CAR vector of targeted gastric cancer cell-specific high-expression protein MSLN and construction method and application thereof
阅读说明：本技术 靶向胃癌细胞特异性高表达蛋白msln的car载体及其构建方法和应用 (CAR vector of targeted gastric cancer cell-specific high-expression protein MSLN and construction method and application thereof ) 是由 田晓丽 于 2021-08-20 设计创作，主要内容包括：本发明属于免疫细胞技术领域,具体涉及靶向胃癌细胞特异性高表达蛋白MSLN的CAR载体及其构建方法和应用。所述的CAR载体包含编码嵌合抗原受体的基因,CAR的结构为SP-MSLNscFv-TM-CD28-4-1BB-CD3ζ。以病毒感染方式获得含有该载体的CAR-T细胞,此特异性CAR-T细胞通过表达此独特的CAR结构,可识别并靶向杀伤高表达肿瘤相关抗原MSLN的胃癌细胞。(The invention belongs to the technical field of immune cells, and particularly relates to a CAR vector of a target gastric cancer cell specific high-expression protein MSLN, and a construction method and application thereof. The CAR vector comprises a gene for coding a chimeric antigen receptor, and the structure of the CAR is SP-MSLNscFv-TM-CD28-4-1BB-CD3 zeta. The CAR-T cell containing the vector is obtained by means of virus infection, and the specific CAR-T cell can recognize and target and kill gastric cancer cells highly expressing tumor associated antigen MSLN by expressing the unique CAR structure.)
1. A CAR vector targeting gastric cancer cell-specific high expression protein MSLN comprising a gene encoding a chimeric antigen receptor,
the chimeric antigen receptor is used for targeting the specific high-expression protein MSLN of gastric cancer cells,
the structure of the chimeric antigen receptor is SP-MSLNscFv-TM-CD28-4-1BB-CD3 zeta, wherein SP is a leader peptide for guiding transmembrane transfer of a newly synthesized protein, MSLNscFv is a single-chain antibody for specifically recognizing the protein MSLNscFv, TM is a transmembrane region for connecting an extracellular antigen binding domain and an intracellular signal domain, CD28-4-1BB is a costimulatory domain, and CD3 zeta is a signal transduction domain.
2. The CAR vector targeting the gastric cancer cell-specific high expression protein MSLN according to claim 1,
the nucleotide sequence of the SP is shown as SEQ ID NO.1,
the MSLNscFv comprises VH, VL and a flexible connecting chain connecting the VH and VL, wherein the nucleotide sequence of the VH is composed of one of SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, the nucleotide sequence of the VL is composed of one of SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, the amino acid sequence of the flexible connecting chain is composed of GGSGGGGSGGGS,
the nucleotide sequence of the TM is shown as SEQ ID NO.8,
in the CD28-4-1BB, the nucleotide sequence of the CD28 is shown as SEQ ID NO.9, the nucleotide sequence of the 4-1BB is shown as SEQ ID NO.10,
the nucleotide sequence of the CD3 zeta is shown in SEQ ID NO. 11.
3. The CAR vector targeting the gastric cancer cell-specific high expression protein MSLN according to claim 1, wherein the recombinant vector is a PUC19 plasmid and a lentiviral vector comprising a gene encoding a chimeric antigen receptor.
4. The CAR vector targeting the gastric cancer cell-specific high expression protein MSLN according to claim 3, wherein the lentiviral vector is pCDH-CMV-MCS-EF 1-Puro.
5. A method for constructing the CAR vector targeting the gastric cancer cell-specific high expression protein MSLN of any of claims 1-4, comprising the steps of:
(1) storing the gene encoding the chimeric antigen receptor on a PUC19 plasmid;
(2) carrying out double enzyme digestion on the PUC19 plasmid in the step (1), and carrying out gel electrophoresis separation on an enzyme digestion product to obtain a target gene fragment containing a gene for encoding the chimeric antigen receptor;
(3) carrying out double enzyme digestion on the lentiviral vector, and carrying out gel electrophoresis separation on an enzyme digestion product to obtain the lentiviral vector subjected to double enzyme digestion;
(4) connecting the target gene segment in the step (2) with the double-enzyme-digested lentiviral vector in the step (3);
(5) and (4) transforming the connection product in the step (4) to obtain a recombinant vector.
6. The method for constructing the CAR vector targeting the gastric cancer cell-specific high expression protein MSLN according to claim 5, wherein the double cleavage sites in steps (2) and (3) are EcoRI and NotI, and the molar ratio of the target gene fragment in step (4) to the lentiviral vector is 5: 1.
