Dual-target CAR vector combining EpCAM and MSLN single-chain antibodies and its construction method and application in ovarian cancer

Abstract

The present invention belongs to the field of medicine and biology, and specifically relates to a dual-target CAR vector combined with EpCAM and MSLN single-chain antibodies, a construction method thereof, and an application in ovarian cancer. The dual-target CAR vector combines EpCAM single-chain antibodies and MSLN single-chain antibodies, and the specific composition structure of CAR is CD8leader-EpCAM scFv-(G4S)5-MSLN scFv-CD8α-CD28-CD137-CD3ζ. CAR-T cells (Chimeric Antigen Receptor T-Cell) containing the therapeutic vector are obtained by viral infection, and by expressing the CAR structure, ovarian cancer cells that highly express EpCAM and MSLN tumor-associated antigens can be targeted for recognition and killing.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and biology, and specifically relates to a dual-target CAR vector combining EpCAM and MSLN single-chain antibodies, a construction method thereof, and an application thereof in ovarian cancer. Background Art

[0002] Ovarian cancer is one of the common malignant tumors of female reproductive organs. Its mortality rate ranks first among female malignant tumors and is the leading cause of death among female cancer patients. Currently, the treatments for ovarian cancer include surgery, chemotherapy, traditional Chinese medicine, immunotherapy, etc. In recent years, with the continuous development of basic scientific research, immunotherapy has received more and more attention.

[0003] LAK cells, DCs, and CIK cells have all been used as immunotherapy for tumors, but all of the above treatment methods lack the ability to specifically recognize tumor associated antigens (TAA) and kill specific tumor target cells; and the process of tumor immunoediting will cause the expression of the major histocompatibility complex (MHC) on the surface of target cells to decrease on the surface of tumor cells, resulting in immune escape, allowing tumor cells to successfully evade T cell attacks.

[0004] Chimeric antigen receptor (CAR)-T cell therapy is to artificially overexpress single-chain antibody variable region gene fragments that can recognize specific tumor surface antigens on the surface of T cells through genetic engineering technology, so that T cells can recognize specific antigens and kill target cells expressing the antigen. At the same time, because CAR-T cells use antibody recognition antigen mode, they are not restricted by MHC.

[0005] Epithelial cell adhesion molecule (EpCAM) 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 has attracted much attention due to its elevated expression in a variety of tumor tissues such as ovarian cancer, gastric cancer, colorectal adenocarcinoma, and breast cancer. Studies have found that the expression of EpCAM is associated with the progression and prognosis of ovarian tumors, and can be used as a target for targeted therapy of ovarian cancer.

[0006] Mesothelin (MSLN) is an anchored glycoprotein present on the cell surface. It has become a tumor cell-specific targeting antigen that has attracted much attention because it is not expressed in normal tissues or is lowly expressed in mesothelial tissues, but is highly expressed in 30% of tumors, such as ovarian cancer, gastric cancer, colon cancer and other solid tumors.

[0007] Off-target effect is the main source of side effects of CAR-T cell therapy. Off-target effect refers to the phenomenon of failure to achieve the pre-set target and deviation. The vast majority of CAR-T cell extracellular recognition domains are tumor-associated antigens (TAA), which are not unique to tumor cells and are also expressed to varying degrees in healthy tissues. This type of CAR-T cell will attack normal tissues while killing tumor cells. This toxicity is called non-tumor targeted toxicity, and the toxic effect caused by this toxic mechanism is called off-target effect.

[0008] Therefore, how to effectively avoid and reduce the occurrence of off-target effects, enhance the killing of tumor cells by T lymphocytes, and thus improve the effect of CAR-T immunotherapy against ovarian cancer has become a technical challenge in CAR-T treatment.

[0009] At present, there is no report on a dual-target treatment plan that can target EpCAM and MSLN to reduce the occurrence of off-target effects. Summary of the invention

[0010] The purpose of the present invention is to solve the above problems. The present invention provides a CAR vector that can target EpCAM and MSLN and reduce the occurrence of off-target effects. The CAR-T technology for EpCAM and MSLN used in the present invention reduces the occurrence of off-target effects of CAR-T cell therapy, enhances the killing of tumor cells by T lymphocytes, and thus improves the effect of CAR-T immunotherapy against ovarian cancer. In addition, it also includes a method for constructing a dual-target CAR vector combined with EpCAM and MSLN single-chain antibodies and its application.

