] 4) The lentiviral expression vector fragment recovered in step 3) and the CAR target fragment are connected and transformed at a molar ratio of 1:5, and the plasmid is extracted to obtain a CAR-T therapeutic vector.

[0016] The third purpose of the present invention is to provide a CAR-T vector for use in the treatment of colon cancer for use in the preparation of CAR-T: the CAR-T treatment vector and the packaging plasmids PSPAX2 and pMD2G are packaged in a three-plasmid packaging system, 293T cells are used as packaging cells for lentivirus, and the virus liquid is collected and concentrated to infect the amplified activated T cells to obtain CAR-T cells. The CAR-T cells of the present invention have a high CD3 positive rate, and CAR-positive T cells account for more than 44% of the total CD3-positive T cells.

[0017] Furthermore, the ratio of the packaging plasmid and the vector plasmid in the system is packaging plasmid (PSPAX2: pMD2G = 8:1): vector plasmid = 3:2.

[0018] The fourth object of the present invention is to provide a CAR-T vector for use in the treatment of colon cancer. The CAR-T obtained by using the CAR-T treatment vector of the present invention has a good killing effect on colon cancer cells. When the effect-target ratio is 2.5:1 and 5:1, the colon cancer cell T84 can be completely killed after adding CAR-T cells for about 60 hours.

[0019] The beneficial effect of the present invention is that, since the recombinant T cells can express the antibody Anti-EpCAM of EpCAM highly expressed on the surface of colon cancer cells, it can specifically recognize and kill colon cancer cells; the antibody Anti-PD-1 can bind to PD-1 on the surface of T cells, blocking the binding of PD-1 and PD-L1, thereby downregulating the effect of PD-1 on T cells, enhancing the killing effect of T cells on tumor cells, and at the same time, enhancing the body's own immune function to achieve anti-tumor effects. In addition, the CAR structure in the present invention gives T cells stronger proliferation. When T cells obtain the vector through viral infection and express the CAR structure, T cells can target and kill colon cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of the CAR structure;

[0021] Figure 2 This is a schematic diagram of the structure of the lentiviral expression vector pCDH-CMV-MCS-EF1-Puro;

[0022] Figure 3 This is the agarose gel electrophoresis image of the EcoRI and NotI double-digestion products of the lentiviral expression vector pCDH-CMV-MCS-EF1-Puro;

[0023] Figure 4This is the agarose gel electrophoresis diagram of the EcoRI and NotI double digestion products of the CAR-T therapeutic vector;

[0024] Figure 5 The result of CD3-positive T cells test;

[0025] Figure 6 It is the detection result of CD4-positive and CD8-positive T cells among CD3-positive T cells;

[0026] Figure 7 The CAR positive cell detection result is CD3 positive T cells;

[0027] Figure 8 RTCA was used to detect the killing efficiency of CART cells on T84;

[0028] Fig. 9 The results of IFN-γ secretion after co-culture of CAR-T cells and T84. DETAILED DESCRIPTION

[0029] The CAR structure of the present invention is composed of CD8leader-EpCAM scfv-CD8α-CD28-CD137-CD3-IRES-PD-1mAb. Among them, the nucleotide sequence of CD8leader is shown in SEQ ID NO.1; the nucleotide sequence of EpCAM scfv is shown in SEQ ID NO.2. Epithelial-specific adhesion molecule (EpCAM, Epithelial cell adhesion moleculer) is an epithelial cell transmembrane glycoprotein that participates in the Wnt signal transduction pathway, regulates target gene transcription, and is related to cell adhesion, migration, proliferation, and differentiation. It was originally discovered as a tumor-associated antigen, and EpCAM has attracted much attention due to its increased expression in various tumor tissues such as colorectal adenocarcinoma, gastric cancer, ovarian cancer, breast cancer, etc. Studies have found that the expression of EpCAM is related to the progression and prognosis of tumors and can be used as a target for tumor targeted therapy. The nucleotide sequence of CD8α is shown in SEQ ID NO.3; the nucleotide sequence of CD28-CD137 is shown in SEQ ID NO.4 and SEQ ID NO.5; the nucleotide sequence of CD3ζ is shown in SEQ ID NO.6; the nucleotide sequence of IRES is shown in SEQ ID NO.7; the nucleotide sequence of PD-1mAb is shown in SEQ ID NO.8. Anti-PD-1 (PD-1 antibody) can bind to PD-1 on the surface of T cells, block the binding of PD-1 and PD-L1, thereby upregulating the growth and proliferation of T cells, enhancing T cells' recognition of tumor cells, activating their attack and killing functions, and achieving anti-tumor effects by mobilizing the body's own immune function.