7. A method for obtaining CAR-T cells targeting gastric cancer cell-specific high expression protein MSLN, which is characterized in that the recombinant vector of any one of claims 1-4 is packaged by lentivirus and centrifugally concentrated to obtain high-titer lentivirus suspension, and the lentivirus suspension is used for infecting T cells to obtain chimeric antigen receptor-T cells.
8. The method for obtaining CAR-T cells targeting gastric cancer cell-specific high expression protein MSLN according to claim 7, wherein the lentiviral packaging employs 293T cells as packaging cells and a three-plasmid packaging system comprising PSPAX2 plasmid, pMD2G plasmid and the recombinant vector, wherein the molar ratio of PSPAX2 plasmid, pMD2G plasmid and recombinant vector is 27:3: 20.
Technical Field
The invention belongs to the technical field of biological medicine immune cells, and particularly relates to a CAR vector of a target gastric cancer cell specific high-expression protein MSLN, and a construction method and application thereof.
Background
The gastric cancer is one of the most common malignant tumors in China at present, has the characteristics of high morbidity and high mortality, and in China, the morbidity and mortality of the gastric cancer account for the third place of the malignant tumors. At present, the treatment modes of the gastric cancer mainly comprise operation treatment, radiotherapy, chemotherapy, targeted therapy and immunotherapy. The operation is the only possible way to cure the gastric cancer, but the simple operation treatment of the gastric cancer in the advanced stage may have naked eyes or residual focus under the microscope, so that the recurrence and metastasis rate is greatly increased; the radiochemical therapy has great toxic and side effects, and the curative effect cannot reach the satisfactory effect; the molecular targeted therapy such as trastuzumab therapy has the problems of drug resistance, no obvious benefit and the like; no significant remission was observed in gastric cancer with immunotherapy, such as the immune checkpoint inhibitor PD-L1 antibody, which may be related to the tumor cells themselves and their microenvironment.
Cellular immunotherapy has been considered as the most promising therapeutic approach, and LAK cells, DCs, and CIK cells have been used as immunotherapies for tumors. Among the numerous cellular immunotherapies, CAR-T therapy is a relatively potential immunotherapy.
However, how to avoid the occurrence of tumor immune escape and enhance the killing of T lymphocytes to tumor cells so as to improve the effect of CAR-T immunotherapy on gastric cancer still remains a problem to be solved for CAR-T therapy.
Mesothelin (MSLN) is a glycoprotein expressed on the cell surface and is expressed only in mesothelial tissue within the body cavity. MSLN is over-expressed in gastric cancer tissues, malignant mesothelioma, ovarian epithelial malignant tumors and pancreatic cancer, and is closely related to the occurrence and development of tumors. MSLN-targeting CAR-T has been demonstrated for mesothelioma, lung, pancreatic, and other cancers. The research group of Guangzhou biomedical and health research institute, namely Lipeng, of Chinese academy of sciences proposes that MSLN can be used as an effective new target for CAR-T to treat gastric cancer, provides a brand-new treatment idea for the treatment of gastric cancer, particularly for the treatment of drug-resistant gastric cancer, and is expected to fundamentally improve the treatment method of gastric cancer so as to improve the prognosis of gastric cancer. The leec group investigator constructed CAR-T vectors that exhibited strong targeted killing and cytokine secretion capacity when co-cultured with target cells. Compared with the CAR-T vector structure, the constructed CAR-T vector structure has essential difference, and the obtained CAR-T cells can be used for clinical experiments.
Disclosure of Invention
The invention aims to solve the problems, and provides a unique CAR vector capable of targeting MSLN, wherein the CAR vector can recognize MSLN positively-expressed gastric cancer cells and enhance the killing capacity of T lymphocytes, so that the effect of CAR-T immunotherapy on gastric cancer treatment is improved. In addition, the invention also provides a construction method of the CAR vector of the target gastric cancer cell-specific high expression protein MSLN.
The invention can be realized by the following technical scheme:
a recombinant vector comprising a gene encoding a chimeric antigen receptor,
the chimeric antigen receptor is used for targeting the specific high-expression protein MSLN of gastric cancer cells,
the structure of the chimeric antigen receptor is SP-MSLNscFv-TM-CD28-4-1BB-CD3 zeta,
wherein, SP is a leader peptide for guiding a newly synthesized protein to perform transmembrane transfer, MSLNscFv is a single-chain antibody for specifically recognizing the protein MSLNscFv, TM is a transmembrane region connecting an extracellular antigen binding domain and an intracellular signal domain, CD28-4-1BB is a costimulatory domain, and CD3 zeta is a signal transduction domain.