[0011] The specific technical scheme of the dual-target CAR vector combined with EpCAM and MSLN single-chain antibodies provided by the present invention is as follows:

[0012] A dual-target CAR vector combining EpCAM and MSLN single-chain antibodies comprises a chimeric antigen receptor and a vector, wherein the chimeric antigen receptor is connected to the vector, and the chimeric antigen receptor comprises two specific structures: an EpCAM single-chain antibody and an MSLN single-chain antibody, wherein the nucleotide sequence of the EpCAM single-chain antibody is shown in SEQ ID NO.2, and the nucleotide sequence of the MSLN single-chain antibody is shown in SEQ ID NO.4,

[0013] The structural composition of the chimeric antigen receptor is CD8leader-EpCAM scFv-(G4S)5-MSLN scFv-CD8α-CD28-CD137-CD3ζ.

[0014] In certain embodiments, it is characterized in that the CD8leader can express a leader peptide that guides the newly synthesized protein to transfer across the membrane, the nucleotide sequence of the CD8leader is shown in SEQ ID NO.1, the EpCAM scFv represents the EpCAM single-chain antibody, the (G4S)5 is the Inner-Linker hinge between the single-chain antibodies, and the nucleotide sequence is shown in SEQ ID NO.3, the EpCAM single-chain antibody and the MSLN single-chain antibody are connected through the (G4S)5, the MSLN scFv represents the MSLN single-chain antibody, the CD8α is a transmembrane region connecting the extracellular antigen binding domain and the intracellular signaling domain, and the nucleotide sequence is shown in SEQ ID NO.5, the CD28-CD137 is a co-stimulatory domain, the CD28 nucleotide sequence is shown in SEQ ID NO.6, the CD137 nucleotide sequence is shown in SEQ ID NO.7, and the CD3ζ is a signal transduction domain, and the nucleotide sequence is shown in SEQ ID NO.8.

[0015] In certain embodiments, the vector comprises a PUC19 plasmid or a lentiviral vector.

[0016] In certain embodiments, the lentiviral vector is pCDH-CMV-MCS-EF1-Puro.

[0017] The present invention also provides a method for constructing a dual-target CAR vector combining EpCAM and MSLN single-chain antibodies, comprising the following steps:

[0018] (1) storing the chimeric antigen receptor on the PUC19 plasmid;

[0019] (2) double-digesting the PUC19 plasmid in step (1) and the lentiviral vector, and separating the digestion products by agarose gel electrophoresis to obtain the double-digested target fragment containing the chimeric antigen receptor structure and the double-digested lentiviral vector,

[0020] (3) Connecting the target fragment containing the chimeric antigen receptor structure in step (2) and the lentiviral vector in step (2), transforming the connection product, extracting the plasmid, and obtaining a recombinant plasmid containing the chimeric antigen receptor structure, wherein the recombinant plasmid is a dual-target CAR vector combined with EpCAM and MSLN single-chain antibodies.

[0021] In certain embodiments, the double enzyme digestion sites in step (2) are EcoRI and NotI, and the molar ratio of the target fragment containing the chimeric antigen receptor structure and the lentiviral vector in step (3) is 5:1.

[0022] The present invention also provides an application of a dual-target CAR vector combined with EpCAM and MSLN single-chain antibodies, wherein the dual-target CAR vector combined with EpCAM and MSLN single-chain antibodies is packaged with a lentivirus, and a high-titer lentivirus suspension is obtained after centrifugation and concentration. The lentivirus suspension is used to infect T cells to obtain CAR-T cells, and the CAR-T cells target and kill ovarian cancer cells by recognizing specific proteins EpCAM and MSLN.

[0023] In certain embodiments, it is characterized in that the lentivirus packaging adopts a three-plasmid packaging system, and the three-plasmid packaging system includes a PSPAX2 plasmid, a pMD2G plasmid and a dual-target CAR vector combined with EpCAM and MSLN single-chain antibodies, the ratio of the PSPAX2 plasmid, the pMD2G plasmid, and the dual-target CAR vector combined with EpCAM and MSLN single-chain antibodies is 27:3:20, and the lentivirus packaging adopts 293T cells as lentivirus packaging cells.