[0030] The specific structure of the lentiviral expression vector mentioned in the present invention is pCDH-CMV-MCS-EF1-Puro, and the structural schematic diagram is shown in the attached Figure 2 As shown. By analyzing the vector with Snap Gene software and searching related literature, it is known that pCDH-CMV-MCS-EF1-Puro is double-digested with EcoRI and NotI to insert the fragment. The expression vector includes: CMV promoter - a mammalian cell-specific promoter with strong driving ability; multiple cloning site (MCS) - contains multiple restriction enzyme sites (restriction site), which is the location for inserting foreign genes; WPRE element - can improve the polyA tailing efficiency of mRNA and improve the expression efficiency of transferred genes; SV40 polyA sequence - can effectively terminate transcription and add PolyA tail to transcribed mRNA; hybrid RSV / 5'LTR - contains regulatory elements such as promoter and enhancer, so that it can express full-length viral transcripts at high levels in 293T cells; genetic elements (cPPT, gag, env, LTRs) - used for packaging, transduction and stable integration of viral expression structures into the host's genomic DNA; SV40origin - allows plasmids to proliferate stably in packaging cells.

[0031] The construction method of the CAR-T vector for use in the treatment of colon cancer of the present invention is as follows: the CAR gene sequence is synthesized and present on the PUC19 plasmid vector; the PUC19 plasmid vector and the lentiviral expression vector are both double-digested with ECoRⅠ and NotⅠ, the digestion products are separated by agarose gel electrophoresis, the target bands are recovered, the concentrations of the vector and the target fragment are obtained, the two are connected and transformed at a molar ratio of 1:5, and then the plasmid is extracted to finally obtain a therapeutic vector containing a specific CAR structure.

[0032] Application of the CAR-T vector for use in the treatment of colon cancer of the present invention: the therapeutic vector adopts a three-plasmid packaging system, composed of PSPAX2, pMD2G, and a therapeutic vector, and the ratio of the three plasmids in the system is packaging plasmid (PSPAX2: pMD2G = 8:1): vector plasmid = 3:2, and 293T cells are used as packaging cells for lentivirus, and the virus liquid is collected after 48h and 72h of culture, respectively, and the virus liquid is concentrated, and the virus titer is measured, and finally the amplified and activated T cells are infected with MOI = 30 to obtain CAR-T cells, and flow cytometry is used to detect the cell typing of CAR-T cells and the positive rate of CAR structure expression. At the cellular level, RTCA technology is used to detect the killing efficiency of CAR-T cells on colon cancer tumor cells, and the ELISA method is used to detect the killing cytokine IFN-γ.

[0033] The present invention is described below in conjunction with specific embodiments:

[0034] The main sources of materials used in various embodiments of the present invention are described as follows:

[0035] 1. Lentiviral expression plasmid pCDH-CMV-MCS-EF1-Puro, lentiviral packaging plasmid pMD2G, and vector plasmid PSPAX2 were purchased from SBI;

[0036] 2.The CAR gene sequence was designed by Shanghai Beautiful Life Medical Technology Co., Ltd., synthesized by Shanghai Bioengineering Co., Ltd., and stored in the form of PUC19 plasmid;

[0037] 3. Restriction endonucleases EcoRI and Not I were purchased from NEB;

[0038] 4. Agarose gel recovery kit (Cat. No.: DP209-02) and plasmid extraction kit (Cat. No.: DP103-03) were purchased from Tiangen Biochemical Technology Co., Ltd.;

[0039] 5.293T cells and T84 cells were purchased from the Cell Bank of the Chinese Academy of Sciences;

[0040] 6. Lipofectamine 2000 was purchased from Gibco;

[0041] 7. CD3 monoclonal antibody (model GMP-A018), CD28 monoclonal antibody (model GMP-A063), CH38 protein (model GMP-CH38), and IL-2 (model GMP-CD66) were purchased from Jinan Protein Technology Co., Ltd.;

[0042] 8. Flow cytometer was purchased from ThermoFisher, ST16R desktop refrigerated centrifuge, Fresco microcentrifuge were purchased from ThermoFisher, HE120 horizontal electrophoresis tank, and Tanon gel imager were purchased from Tanon.