The nucleotide sequence of the SP is shown as SEQ ID NO.1,
the MSLNscFv consists of VH, VL and a flexible connecting chain connecting the VH and VL, the nucleotide sequence of the VH consists of one of SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, the nucleotide sequence of the VL consists of one of SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, the amino acid sequence of the flexible connecting chain consists of GGGGSGGGGSGGGS,
preferably, in the present invention, the single-chain antibody nucleotide sequence of MSLN consists of the heavy chain variable region (VH) shown in SEQ ID NO.2 and the light chain variable region (VL) shown in SEQ ID NO. 5.
Further, according to the process rules of scFv, VH and VL sequences are linked together via a flexible linker amino acid sequence of GGGGSGGGGSGGS to form scFv sequences.
The nucleotide sequence of the TM is shown as SEQ ID NO.8,
in the CD28-4-1BB, the nucleotide sequence of the CD28 is shown as SEQ ID NO.9, the nucleotide sequence of the 4-1BB is shown as SEQ ID NO.10,
the nucleotide sequence of the CD3 zeta is shown in SEQ ID NO. 11.
The recombinant vector of the invention is a PUC19 plasmid or a lentiviral vector comprising a gene encoding a chimeric antigen receptor.
In certain embodiments, the recombinant vector is a PUC19 plasmid, in certain embodiments a lentiviral vector, comprising a gene encoding a chimeric antigen receptor.
Preferably, in certain embodiments, the lentiviral vector is pCDH-CMV-MCS-FF 1-Puro.
The invention also provides a construction method of the CAR vector of the target gastric cancer cell specificity high expression protein MSLN, which comprises the following steps:
(1) storing the gene encoding the chimeric antigen receptor on a PUC19 plasmid;
(2) carrying out double enzyme digestion on the PUC19 plasmid in the step (1), separating the enzyme digestion product through gel electrophoresis to obtain a target gene fragment containing the gene of the coding chimeric antigen receptor,
(3) carrying out double enzyme digestion on the lentiviral vector, carrying out gel electrophoresis separation on the enzyme digestion product to obtain the lentiviral vector after double enzyme digestion,
(4) connecting the target gene segment in the step (2) with the double-enzyme-cut lentiviral vector in the step (3),
(5) and (4) transforming the connection product in the step (4) to obtain a recombinant vector.
In certain embodiments, the double cleavage sites in step (2) above are EcoRI and NotI, and the molar ratio of the fragment of the gene of interest comprising the chimeric antigen receptor and the lentiviral vector in step (4) is 5: 1.
The invention also provides application of the CAR vector of the target gastric cancer cell specific high expression protein MSLN, and CAR-T cells are obtained by packaging infection. And (3) carrying out lentivirus packaging on any CAR vector obtained in the above steps, carrying out centrifugal concentration to obtain a high-titer lentivirus suspension, infecting T cells with the lentivirus suspension to obtain CAR-T cells, and killing gastric cancer cells by the CAR-T cells through recognizing specific protein MSLN in a targeted manner.
In certain embodiments, the lentiviral packaging employs 293T cells as packaging cells and a three-plasmid packaging system comprising a PSPAX2 plasmid, a pMD2G plasmid and the recombinant CAR vector in a ratio of 27:3:20 of the PSPAX2 plasmid, the pMD2G plasmid and the recombinant CAR vector.
The invention has the following beneficial effects:
the CAR vector of the target gastric cancer cell-specific high expression protein MSLN provided by the invention comprises an MSLN single-chain antibody; the MSLN single-chain antibody is a specific structure determined according to the high expression of the MSLN in the stomach cancer tissue, and the antibody Anti-MSLN expressed by the structure is responsible for identifying the high expression of the MSLN in the stomach cancer tissue, and on one hand, the MSLN single-chain antibody can be combined with the stomach cancer cell in a targeting way; on the other hand, the T cells are endowed with new antigen specificity, and the immune escape mechanism of tumor cell MHC expression down regulation can be effectively avoided. When the T cell obtains the vector through a virus infection mode and expresses the CAR structure, the T cell can identify and kill gastric cancer cells in a targeting way.
Compared with the prior art, in an in vitro killing experiment, when the effective-target ratio is 2.5:1, the invention can rapidly kill tumor cells in a short time; in an in vivo killing experiment, the effectiveness and safety of the CAR-T cell also obtain good effects; the invention is capable of completely eliminating tumors by injecting CAR-T cells in a mouse tumor model. Meanwhile, the CAR-T cells with low target ratio in the invention have killing effect and cytokine release which can achieve the same effect as the CAR-T cells constructed by Lepeng group researchers.