[0024] The present invention has the following beneficial effects: the CAR structure for the therapeutic carrier of the present invention includes two specific structures, EpCAM single-chain antibody and MSLN single-chain antibody; EpCAM single-chain antibody and MSLN single-chain antibody are specific structures determined based on the expression of tumor-associated antigens EpCAM and MSLN on the surface of various ovarian cancer cells. The two specific antibodies Anti-EpCAM and Anti-MSLN expressed by the structure are responsible for identifying TAA, which can target and kill tumor cells on the one hand; on the other hand, it gives T cells new antigen specificity, which can effectively avoid the immune escape mechanism of downregulation of MHC expression of tumor cells. In addition, the design of dual targets can effectively reduce the off-target effects produced during CAR-T treatment, so that CAR-T cells can better identify ovarian cancer cells with dual positive targets, improve the killing efficiency, and reduce the risks brought by CAR-T treatment. When T cells obtain the vector and express the CAR structure through viral infection, T cells can target and kill ovarian cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the structure of the lentiviral expression vector pCDH-CMV-MCS-EF1-Puro of the present invention;

[0026] Figure 2 is a schematic diagram of the CAR structure of the present invention;

[0027] Figure 3 It is an agarose gel electrophoresis diagram of the double digestion product of EcoRI and NotI of the lentiviral expression vector pCDH-CMV-MCS-EF1-Puro of the present invention;

[0028] Figure 4 This is an agarose gel electrophoresis diagram of the EcoRI and NotI double-digestion products of the dual-target CAR vector of the present invention combining EpCAM and MSLN single-chain antibodies;

[0029] Figure 5 is a flow cytometry detection diagram of the CAR-T cells of the present invention;

[0030] Figure 6 It is a curve diagram of the killing efficiency of SK-OV-3 detected by RTCA of the present invention by CAR-T cells;

[0031] Figure 7 It is a bar graph of ELISA detection of IFNgamma factor after 24 hours of killing SK-OV-3 by CAR-T cells of the present invention. DETAILED DESCRIPTION

[0032] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0033] The present invention provides a dual-target CAR vector combining EpCAM and MSLN single-chain antibodies, and the specific scheme is as follows: comprising a chimeric antigen receptor and a vector, the chimeric antigen receptor is connected to the vector, and the chimeric antigen receptor comprises two specific structures: EpCAM single-chain antibody and MSLN single-chain antibody, the nucleotide sequence of EpCAM single-chain antibody is shown in SEQ ID NO.2, and the nucleotide sequence of MSLN single-chain antibody is shown in SEQ ID NO.4. The structure of the chimeric antigen receptor is CD8leader-EpCAM scFv-(G4S)5-MSLN scFv-CD8α-CD28-CD137-CD3ζ.

[0034] The structure of the chimeric antigen receptor is CD8leader-EpCAM scFv-(G4S)5-MSLN scFv-CD8α-CD28-CD137-CD3ζ (such as Figure 2). CD8leader can express a leader peptide that guides newly synthesized proteins to transfer across the membrane. The nucleotide sequence of CD8leader is shown in SEQ ID NO.1. EpCAM scFv represents EpCAM single-chain antibody. (G4S)5 is an inner-linker hinge between single-chain antibodies, and the nucleotide sequence is shown in SEQ ID NO.3. EpCAM single-chain antibody and MSLN single-chain antibody are connected through (G4S)5. MSLN scFv represents MSLN single-chain antibody. CD8α is a transmembrane region that connects the extracellular antigen binding domain and the intracellular signaling domain, and the nucleotide sequence is shown in SEQ ID NO.5. CD28-CD137 is a co-stimulatory domain. The nucleotide sequence of CD28 is shown in SEQ ID NO.6. The nucleotide sequence of CD137 is shown in SEQ ID NO.7. CD3ζ is a signal transduction domain, and the nucleotide sequence is shown in SEQ ID NO.8. Antibodies Anti-EpCAM and Anti-MSLN that express tumor-associated antigens EpCAM and MSLN on the surface of ovarian cancer cells enable them to more accurately identify and kill ovarian cancer cells that express both tumor-associated antigens, greatly increasing the targeting of CAR-T cells and reducing off-target effects. The CAR structure mentioned in the present invention gives T cells stronger proliferation and lasting vitality, allowing them to exhibit stronger tumor cell killing ability.

[0035] The vector includes a PUC19 plasmid and a lentiviral vector. The lentiviral vector is pCDH-CMV-MCS-EF1-Puro. By analyzing the vector with Snap Gene software and searching related literature, it is known that the insertion fragment of pCDH-CMV-MCS-EF1-Puro is double-digested with EcoRI and NotI. The expression vector includes: CMV promoter, which is a mammalian cell-specific promoter with strong driving ability; multiple cloning site (MCS), which contains multiple restriction enzyme cutting sites and is the position for inserting foreign genes; WPRE element, which can improve the polyA tailing efficiency of mRNA and improve the expression efficiency of transferred genes; SV40 polyA sequence, which can effectively terminate transcription and add PolyA tail to transcribed mRNA; hybrid RSV / 5'LTR, which 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), which are used for packaging, transduction and stably integrating viral expression structures into the host's genomic DNA; SV40 origin, which enables plasmids to stably proliferate in packaging cells.