[0043] Example 1: Construction of therapeutic vector for CAR-T preparation

[0044] (1) Plasmid extraction:

[0045] Take out the glycerol bacteria containing the lentiviral expression plasmids pCDH-CMV-MCS-EF1-Puro and PUC19 plasmid from the -80°C refrigerator, take 5 μL of each and inoculate it into 5 mL LB liquid culture medium (AMP resistance), and culture it in a constant temperature shaker for 12 to 16 hours at 37°C and 250 rpm.

[0046] Plasmid extraction was performed according to the instructions of Tiangen's plasmid extraction kit.

[0047] (2) Enzyme digestion, ligation, and transformation

[0048] The extracted lentiviral expression plasmid pCDH-CMV-MCS-EF1-Puro and PUC19 plasmid were simultaneously digested with EcoRI and NotI, and the digestion products were subjected to agarose gel electrophoresis respectively. The results were observed and recorded using a gel imager. The agarose gel electrophoresis results of pCDH-CMV-MCS-EF1-Puro are shown in Figure 3 shown.

[0049] The target band was recovered according to the instructions of the agarose gel recovery kit, and the recovered fragments were connected according to the molar ratio of CAR structure: pCDH-CMV-MCS-EF1-Puro = 5:1, and then transformed. The transformation reaction conditions were 16°C, 4h connection in the ProFlex PCR instrument, and the transformation reaction system was as follows:

[0050] The connection system is as follows:

[0051]

[0052] The specific conversion process is as follows:

[0053] ①Take out the competent cells from the -80℃ refrigerator and thaw them on ice, and take out the solid culture medium from 4℃ and preheat it in a 37℃ incubator;

[0054] ② Transfer the ligation product into a 1.5 mL centrifuge tube;

[0055] ③ Slowly add 50 μL of competent cells, stirring while adding to mix the two;

[0056] ④ Place the centrifuge tube in 3 on ice for 30 minutes, and then heat shock in a 37°C water bath for 90 seconds;

[0057] ⑤After heat shock, take out immediately and place on ice for 2 minutes;

[0058] ⑥ Add 950 μL of liquid culture medium and culture in a constant temperature shaker for 40 to 60 minutes at 37°C and 200 rpm;

[0059] ⑦ Centrifuge at 6000rpm for 2min, discard most of the supernatant, leaving only 40-60μL;

[0060] ⑧ Use the pipette tip to blow and resuspend the bacterial solution, drop the bacterial solution on the preheated solid culture medium, mark it, and incubate it in a 37℃ incubator overnight.

[0061] (3) Sequencing verification of transformation results and extraction of therapeutic vectors

[0062] Some of the colonies cultured in step (2) were picked and placed in 5 mL LB liquid medium (AMP resistance), cultured on a constant temperature shaker, and plasmids were extracted using Tiangen's plasmid extraction kit. Before plasmid extraction, 500 μL was taken out and placed in a 1.5 mL Eppendorf tube for strain preservation and stored at -80°C. The extracted product was double-digested with EcoRI and NotI and then verified by agarose gel electrophoresis. The results were as follows: Figure 4 As shown. Take 1 μg of plasmid with correct band and send it to Shenggong Biotechnology (Shanghai) Co., Ltd. for sequencing. The plasmid with abnormal band and the remaining bacterial solution are discarded. The plasmid with correct sequencing result is extracted to obtain the therapeutic vector plasmid.

[0063] Example 2: Preparation of concentrated virus solution

[0064] The three plasmid packaging system was used for lentiviral packaging. The three plasmids were the therapeutic vector plasmid obtained in Example 1, the lentiviral packaging plasmid pMD2G and the vector plasmid PSPAX2, and the cells were 293T cells.