Drawings
FIG. 1 is a schematic structural diagram of a lentiviral expression vector pCDH-CMV-MCS-FF1-Puro of the present invention;
FIG. 2 is a schematic diagram of the structure of the CAR of the present invention;
FIG. 3 is a graph of MSLN target expression in gastric cancer samples;
FIG. 4 is a graph showing the results of measurement of expression of MSLN on a gastric cancer cell line AGS;
FIG. 5 is a graph of the killing effect of MSLN-CAR-T cells on gastric cancer cell line AGS;
FIG. 6 is a graph comparing the killing results of MSLN-CAR-T cells on different cell lines at an effective target ratio of 2.5: 1;
FIG. 7 is a graph of the killing effect of MSLN-CAR-T cells on gastric cancer cell line AGS;
FIG. 8 is a graph showing the effect of MSLN-CAR-T cells on the drug effect of gastric cancer PDX animal model.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to specific embodiments and the accompanying drawings.
The invention provides a CAR vector of a target gastric cancer cell specificity high expression protein MSLN, and the specific scheme is as follows: the chimeric antigen receptor comprises an MSLN single-chain antibody, wherein the MSLN single-chain antibody is composed of nucleotide sequences shown as SEQ ID NO.2 and SEQ ID NO.5 and a flexible Linker. The Anti-MSLN is used for identifying the MSLN with high expression in gastric cancer tissues, greatly increases the targeting property of CAR-T cells and reduces the tumor immune escape.
The structural composition of the chimeric antigen receptor was SP-MSLNscFv-TM-CD28-4-1BB-CD3 ζ (shown in FIG. 2). SP can express a leader peptide for guiding newly synthesized protein to perform transmembrane transfer, the nucleotide sequence of SP is shown in SEQ ID NO.1, MSLNscFv is an MSLN single-chain antibody, TM is a transmembrane region and is used for connecting an extracellular antigen binding domain and an intracellular signal domain to anchor a CAR structure on a T cell membrane, the nucleotide sequence is shown in SEQ ID NO.8, CD28-4-1BB is a costimulatory domain and is used for transducing a proliferation signal and inducing cytokine generation and stimulating T cell activation, the nucleotide sequence of CD28 is shown in SEQ ID NO.9, the nucleotide sequence of 4-1BB is shown in SEQ ID NO.10, and CD3 zeta is a signal transduction domain, and when the extracellular region is combined with a target antigen, a TCR-like signal is conducted to the intracellular to activate T cells, and the nucleotide sequence is shown in SEQ ID NO. 11. The CAR structure provided by the invention endows T cells with stronger proliferation and durable vitality, so that the T cells have stronger tumor cell killing capacity.
The vector comprises a PUC19 plasmid and a lentivirus vector. The lentiviral vector is pCDH-CMV-MCS-EF1-Puro, and the structure of the lentiviral vector is shown in FIG. 1. The vector was analyzed by Snap Gene software and relevant literature was searched to find that pCDH-CMV-MCS-EF1-Puro cleaved the insert with EcoRI and NotI in a double-restriction manner. The expression vector comprises: the CMV promoter is a mammalian cell specific promoter, and has strong driving capability; multiple Cloning Site (MCS) -containing multiple restriction sites (restriction sites), is the location of insertion of a foreign gene; WPRE element can improve polyA tailing efficiency of mRNA and improve expression efficiency of transferred gene; SV40 polyA sequence-effective to terminate transcription and add polyA tail to transcribed mRNA; hybrid RSV/5 'LTR-contains regulatory elements such as promoter and enhancer, and enables the hybrid RSV/5' LTR to express full-length viral transcripts in 293T cells at high level; genetic elements (cPPT, gag, env, LTRs) -used for packaging, transduction, and stable integration of viral expression constructs into the genomic DNA of a host; SV40 origin-allows the plasmid to proliferate stably in packaging cells.
The lentivirus expression vector can be used as the most effective vector for expressing target genes in almost all mammalian cells including non-dividing cells and dividing cells, has large capacity of carrying exogenous gene fragments and high transfection efficiency, and can achieve satisfactory transfection effect on T cells.