[0036] This lentiviral expression vector can be used as the most effective vector to express the target gene in almost all mammalian cells, including non-dividing cells and dividing cells. It can accommodate large exogenous gene fragments, has a high transfection efficiency, and can also achieve satisfactory transfection effects on T cells.

[0037] 1. Experimental Materials

[0038] 1. Lentiviral expression plasmid pCDH-CMV-MCS-EF1-Puro, lentiviral packaging plasmid pMD2G, and vector plasmid PSPAX2 were purchased from SBI; the structure of lentiviral expression plasmid pCDH-CMV-MCS-EF1-Puro is as follows Figure 1 As shown;

[0039] 2. The CAR structure sequence was designed by Shanghai Beautiful Life Medical Technology Co., Ltd., synthesized by Sangon Biotech (Shanghai) Co., Ltd., and stored in the form of PUC19 plasmid;

[0040] 3. Restriction endonucleases EcoRI and NotI were purchased from NEB;

[0041] 4.T4 DNA ligase and Free H2O were purchased from Bao Biotechnology;

[0042] 5. Competent cells were purchased from Trans;

[0043] 6. Gel recovery kit and plasmid extraction kit were purchased from Tiangen Biochemical Technology Co., Ltd.;

[0044] 7.293T cells and SK-OV-3 cells were purchased from the Cell Bank of the Chinese Academy of Sciences;

[0045] 8. FBS, DMEM, 1640 medium, PBS, Opti-MEM, lipofectamine 2000 were purchased from Gibco;

[0046] 8. FBS, DMEM, 1640 medium, PBS, Opti-MEM, and lipofectamine 2000 were purchased from Gibco;

[0047] 9. CD3 monoclonal antibody, CD28 monoclonal antibody, CH38 protein, and IL-2 were purchased from Shanghai Jinan Protein Technology Co., Ltd.;

[0048] 10.Multiskan GO microplate reader + uDrop ultra-microplate and flow cytometer were purchased from ThermoFisher;

[0049] 11.HE120 horizontal electrophoresis tank and Tanon gel imager were purchased from Tanon;

[0050] 12. Water-proof constant temperature incubator and constant temperature incubator shaker were purchased from Shanghai Yiheng Technology Co., Ltd.;

[0051] 13. Bio-Rad Mini-PROTEAN Tetra Cell mini electrophoresis system was purchased from Bio-Rad;

[0052] 14.Olympus microscope was purchased from Olympus;

[0053] 15. Inoculation loop and coating rod were purchased from Clean Biofiltration Co., Ltd.;

[0054] 16. Syringes, 0.45 μm filter membranes, culture dishes of various specifications, culture bottles, multi-well culture plates, and centrifuge tubes of various specifications were purchased from Corning.

[0055] 2. Construction method of dual-target CAR vector combining EpCAM and MSLN single-chain antibodies

[0056] (I) Plasmid extraction

[0057] Preparation method of LB liquid culture medium: weigh 5g of liquid culture medium powder into a 500mL conical flask using an electronic balance, add 100mL of ultrapure water, seal with tin foil, and sterilize in a high-pressure steam sterilizer. When cooled to 40℃-50℃, add 0.2% ampicillin (AMP) at a ratio of 1000:1, mix carefully, and transfer to a clean 500mL reagent bottle for use. The storage condition is 4℃.

[0058] Preparation method of LB solid culture medium: weigh 5g of solid culture medium powder into a 500mL conical flask using an electronic balance, add 100mL of ultrapure water, seal with tin foil, and sterilize in a high-pressure steam sterilizer. When cooled to 40℃-50℃, add 0.2% ampicillin at a ratio of 1000:1, mix carefully, pour into a plate, and after solidification, cover with sealing film and store at 4℃.

[0059] 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 12-16 hours at 37°C and 250 rpm.

[0060] Plasmid extraction was performed according to the instructions of the plasmid extraction kit (Cat. No.: DP103-03) purchased from Tiangen Biochemical Technology Co., Ltd. to obtain the lentiviral expression plasmid pCDH-CMV-MCS-EF1-Puro and the PUC19 plasmid containing the dual-target (EpCAM single-chain antibody and MSLN single-chain antibody) CAR structure.

[0061] The extracted lentiviral expression plasmid pCDH-CMV-MCS-EF1-Puro and the PUC19 plasmid containing the dual-target (E EpCAM single-chain antibody and MSLN single-chain antibody) CAR structure were simultaneously digested with EcoRI and NotI, and the digestion products were subjected to agarose gel electrophoresis, and the results were observed and recorded using a gel imager. The test results are shown in the attached Figure 3 .