[0065] The specific implementation steps are as follows:

[0066] (1) Plating within 24 hours before transfection: Select cells with a passage number of less than 3 generations, adjust the cell density according to the cell growth density and state, and select 293T cells with a growth density of 80% for plating. In this example, a 6 cm dish is used;

[0067] (2) When the cell growth density reaches 60-90% and the cell state is good, virus packaging can be carried out. The ratio of lentiviral packaging mixture to vector plasmid is 3:2. The required three-plasmid mixture ratio is as follows:

[0068] Vector plasmid 3μg

[0069] pMD2G 0.5 μg

[0070] PSPAX2 4 μg

[0071] (3) Mix the plasmid mixture with 15 μL of transfection reagent lipofectamine 2000 in a tube, let it stand at room temperature for 20 min, then slowly add it to the 293T cells that have been replaced with culture medium and continue culturing;

[0072] (4) Collect the virus liquid at 48 h and 72 h, filter it through a 0.45 μm filter membrane, and store it at -80°C;

[0073] (5) The collected virus solution was concentrated using the PEG8000 concentration method, and the virus titer was determined and stored at -80°C for future use. The virus titer was determined using the following method:

[0074] ① Take 293T cells with good growth status, digest them and count them according to 2×10 5 Cells / well: Evenly plate cells into 24-well cell culture plates and culture for 6-10 hours until cells adhere to the wall;

[0075] ② Add different concentrations of concentrated virus solution to the cell culture supernatant in the 24-well plate, gently tap the edge of the 24-well plate to mix the virus solution and culture medium evenly, and infect in the incubator for 48 hours;

[0076] ③ 48 hours after infection, digest and collect cells, detect the percentage of positive cells by flow cytometry, and calculate the virus titer according to the following formula, in TU / mL:

[0077] T = (number of cells at the time of plating × percentage of positive cells × 1000) / volume of virus solution added (μL).

[0078] Example 3: Acquisition of CAR-T cells

[0079] 1. PBMC (Peripheral Blood Mononuclear Cell) Isolation

[0080] (1) Collect approximately 6 mL of fresh human peripheral blood using a vacuum blood collection tube containing heparin;

[0081] (2) Dilution: Add an equal volume of PBS at room temperature and mix gently by pipetting;

[0082] (3) Sample addition: Take a 50 mL centrifuge tube and pipette 6 mL of Ficoll (lymphocyte separation fluid) into the centrifuge tube (the volume ratio of Ficoll to blood before dilution is 1:1). Tilt the tube at 45° and slowly add the diluted blood onto the Ficoll along the tube wall at about 1 cm above the Ficoll liquid surface.

[0083] (4) Centrifugation: 18-20°C, 2000 rpm, 30 min. After centrifugation, four layers are separated from the bottom of the tube to the liquid surface, namely, red blood cell and granulocyte layer, stratified liquid layer, mononuclear cell layer, and plasma layer.

[0084] (5) Recovery: Insert the pipette directly into the cloud layer (or first aspirate the upper plasma), gently aspirate the cloud layer, and place it into a new centrifuge tube;

[0085] (6) Washing: add PBS at least 3 times the volume of PBMC, 18-20°C, 2000 rpm, 10 min, twice;

[0086] (7) Cell counting: discard the supernatant, add 1 mL of RPMI-1640 medium (containing 10% fetal bovine serum), mix by pipetting, and prepare a PBMC cell suspension. Count using a blood cell counting plate: take a drop of PBMC suspension and a drop of 2% trypan blue dye solution, mix well, add to the blood cell counting plate, and count the total number of cells in 4 large squares under a microscope. Number of cells / mL = (total number of cells in 4 large squares / 4) × 10 4 ×2 (dilution factor).

[0087] 2. T cell activation and lentiviral infection to obtain CAR-T cells

[0088] (1) Preparation of relevant reagents:

[0089] ① Preparation of Anti-CD3 monoclonal antibody solution: 50μg of CD3 monoclonal antibody was dissolved in 5ml PBS solution to prepare a solution with a concentration of 10μg / ml. After dissolution, it was divided into 400μl per EP tube and stored in a -80℃ refrigerator. (At this concentration, 175μl of antibody solution was added to each 24-well plate);

[0090] ② Preparation of Anti-CD28 monoclonal antibody solution: 50 μg of CD28 monoclonal antibody was dissolved in 5 ml of PBS solution to prepare a solution with a concentration of 10 μg / ml. After dissolution, it was divided into 400 μl per EP tube and stored in a -80°C refrigerator. (At this concentration, 175 μl of antibody solution was added to each 24-well plate);