First, experimental material
1. The lentiviral expression plasmid pCDH-CMV-MCS-EF1-Puro, the lentiviral packaging plasmid pMD2G, the vector plasmid PSPAX2 was purchased from SBI; the structure of the lentivirus expression plasmid pCDH-CMV-MCS-EF1-Puro is shown in figure 1;
CAR structural sequence was designed by Hai Biotechnology Inc. of Shanghai, synthesized by Biotechnology engineering (Shanghai), Inc., and stored as PUC19 plasmid;
3. restriction endonucleases EcoRI, NotI were purchased from NEB;
4.T4 DNA ligase、Free H2o is purchased from Boehringer;
5. competent cells were purchased from Trans;
6. the gel recovery kit and the plasmid small-extraction kit are purchased from Tiangen Biotechnology limited company;
7.293T cells and AGS cells were purchased from the cell bank of Chinese academy of sciences;
FBS, DMEM, 1640 medium, PBS, Opti-MEM, lipofectamine 2000 from Gibco;
CD3 monoclonal antibody, CD28 monoclonal antibody, CH38 protein, IL-2 from Shanghai inshore protein science and technology limited;
multiskan GO microplate reader + uDrop ultramicro plate, flow cytometer purchased from ThermoFisher;
HE120 horizontal electrophoresis tank, Tanon gel imager from Tanon;
12. the water-proof constant-temperature incubator and the constant-temperature culture shaking table are purchased from Shanghai-Hengshi Co., Ltd;
Bio-Rad Mini-PROTECTAN Tetra Cell Mini electrophoresis System purchased from Bio-Rad;
14. olympus microscope purchased from olympus;
15. the inoculating loop and the coating rod are purchased from self-cleaning special biological filtration corporation;
16. syringes, 0.45 μm filters, petri dishes of various sizes, flasks, multi-well plates, and centrifuge tubes of various sizes were purchased from Corning.
Second, construction method of CAR vector of target gastric cancer cell specificity high expression protein MSLN
(one) plasmid extraction
The preparation method of the LB liquid culture medium comprises the following steps: weighing 5g of dry liquid culture medium powder in a 500mL conical flask by an electronic balance, adding 100mL of ultrapure water, sealing by tin foil paper, sterilizing in a high-pressure steam sterilization pot, cooling to 40-50 ℃, adding 0.2% Ampicillin (AMP) at a ratio of 1000:1, carefully mixing uniformly, transferring into a clean 500mL reagent bottle for later use, and storing at 4 ℃.
The preparation method of the LB solid medium comprises the following steps: weighing 5g of dry solid culture medium powder in a 500mL conical flask by an electronic balance, adding 100mL of ultrapure water, sealing by tin foil paper, sterilizing in a high-pressure steam sterilization pot, cooling to 40-50 ℃, adding 0.2% AMP (adenosine monophosphate) at a ratio of 1000:1, carefully mixing uniformly, pouring, solidifying, sticking a sealing film, and storing at 4 ℃.
Glycerol bacteria containing lentiviral expression plasmids pCDH-CMV-MCS-EF1-Puro and PUC19 were removed from a refrigerator at-80 ℃ and inoculated in 5. mu.L each into 5mL LB liquid medium (AMP-resistant) and shake-cultured on a constant temperature shaker for 12-16 hours at 37 ℃ and 250 rmp.
Plasmid extraction was carried out according to the instructions of a plasmid Mini kit (cat # DP103-03) purchased from Tiangen Biochemical technology Ltd to obtain a lentiviral expression plasmid pCDH-CMV-MCS-EF1-Puro and a PUC19 plasmid containing a target (MSLN single-chain antibody) CAR structure.
(II) digestion, ligation, transformation
EcoRI and NotI double enzyme digestion is carried out on the extracted slow virus expression plasmid pCDH-CMV-MCS-EF1-Puro and PUC19 plasmid with a target (MSLN single-chain antibody) CAR structure at the same time, the enzyme digestion products are respectively subjected to agarose gel electrophoresis, and the result is observed and recorded by a gel imager.
The target band was recovered using the Tiangen agarose gel recovery kit (cat # DP209-02) instructions and the fragments recovered were assigned to the CAR structure: pCDH-CMV-MCS-EF1-Puro is ligated at a molar ratio of 5:1, transformed into competent cells, and the transformed competent cells are dropped onto a preheated solid medium, labeled, and incubated overnight at 37 ℃.
(III) plasmid extraction, enzyme digestion verification and sequencing
Selecting a part of colonies in a 5mL LB liquid medium (AMP resistance), performing enrichment culture in a constant temperature shaking table, performing plasmid extraction, taking 500 mu L of the colonies in a 1.5mL Ep tube before plasmid extraction, preserving the strains, and storing the strains at-80 ℃; and (3) carrying out double enzyme digestion verification on the extracted product by using EcoRI and NotI, taking 1 mu g of plasmid with correct band, sending the plasmid to the biological engineering (Shanghai) company Limited for sequencing, and discarding the plasmid with abnormal band and the retained bacterial liquid. And extracting the plasmid with the correct sequencing result, and discarding the plasmid with the wrong sequencing result and the bacterial liquid thereof.
Application of CAR vector of target gastric cancer cell-specific high expression protein MSLN
Preparation of concentrated virus liquid
The lentivirus packaging was performed using a three plasmid packaging system. The three plasmids are respectively a lentiviral expression plasmid pCDH-CMV-MCS-EF1-Puro containing CAR structure, a lentiviral packaging plasmid pMD2G and a vector plasmid PSPAX 2. The cells were 293T cells.