[0062] The target band was recovered according to the instructions of Tiangen Agarose Gel Recovery Kit (Cat. No.: DP209-02), and the recovered fragments were connected according to the molar ratio of CAR structure: pCDH-CMV-MCS-EF1-Puro = 5:1, and transformed into competent cells. The transformed competent cells were dropped onto the preheated solid culture medium, marked, and incubated at 37°C overnight.

[0063] (III) Plasmid extraction, enzyme digestion verification, and sequencing

[0064] Pick some colonies in 5mL LB liquid medium (AMP resistance), culture them in a constant temperature shaker and extract plasmids. Before plasmid extraction, take out 500μL in a 1.5mL Eppendorf tube for strain preservation and store at -80℃. Use EcoRI and NotI double enzyme digestion to verify the extraction product, and take 1μg of plasmid with correct bands and send it to Shenggong Biotechnology (Shanghai) Co., Ltd. for sequencing (such as Figure 4 As shown in the figure, the target fragment is the PUC19 plasmid containing the CAR structure), and the plasmid with abnormal bands and the retained bacterial solution are discarded. The plasmid with the correct sequencing result is extracted, and the plasmid with the wrong sequencing result and its bacterial solution are discarded.

[0065] 3. Application of dual-target CAR vectors combining EpCAM and MSLN single-chain antibodies

[0066] (I) Preparation of concentrated virus solution

[0067] A three-plasmid packaging system was used for lentiviral packaging. The three plasmids were the lentiviral expression plasmid pCDH-CMV-MCS-EF1-Puro containing the CAR structure, the lentiviral packaging plasmid pMD2G, and the vector plasmid PSPAX2. The cells were 293T cells.

[0068] The specific implementation steps are as follows:

[0069] (1) Plate the cells within 24 hours before transfection: Generally, cells with a passage number of less than 3 generations are selected, and the cell density is adjusted according to the cell growth density and state. 293T cells with a growth density of 80% are plated;

[0070] (2) When the growth density reaches 60-90% and the cells are in good condition, virus packaging can be performed;

[0071] (3) Virus packaging was performed according to the ratio of PSPAX2 plasmid, pMD2G plasmid, and the dual-target CAR vector (recombinant plasmid) combined with EpCAM and MSLN single-chain antibody at 27:3:20. Taking a 6 cm dish as an example, the required three-plasmid mixture ratio is as follows: The amount of plasmid added is determined according to the concentration of each plasmid.

[0072] Recombinant plasmid 3 μg

[0073] pMD2G 0.5 μg

[0074] PSPAX2 4 μg

[0075] (4) The transfection reagent selected was lipofectamine 2000 (stored at 4°C) and the added amount was 2 μL / μg plasmid;

[0076] (5) Mix the plasmid mixture in (3) and the transfection reagent mixture in (4) in one tube, let it stand at room temperature for 20 min, then add the replaced cells and continue culturing;

[0077] (6) Collect the culture supernatant after 48 h and 72 h, respectively, and filter through a 0.45 μm filter membrane;

[0078] (7) The collected virus solution was concentrated using the PEG8000 concentration method, the virus titer was determined, and the solution was stored at -80°C for future use.

[0079] (II) Virus fluid infects T cells

[0080] 1. PBMC Isolation

[0081] 1) Collect about 6 mL of fresh human peripheral blood using a vacuum blood collection tube containing heparin;

[0082] 2) Dilution: Add an equal volume of PBS at room temperature and mix gently by pipetting;

[0083] 3) Sample addition: Take a 50mL centrifuge tube, draw 6mL Ficoll (lymphocyte separation solution) into the centrifuge tube (the volume ratio of Ficoll to blood before dilution is 1:1), tilt the centrifuge tube at 45°, and slowly add the diluted blood onto the Ficoll along the tube wall about 1cm above the Ficoll liquid surface;

[0084] 4) Centrifugation: 18-20°C, 2000rpm, 30min. 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;

[0085] 5) Recovery: Insert the pipette directly into the cloud layer (or first suck out the upper plasma), gently suck out the cloud layer, and put it into a new centrifuge tube;

[0086] 6) Washing: add PBS at least 3 times the volume of PBMC (peripheral blood mononuclear cells), 18-20°C, 2000 rpm, 10 min, twice;

[0087] 7) Cell counting: discard the supernatant, add 1 mL of lymphocyte culture medium, mix by pipetting, and prepare 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).