[0091] ③ Preparation of CH-38 protein solution: 500μg CH38 protein was dissolved in 10ml PBS solution to prepare a 50μg / ml solution. After dissolution, it was divided into 400μl per EP tube and stored in a -80℃ refrigerator. (At this concentration, 175μl antibody solution was added to each 24-well plate);

[0092] ④ IL-2 factor preparation: IL-2 protein solid was 1×10 7 U / mg was prepared by adding 500 μl of PBS solution to every 50 μg of IL-2 protein to prepare 10 3 U / μl concentration, add 32μl to each EP, and store in a -80℃ refrigerator;

[0093] ⑤ Preparation of lymphocyte culture medium: Add 30 μl of the pre-packaged IL-2 solution, 0.5 ml of double antibody (penicillin-streptomycin mixture (100X)), and 250 μl of autologous serum to every 50 ml of Takara-551h3 lymphocyte culture medium.

[0094] (2) Operation steps:

[0095] ①Day-1: 24-well plate coating: Take a Corning 24-well plate, take 2 wells as an example, add 175μl CD3 monoclonal antibody solution, 175μl CD28 monoclonal antibody solution, and 175μl CH-38 protein solution to each well. After adding, shake gently to mix, seal the well plate with sealing film, and place it in a 4℃ refrigerator overnight. Cell recovery: Take PBMC cells in liquid nitrogen and recover;

[0096] ②Day 0: Clean the coated plate: Take out the 24-well plate coated on Day 1, discard the supernatant, wash twice with PBS, and add PBS for standby;

[0097] PBMC plating: Collect PBMC cells, count and adjust the final concentration to 0.7 × 10 6 cells / ml, add 400 μl of cell suspension to each well, that is, add 2.8×10 5 cells;

[0098] Virus infection: The virus concentrate in Example 2 was used to infect PMBC cells at an MOI of 30, and 1 ml of virus culture medium suspension was prepared and added to a 24-well plate. The well plate was placed in a centrifuge and centrifuged at 1000 g for 30 min. The centrifuge temperature was adjusted to 32° C.

[0099] ③Day1-Day2: observe cell status;

[0100] ④Day 3: Transfer all cells in the 24-well plate to a 25 cm 2 In the culture flask, observe the cell status;

[0101] ⑤Day4-Day7: Observe the cell status and cell number. If the cells begin to proliferate significantly and the cell density in the local area is high, add 10 ml of culture medium;

[0102] ⑥Day 8: 25cm 2 The cells in the culture flask have grown to full size and were transferred to a 75 cm 2 Continue to culture in the culture bottle;

[0103] ⑦Day9-Day10: Observe the cell status. When the cells are at 75cm 2 When the culture flask is full, stop growing, enrich the cells and calculate the expansion ratio, perform flow cytometry on cell typing, and perform subsequent experiments such as cell killing detection or cell freezing.

[0104] Example 4: Detection of positive expression rate of CAR structure by flow cytometry

[0105] 1. Detect the positive rate of CD3, CD4 and CD8 cells

[0106] ① The obtained PBMC cells of the NC group (not infected with the virus) and the cells of the sample group (infected with the virus) were gently washed twice with PBS + 2% BSA, and the centrifugation conditions were 1500 rpm and 3 min;

[0107] ② Add 1000μl PBS + 2% BSA to the NC tube and divide it into 5 tubes, namely NC, NC-CD3, NC-CD4, NC-CD8, and NC-CD3 / 4 / 8. Add 200μl PBS to the sample tube, marked as sample-CD3 / 4 / 8, and add 5μl / 20μl / 20μl of CD3 / 4 / 8 antibody respectively, and mix gently;

[0108] ③ After incubation at room temperature and away from light for 30 minutes, incubate at 1500 rpm for 3 minutes and discard the waste liquid;

[0109] ④ Add 200 μl PBS + 2% BSA, gently mix and resuspend, 1500 rpm, 3 min, and discard the waste liquid;

[0110] ⑤ Add 100 μl PBS + 2% BSA, mix gently to resuspend and then detect on the instrument.