The specific implementation steps are as follows:
(1) plating was performed within 24h before transfection: generally selecting cells with passage times within 3 generations, adjusting the cell density according to the cell growth density and state, and paving 293T cells with the growth density reaching 80%;
(2) when the growth density reaches 60-90%, the cell state is good, and then the virus packaging can be carried out;
(3) the virus packaging is carried out according to the proportion of a PSPAX2 plasmid, a pMD2G plasmid and a CAR vector (recombinant plasmid) targeting a gastric cancer cell specific high expression protein MSLN as 27:3:20, and the required three-plasmid mixed liquid is prepared as follows by taking a 6cm dish as an example: the amount of plasmid added was determined according to the concentration of each plasmid.
Recombinant plasmid 3. mu.g
pMD2G 0.5μg
PSPAX2 4μg
(4) The transfection reagent is lipofectamine 2000 (stored at 4 ℃), and the adding amount is 2 muL/mug plasmid;
(5) mixing the plasmid mixture in the step (3) and the transfection reagent mixture in the step (4) in a tube, standing at room temperature for 20min, adding the mixture into the liquid change cells, and continuing culturing;
(6) collecting culture supernatants after 48h and 72h, respectively, and filtering through 0.45 μm filter membrane;
(7) concentrating the collected virus solution by PEG8000 concentration method, determining virus titer, and storing at-80 deg.C for use.
(II) viral fluid infection of T cells
1. PBMC isolation
1) Collecting about 6mL of human peripheral fresh blood with a heparin-containing vacuum blood collection tube;
2) diluting: adding PBS with the same volume at room temperature, lightly blowing, beating and uniformly mixing;
3) sample adding: taking a 50mL centrifuge tube, sucking 6mL of Ficoll (lymphocyte separation fluid) into the centrifuge tube (the volume ratio of the Ficoll to the blood before dilution is 1:1), inclining the centrifuge tube at 45 degrees, and slowly adding the diluted blood to the Ficoll along the tube wall at a position which is about 1cm above the Ficoll liquid surface;
4) centrifuging: centrifuging at 18-20 deg.C at 2000rpm for 30min, and separating into four layers from tube bottom to liquid surface, including erythrocyte and granulocyte layer, layered liquid layer, mononuclear cell layer, and plasma layer;
5) and (3) recovering: directly inserting a pipette into the cloud layer (or sucking the upper plasma layer first), slightly sucking out the cloud layer, and putting into a new centrifuge tube;
6) washing: adding PBS to a volume of less than 3 times the volume of PBMC (peripheral blood mononuclear cells), at 18-20 deg.C, 2000rpm, for 10min twice;
7) cell counting: the supernatant was discarded, 1mL of lymphocyte culture medium was added, and the mixture was blown and homogenized to prepare a PBMC cell suspension. Counting with a blood cell counting plate: a drop of PBMC suspension was mixed with a drop of 2% Trypan blue stain and applied to a blood cell counting plate, and the total number of cells in 4 grids was counted under a microscope. Cell count/mL-4 total large square lattice cells/4 × 104X2 (dilution factor).
2. Activation of T cells and lentivirus infection
(1) Preparation before experiment:
1) preparing Anti-CD3 monoclonal antibody solution: 50 mu g of CD3 monoclonal antibody is dissolved in 5mL PBS solution to prepare solution with the concentration of 10 mu g/mL, and the solution is subpackaged according to 400 mu L of each EP tube and is stored in a refrigerator at the temperature of 80 ℃ below zero. (175. mu.L of antibody solution was added to each 24-well plate at this concentration);
2) preparing Anti-CD28 monoclonal antibody solution: 50 mu g of CD28 monoclonal antibody is dissolved in 5mL PBS solution to prepare solution with the concentration of 10 mu g/mL, and the solution is subpackaged according to 400 mu L of each EP tube and is stored in a refrigerator at the temperature of 80 ℃ below zero. (175. mu.L of antibody solution was added to each 24-well plate at this concentration);
3) preparing a CH-38 protein solution: dissolving 500 μ g of CH38 protein in 10mL of PBS solution to obtain 50 μ g/mL solution, packaging with 400 μ L per EP tube, and storing in refrigerator at-80 deg.C. (175. mu.L of antibody solution was added to each 24-well plate at this concentration);
4) IL-2 factor preparation: IL-2 protein solid at 1X 107U/mg, 500. mu.L of PBS solution is added to 50. mu.g of IL-2 protein to prepare 103U/Mu L, preparing, adding 32 mu L into each EP, subpackaging, and storing in a refrigerator at-80 ℃;
5) preparing a lymphocyte culture medium: takara-551h3 lymphocyte culture medium every 50mL was added with 30. mu.L of the dispensed IL-2 solution, 0.5mL of the double antibody, 250. mu.L of the autologous serum.