[0088] 2. T cell activation and lentiviral infection

[0089] (1) Preparation before the experiment:

[0090] 1) Preparation of Anti-CD3 Monoclonal Antibody Solution: Dissolve 50 μg of CD3 Monoclonal Antibody in 5 mL of PBS solution to prepare a solution with a concentration of 10 μg / mL. After dissolution, dispense 400 μL per EP tube and store in a -80°C refrigerator. (At this concentration, add 175 μL of antibody solution to each 24-well plate);

[0091] 2) Preparation of Anti-CD28 Monoclonal Antibody Solution: Dissolve 50 μg of CD28 Monoclonal Antibody in 5 mL of PBS solution to prepare a solution with a concentration of 10 μg / mL. After dissolution, dispense 400 μL per EP tube and store in a -80°C refrigerator. (At this concentration, add 175 μL of antibody solution to each 24-well plate);

[0092] 3) Preparation of CH-38 protein solution: 500 μg of CH38 protein was dissolved in 10 mL of PBS solution to prepare a 50 μg / mL solution. After dissolution, 400 μL of the solution was dispensed into each EP tube and stored in a -80°C refrigerator. (At this concentration, 175 μL of antibody solution was added to each 24-well plate);

[0093] 4) IL-2 factor preparation: IL-2 protein solid was prepared at 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 32 μL to each EP, and store in a -80℃ refrigerator;

[0094] 5) Preparation of lymphocyte culture medium: Add 30 μL of the pre-packaged IL-2 solution, 0.5 mL of dual antibody, and 250 μL of autologous serum to every 50 mL of Takara-551h3 lymphocyte culture medium.

[0095] (2) Experimental process:

[0096] Day-1: 24-well plate coating: Take a Corning 24-well plate, take 2 wells as an example, add 175μL of CD3 monoclonal antibody solution, 175μL of CD28 monoclonal antibody solution, and 175μL of 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;

[0097] Day 0: Cleaning the coated plate: Take out the 24-well plate coated yesterday, discard the supernatant, wash twice with PBS, and add PBS for standby;

[0098] PBMC plating: Collect PBMC cells, count and adjust the final concentration to 0.7 × 10 6 cells / mL, 400 μL of cell suspension was added to each well, that is, 2.8×10 5 cells;

[0099] Virus infection: Infect with MOI=30, prepare 1 mL of virus culture medium suspension, add to 24-well plate, centrifuge at 1000 g for 30 min, and adjust the centrifuge temperature to 32 °C;

[0100] Day1-Day2: Observe cell status;

[0101] Day 3: Transfer all cells in the 24-well plate to a 25 cm 2 In the culture flask, observe the cell status;

[0102] Day 4-Day 7: Observe the cell status and cell number. If the cells begin to proliferate significantly and the cell density in a local area is high, add 10 mL of culture medium.

[0103] Day 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;

[0104] Day 9-Day 10: 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, detect cell typing by flow cytometry, and perform subsequent experiments such as cell killing detection or cell freezing.

[0105] 4. Identification and Detection of CAR-T Cells

[0106] (I) Detection of CAR structure positive expression rate by flow cytometry

[0107] 1. Detect the positive rate of CD3, CD4 and CD8 cells

[0108] 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, and the centrifugation condition was 1500 rpm for 3 min;

[0109] 2) Add 1000 μL PBS + 2% BSA to the NC tube and divide into 5 tubes, namely NC, NC-CD3, NC-CD4, NC-CD8, and NC-CD3 / 4 / 8. Add 200 μL PBS to the sample tube, marked as sample-CD3 / 4 / 8, and add 5 μL / 20 μL / 20 μL of CD3 / 4 / 8 antibody respectively, and mix gently;

[0110] 3) After incubation at room temperature in the dark for 30 min, spin at 1500 rpm for 3 min, and discard the waste liquid;

[0111] 4) Add 200 μL PBS + 2% BSA, gently mix and resuspend, 1500 rpm / 3 min, and discard the waste liquid;

[0112] 5) Add 100 μL PBS + 2% BSA, mix gently and resuspend, then it can be tested on the instrument;

[0113] like Figure 5 As shown, CD3 + The cells accounted for 98.18% of the total cells detected, and CD4 - CD8 + CD3 + 60.40% of the total cells, CD4 + CD8 - CD3 + 32.56% of the total cells, consistent with the phenotype of T cells.