[0111] The results of CD3 positive T cell detection are as follows Figure 5 As shown in Figure 2, the proportion of CD3-positive T cells reached 94.85%. The proportion of CD4-positive and CD8-positive T cells was Figure 6 As shown, CD4-negative CD8-positive T cells accounted for 51.20% of the total CD3-positive T cells, CD4-positive CD8-positive T cells accounted for 1.08% of the total CD3-positive T cells, and CD4-positive CD8-negative T cells accounted for 37.44% of the total CD3-positive T cells.

[0112] 2. Detection of CAR structure positive expression rate

[0113] ① The obtained NC group cells (not infected with virus) and sample group cells (infected with virus) were gently washed twice with PBS + 2% BSA, centrifuged at 1500 rpm for 6 min, and the waste liquid was discarded;

[0114] ② Add 200 μl PBS to the NC tube and resuspend; add 100 μl PBS to the sample tube and resuspend, then add 100 μl primary antibody working solution (3 μg / ml) and mix well;

[0115] ③ Incubate at room temperature for 1 hour, centrifuge at 1500 rpm for 3 minutes, and discard the waste liquid;

[0116] ④ Add 200 μl PBS, gently mix and resuspend, 1500 rpm / 3 min, and discard the waste liquid;

[0117] ⑤ Add 200μl PBS to the NC tube and resuspend; add 200μl PBS to the sample tube and resuspend, then add 5μl secondary antibody working solution and mix well;

[0118] ⑥ After incubation at room temperature in the dark for 1 hour, centrifuge at 1500 rpm for 3 minutes, discard the waste liquid, wash gently 3 times with PBS + 2% BSA, centrifuge at 1500 rpm for 3 minutes, and discard the waste liquid;

[0119] ⑦ Add 100 μl PBS, mix gently to resuspend and then test on the instrument.

[0120] The results are as follows Figure 7 As shown, CAR-positive T cells account for 44.10% of the total CD3-positive T cells. 3. RTCA real-time cell killing effect detection

[0121] ① Digest colon cancer cells T84 and prepare a cell suspension. Mix by pipetting and perform RTCA cell counting. Dilute the cell suspension to 5×10 4 cells / ml concentration, placed on ice for later use;

[0122] ②Take out the RTCA test plate, add 50 μl of culture medium, and select the test program for this test in the RTCA tester program;

[0123] ③ Place the RTCA test board into the tester and observe whether the Messege item in the program is normal. If it is normal, start the experimental program 1;

[0124] ④After the program is finished, take out the test plate and add 100 μl of colon cancer cell suspension to the corresponding wells. Mix each tube of cell suspension before adding;

[0125] ⑤ Place the test plate in the incubator and let it stand for 30 minutes to allow the cells to settle naturally;

[0126] ⑥After 30 minutes, place the test plate into the tester and run program 2;

[0127] ⑦After 24 hours, observe the cell growth curve. When the cells are in the logarithmic growth phase, prepare to add the CAR-T cells obtained in Example 3:

[0128] The effector T cells were removed from the culture flask, centrifuged, washed, counted, and the concentrations of the effector group cells were prepared at effector-target ratios (CAR-T cells: colon cancer cell T84) of 5:1, 2.5:1, and 1:1, respectively. The control group was prepared with uninfected T cells at a ratio of T cells: colon cancer cell T84 = 1.25:1.

[0129] ⑧ Pause the program, take out the test plate, add 50 μl of effector cells to the corresponding position, put it back into the detector, continue the program, and observe every day.

[0130] The experimental results are as follows Figure 8 As shown in the results, it can be seen that when the CAR-T cells of the present invention are added for about 30 hours, they can have a significant killing effect on colon cancer cells T84. The higher the addition ratio of CAR-T cells, the better the killing effect on colon cancer cells T84. When the effector-target ratio is 2.5:1 and 5:1, colon cancer cells T84 can be completely killed about 60 hours after the addition of CAR-T cells, while the T lymphocytes in the control group have no killing effect on colon cancer cells T84.