(2) The experimental process comprises the following steps:
day 0: coating with 24-hole plate: a Corning 24-well plate was taken, and 175. mu.L of a CD3 monoclonal antibody solution, 175. mu.L of a CD28 monoclonal antibody solution, and 175. mu.L of a CH-38 protein solution were added to each of 2 wells, for example, 2 wells. After adding, shaking gently and mixing uniformly, sealing the pore plate with a sealing film, and putting the sealed pore plate into a refrigerator at 4 ℃ overnight. Cell recovery: taking PBMC cells in liquid nitrogen, and recovering;
day 1: cleaning a coating plate: taking out a 24-hole plate coated yesterday, discarding the supernatant, washing for 2 times by PBS, and adding PBS for later use;
PBMC (plated nucleic acid) plating: PBMC cells were collected, counted and the concentration finally adjusted to 0.7X 106cells/mL, 400. mu.L of cell suspension per well, i.e., 2.8X 10 per well5Cells;
viral infection: infecting with MOI of 30, preparing 1mL of virus culture medium suspension, adding into a 24-well plate, centrifuging for 30min at 1000g, and adjusting the temperature of a centrifuge to 32 ℃;
day1-Day 2: observing the state of the cells;
day 3: all cells in a 24-well plate were transferred to 25cm with 10mL of medium added2Observing the state of the cells in the culture flask;
day4-Day 7: observing the state and the number of the cells, and if the cells begin to be obviously amplified and the cell density of a local area is high, adding 10mL of culture medium;
day 8: at this time 25cm2The cells in the flask were confluent and transferred to 75cm of medium added to 20mL2Continuing culturing in a culture bottle;
day9-Day 10: observing the state of the cells when the cells are at 75cm2And when the culture bottle is in a full state, stopping continuously growing, enriching cells, calculating an amplification ratio, detecting cell typing by flow detection, and performing subsequent experiments such as cell killing detection or cell cryopreservation.
Fourth, identification and detection of CAR-T cells
(I) flow cytometry detection of Positive expression rates of CAR Structure
1. Detecting the positive rate of cells CD3, CD4, CD8, CCR7 and CD45RA
1) The obtained NC group cells (not infected by virus) and sample group cells (infected by virus) were gently washed 2 times with PBS + 2% BSA, and centrifuged at 1500rpm for 3 min;
2) adding 1000 mu L of PBS + 2% BSA into an NC tube, subpackaging into 5 tubes, namely NC, NC-CD3, NC-CD4, NC-CD8 and NC-CD3/4/8, adding 200 mu L of PBS into a sample tube, marking as sample-CD 3/4/8, adding 5 mu L/20 mu L of CD3/4/8 antibody, and mildly and uniformly mixing;
3) incubating at room temperature in dark for 30min, then, 1500rpm for 3min, and discarding waste liquid;
4) adding 200 μ L PBS + 2% BSA, gently mixing, re-suspending at 1500rpm/3min, and discarding the waste liquid;
5) adding 100 mu L PBS + 2% BSA, gently mixing, uniformly mixing and re-suspending, and then carrying out on-machine detection;
2. detection of Positive expression rates of CAR Structure
1) Gently washing the obtained NC group cells (not subjected to virus infection) and sample group cells (subjected to virus infection) with PBS + 2% BSA for 2 times at 1500rpm/6min, and discarding the waste liquid;
2) adding 200 mu L PBS into an NC tube, and resuspending; adding 100 mu L PBS into the sample tube, resuspending, adding 100 mu L primary antibody working solution (3 mu g/mL), and mixing;
3) incubating at room temperature for 1h at 1500rpm/3min, and discarding waste liquid;
4) adding 200 mu LPBS, gently mixing, re-suspending at 1500rpm/3min, and discarding waste liquid;
5) adding 200 mu L PBS into an NC tube, and resuspending; adding 200 mu L PBS into the sample tube, resuspending, adding 5 mu L secondary antibody working solution, and mixing uniformly;
6) incubating at room temperature in a dark place for 1h, removing waste liquor at 1500rpm/3min, mildly washing for 3 times by using PBS and 2% BSA, centrifuging at 1500rpm for 3min, and removing waste liquor;
7) add 100. mu.L PBS, mix gently, resuspend and then test on the machine (FIG. 4).