[0114] 2. Detection of CAR structure positive expression rate

[0115] 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 at 1500 rpm / 6 min, and the waste liquid was discarded;

[0116] 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;

[0117] 3) Incubate at room temperature for 1 h, 1500 rpm / 3 min, and discard the waste liquid;

[0118] 4) Add 200 μL PBS, gently mix and resuspend, 1500 rpm / 3 min, and discard the waste liquid;

[0119] 5) Add 200 μL PBS to the NC tube and resuspend; add 200 μL PBS to the sample tube and resuspend, then add 5 μL secondary antibody working solution and mix well;

[0120] 6) After incubation at room temperature in the dark for 1 hour, centrifuge at 1500 rpm / 3 min, discard the waste liquid, wash gently with PBS + 2% BSA 3 times, centrifuge at 1500 rpm, 3 min, and discard the waste liquid;

[0121] 7) Add 100 μL PBS, gently mix and resuspend, and then test on the instrument;

[0122] like Figure 5 As shown, CAR-T cells accounted for 49.58% of the total T cells

[0123] (II) RTCA real-time cell killing detection

[0124] 1) Taking ovarian cancer cell SK-OV-3 as an example, prepare a cell suspension after digestion, mix by pipetting, and then count the cells;

[0125] 2) Dilute the cell suspension to 5×10 4 cells / mL concentration, placed on ice for later use;

[0126] 3) Take out the RTCA test plate and add 50 μL of culture medium;

[0127] 4) Select the test procedure for this test in the RTCA detector program;

[0128] 5) 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;

[0129] 6) After program 1 is completed, remove the test plate and add 100 μL of tumor cell suspension to the corresponding wells. Mix each tube of cell suspension before adding;

[0130] 7) After adding the cell suspension, place the test plate in an incubator and let it stand for 30 minutes to allow the cells to settle naturally;

[0131] 8) After 30 minutes, place the test plate into the tester and run program 2;

[0132] 9) Observe the cell growth curve after 24 hours, and prepare to add effector T cells when the cells are in the logarithmic growth phase;

[0133] 10) The effector T cells were taken out from the culture flask, centrifuged, washed, counted, and the concentration of effector group cells was prepared according to different effector-target ratios;

[0134] 11) 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.

[0135] Figure 6 RTCA detection of the killing efficiency of dual-target (EpCAM single-chain antibody and MSLN single-chain antibody) CAR-T cells on SK-OV-3 cells. In the figure, 5:1 means: effector cell: target cell = 5:1; 2.5:1 means: effector cell: target cell = 2.5:1; 1.25:1 means: effector cell: target cell = 1.25:1; NCT5:1 means: effector cell is uninfected T cell, effector cell: target cell = 5:1; NCT2.5:1 means: effector cell is uninfected T cell, effector cell: target cell = 2.5:1; NCT1.25:1 means: effector cell is uninfected T cell, effector cell: target cell = 1.25:1. As shown in the figure, the killing effect of CAR-T cells is significantly stronger than that of uninfected T cells, and its killing effect is concentration-dependent. The higher the proportion of effector cells, the better the killing effect.

[0136] (III) ELISA detection of cytokine secretion

[0137] 1) Dilute 10× coating buffer with ddH2O and prepare 250× coating protein according to the proportion, for example, add 8μL 250× coating protein to 2mL coating buffer;

[0138] 2) Add 100 μL / well of the coating solution prepared in 1) to the Corning 9018 Elisa high affinity 96-well plate, seal and place in a 4°C refrigerator overnight;

[0139] 3) Wash the coated 96-well plate with PBST (0.05% Tween 20) three times;

[0140] 4) Prepare 5× blocking solution with ddH2O, add 200 μL / well, and block at room temperature for 1 hour;

[0141] 5) Add 1× blocking solution according to the requirements of the bottled standard, perform 7 times of dilution, and at the same time, dilute the sample (the supernatant after 24 hours of killing SK-OV-3 by CAR-T cells in the RTCA detection experiment) 5 times;

[0142] 6) Wash the blocked plate 5 times with PBST, add the standard and diluted sample solution, and incubate at room temperature for 2 hours or at 4°C overnight;

[0143] 7) Wash with PBST 4 times;

[0144] 8) Dilute 250× detection antibody with 1× blocking solution, add 100 μL / well, and incubate at room temperature for 1 hour;

[0145] 9) Wash 4 times with PBST, dilute 250×HRP with 1× blocking solution, add 100 μL / well, and incubate at room temperature for 30 min;

[0146] 10) Wash with PBST five times, add 100 μL of 1× TMB reagent to each well, and incubate at room temperature for 15 min;

[0147] 11) Add 50 μL / well stop solution to stop color development;

[0148] 12) Detect OD value with an enzyme reader at 450nm.

[0149] Figure 7 This is the ELISA test of IFNgamma factor after CAR-T cells killed SK-OV-3 for 24 hours. In the figure, 5:1 means: effector cell: target cell = 5:1; 2.5:1 means: effector cell: target cell = 2.5:1; 1.25:1 means: effector cell: target cell = 1.25:1. After CAR-T cells and SK-OV-3 cells were co-cultured for 24 hours, there was obvious release of cytokine IFNgamma. When the effector cell: target cell ratio was 5:1, the cytokine IFNgamma was released the most.