[0131] 4.ELISA detection of cytokine secretion

[0132] ① Dilute 10× coating buffer with ddH2O and add 8μl 250× coating protein to 2ml coating buffer;

[0133] ② Add 100 μl / well of the coating solution prepared in step ① to the Corning 9018 Elisa high affinity 96-well plate, seal and place in a 4°C refrigerator overnight;

[0134] ③ Wash the coated 96-well plate three times with PBST (0.05% Tween 20);

[0135] ④ Prepare 5× blocking solution with ddH2O, add 200 μl / well, and block at room temperature for 1 hour;

[0136] ⑤ Add 1× blocking solution to the bottled standard according to the requirements, and dilute it 7 times. At the same time, dilute the sample 5 times;

[0137] ⑥ Wash the blocked plate 5 times with PBST, add the standard and diluted sample solution, incubate at room temperature for 2 hours and then at 4°C overnight;

[0138] ⑦ Wash with PBST 4 times;

[0139] ⑧ Use 1× blocking solution to dilute 250× detection antibody, add 100 μl / well, and incubate at room temperature for 1 hour;

[0140] ⑨Wash 4 times with PBST, dilute 250×HRP with 1× blocking solution, add 100 μl / well, and incubate at room temperature for 30 min;

[0141] ⑩Wash with PBST 5 times, add 100 μl of 1× TMB reagent to each well, and incubate at room temperature for 15 min;

[0142] Add 50 μl / well stop solution to stop color development;

[0143] The OD value was detected by an enzyme-labeled instrument at 450 nm.

[0144] The results are as follows Fig. 9 As shown, after 24 hours of co-culture of CAR-T cells and colon cancer cells T84 in the "RTCA real-time cell killing effect detection" of this embodiment, obvious cytokine IFN-γ release can be detected. The higher the proportion of effector CAR-T cells added, the more IFN-γ is released.

[0145] The above implementation modes are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the content of the present invention and implement it, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a CAR-T vector for use in the treatment of colon cancer in the preparation of a colon cancer treatment product, characterized in that: The CAR-T vector used in the treatment of colon cancer includes a lentiviral expression vector and a CAR gene sequence connected to the lentiviral expression vector, wherein the CAR gene sequence includes an EpCAM single-chain antibody gene sequence and a PD-1 monoclonal antibody gene sequence. The structural composition of the CAR is CD8leader-EpCAMscfv-CD8 α-CD28-CD137-CD3ζ-IRES-PD-1mAb; The gene sequence of the CAR is: The nucleotide sequence of CD8leader is shown in SEQ ID NO.1; The nucleotide sequence of EpCAMscfv is shown in SEQ ID NO.2; The nucleotide sequence of CD8α is shown in SEQ ID NO. 3; The nucleotide sequences of CD28-CD137 are shown in SEQ ID NO.4 and SEQ ID NO.5; The nucleotide sequence of CD3ζ is shown in SEQ ID NO.6; The nucleotide sequence of IRES is shown in SEQ ID NO.7; The nucleotide sequence of PD-1mAb is shown in SEQ ID NO.8; The lentiviral expression vector is pCDH-CMV-MCS-EF1-Puro.

2. The use according to claim 1, characterized in that: The lentiviral expression vector pCDH-CMV-MCS-EF1-Puro is double-digested with EcoRI and NotI to insert the CAR gene sequence.

3. The use according to claim 2, characterized in that: The following steps are involved: 1) The CAR gene sequence was synthesized and stored on the PUC19 plasmid; 2) After the strain containing the lentiviral expression vector and the glycerol bacteria containing the PUC19 plasmid obtained in step 1) are cultured, the plasmids are extracted and double-digested with EcoRI and NotI respectively; 3) The enzyme digestion products obtained in step 2) are separated by agarose gel electrophoresis, and the target fragments are recovered; 4) The lentiviral expression vector fragment recovered in step 3) and the CAR target fragment are connected and transformed at a molar ratio of 1:5, and the plasmid is extracted to obtain a CAR-T therapeutic vector.

4. The use according to claim 3: the CAR-T therapeutic vector and the packaging plasmids PSPAX2 and pMD2G are packaged using a three-plasmid packaging system, 293T cells are used as lentivirus packaging cells, the virus solution is collected and concentrated, and then infects the amplified and activated T cells to obtain CAR-T cells.

5. The use according to claim 4, characterized in that: The ratio of the packaging plasmid to the vector plasmid in the system is 3:2, and in the packaging plasmid, PSPAX2:pMD2G=8:1.

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