(II) RTCA real-time cell killing assay
1) Taking gastric cancer AGS cells as an example, preparing a cell suspension after digestion, blowing, uniformly mixing and counting the cells;
2) diluting the cell suspension to 5X 104cells/mL, placed on ice for use;
3) taking out the RTCA detection plate, and adding 50 mu L of culture medium;
4) selecting a test program of the test in an RTCA detector program;
5) placing the RTCA detection board into a detector, observing whether the Messege item in the program is normal, and starting an experimental program after the Messege item is normal;
6) taking out the detection plate after the program 1 is operated, adding 100 mu L of tumor cell suspension into corresponding holes, and uniformly mixing each tube of cell suspension before adding;
7) after the cell suspension is added, placing the detection plate in an incubator and standing for 30min to enable cells to naturally settle;
8) after 30min, putting the detection plate into a detector, and operating the program 2;
9) observing a cell growth curve after 24h, and preparing to add effector T cells when the cells are in a logarithmic growth phase;
10) taking out the effector T cells from the culture flask, centrifuging, cleaning, counting, and preparing effector group cell concentrations according to different effector target ratios;
11) the procedure was suspended, the plate removed, 50 μ L of effector cells added to the corresponding site, returned to the meter, and the procedure continued for daily observation (fig. 5, 6).
(III) ELISA for detecting cytokine secretion
1) By ddH2Diluting 10 times of coating solution buffer by O, preparing 250 times of coating protein according to the proportion, for example, adding 8 mu L of 250 times of coating protein into 2mL of coating solution buffer;
2) adding the coating solution prepared in the step 1) into a Corning 9018Elisa high-affinity 96-well plate by 100 mu L/well, sealing and putting the plate into a refrigerator at 4 ℃ for overnight;
3) the coated 96-well plates were washed 3 times with PBST (0.05% Tween 20);
4) by ddH2Preparing 5 Xsealing liquid by O, adding 200 mu L of sealing liquid into the hole, and sealing the liquid for 1 hour at room temperature;
5) adding 1 × sealing solution according to the requirements of bottled standard products to prepare, diluting by 7 times, and simultaneously diluting by 5 times a sample (taking supernatant of CAR-T cells in an RTCA detection experiment after killing AGS for 24 hours);
6) PBST washes the plate after closing 5 times, add the sample solution after the standard substance and dilution, incubate 2h or 4 duC overnight at room temperature;
7) PBST cleaning for 4 times;
8) diluting 250 × detection antibody with 1 × blocking solution, adding 100 μ L/well, and incubating at room temperature for 1 h;
9) PBST was washed 4 times, diluted 250 XHRP with 1 Xblocking solution, added to 100. mu.L/well and incubated at room temperature for 30 min;
10) PBST is washed for 5 times, 100 mu L of 1 XTMB reagent is added into each hole, and the mixture is incubated for 15min at room temperature;
11) adding 50 mu L/hole stop solution to stop color development;
12) detecting OD value with a microplate reader at 450 nm.
FIG. 7 shows the ELISA detection of IFN-gamma factor 24h after killing of AGS by CAR-T cells, where 5:1 is expressed as effector cells: target cells 5: 1; 2.5:1 expressed as effector cells: target cells 2.5: 1; 1.25:1 expressed as effector cells: target cells 1.25: 1; NC is indicated as blank control. After the CAR-T cells and the AGS cells are co-cultured for 24 hours, the obvious cytokine IFN-gamma is released, and effector cells: the cytokine IFN-gamma is released most when the target cells are at 5: 1.
In conclusion, the CAR vector of the targeted gastric cancer cell specificity high expression protein MSLN provided by the invention is applied to infected T cells to obtain an antibody Anti-MSLN of the CAR-T cells expressing the tumor-associated antigen MSLN on the surface of the gastric cancer cells, so that the CAR-T cells can more accurately recognize and kill the gastric cancer cells expressing the tumor-associated antigen; in addition, the CAR structure provided by the invention endows T cells with stronger proliferation and durable vitality, so that the T cells can show stronger tumor cell killing capacity.
(IV) gastric cancer PDX mouse model
1) Using a clinical gastric cancer sample to establish a PDX animal model, and preparing an experiment when an animal model tumor can be detected;
2) randomly grouping PDX animal models into a pharmacodynamic group and a control group;
3) CAR-T cell and NC-T cell injections were performed separately for each group. As shown in FIG. 8, at time D1, 2.5X 10 injections were administered to the pharmacophore6MSLN-CAR-T cells/control group injected with NC-T cells of the same total cell number.
The above description is only for the purpose of illustrating a practical embodiment of the present invention, and is not intended to limit the present invention, and those skilled in the art should also be able to make various changes, modifications, additions or substitutions within the spirit and scope of the present invention.