[0150] In summary, the dual-target CAR vector combined with EpCAM and MSLN single-chain antibodies provided by the present invention is used to infect T cells, and the obtained CAR-T cells can simultaneously express antibodies Anti-EpCAM and Anti-MSLN against the tumor-associated antigens EpCAM and MSLN on the surface of ovarian cancer cells, so that it can more accurately identify and kill ovarian cancer cells that simultaneously express the above two tumor-associated antigens, greatly increasing the targeting of CAR-T cells and reducing off-target effects; in addition, the CAR structure mentioned in the present invention gives T cells stronger proliferation and lasting vitality, enabling them to exhibit stronger tumor cell killing ability.

[0151] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. The present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by technicians in this technical field within the essential scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. Application of a dual-target CAR vector combined with EpCAM and MSLN single-chain antibodies in the preparation of a drug for treating ovarian cancer, characterized in that: comprising a chimeric antigen receptor and a carrier, wherein the chimeric antigen receptor is connected to the carrier, The chimeric antigen receptor includes two specific structures, EpCAM single-chain antibody and MSLN single-chain antibody. The nucleotide sequence of the EpCAM single-chain antibody is shown in SEQ ID NO.2, and the nucleotide sequence of the MSLN single-chain antibody is shown in SEQ ID NO.

4. The chimeric antigen receptor structure consists of CD8leader-EpCAM scFv-(G4S) 5-MSLNscFv-CD8α-CD28-CD137-CD3ζ; The CD8leader expresses a leader peptide that guides newly synthesized proteins to transfer across the membrane. The nucleotide sequence of the CD8leader is shown in SEQ ID NO.

1. The EpCAM scFv represents a single-chain antibody of EpCAM, The (G4S) 5 is the inner-linker of the single-chain antibody, and the nucleotide sequence is shown in SEQ ID NO.

3. The EpCAM single-chain antibody and the MSLN single-chain antibody are connected via the (G4S)5. The MSLN scFv represents a single-chain antibody of MSLN, The CD8α is a transmembrane region connecting the extracellular antigen binding domain and the intracellular signaling domain, and the nucleotide sequence is shown in SEQ ID NO.

5. The CD28-CD137 is a co-stimulatory domain, the CD28 nucleotide sequence is shown in SEQ ID NO.6, the CD137 nucleotide sequence is shown in SEQ ID NO.7, The CD3ζ is a signal transduction domain, and the nucleotide sequence is shown in SEQ ID NO.8; The vector is selected from a PUC19 plasmid and a lentiviral vector; The lentiviral vector is pCDH-CMV-MCS-EF1-Puro; The dual-target CAR vector combined with EpCAM and MSLN single-chain antibodies was packaged with lentivirus, and a high-titer lentivirus suspension was obtained after centrifugation and concentration. The lentivirus suspension was used to infect T cells to obtain CAR-T cells.

2. The use according to claim 1, characterized in that: The lentiviral packaging adopts a three-plasmid packaging system, which includes PSPAX2 plasmid, pMD2G plasmid and the dual-target CAR vector combined with EpCAM and MSLN single-chain antibodies. The ratio of the PSPAX2 plasmid, the pMD2G plasmid and the dual-target CAR vector combined with EpCAM and MSLN single-chain antibodies is 27:3:

20. The lentiviral packaging adopts 293T cells as lentiviral packaging cells.

3. The use according to claim 1, characterized in that: The method for constructing a dual-target CAR vector combining EpCAM and MSLN single-chain antibodies comprises the following steps: (1) storing the chimeric antigen receptor on the PUC19 plasmid; (2) double-digesting the PUC19 plasmid in step (1) and the lentiviral vector, and separating the digestion products by agarose gel electrophoresis to obtain the double-digested target fragment containing the chimeric antigen receptor structure and the double-digested lentiviral vector, (3) Connecting the target fragment in step (2) and the double-enzyme-cut lentiviral vector in step (2), transforming the connection product, extracting the plasmid, and obtaining a recombinant plasmid containing a chimeric antigen receptor structure, wherein the recombinant plasmid is a dual-target CAR vector combining EpCAM and MSLN single-chain antibodies.

4. The use according to claim 3, characterized in that: The double restriction enzyme sites in step (2) are EcoRI and NotI, and the molar ratio of the target fragment containing the chimeric antigen receptor structure and the double restriction enzyme-cut lentiviral vector in step (3) is 5:1.

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