Carrying targeting claudin 16 which is specifically highly expressed in ovarian cancer cells and construction method and application thereof

Abstract

The application discloses a CAR vector targeting Claudin 16 which is specifically highly expressed in ovarian cancer cells, a construction method and application thereof, wherein the CAR vector comprises a chimeric antigen receptor and a carrier, the chimeric antigen receptor is connected to the carrier, the chimeric antigen receptor comprises a specific structure of Claudin 16 single-chain antibody, and the specific structure of the Claudin 16 single-chain antibody is composed of VH, VL and a flexible Linker connecting the VH and the VL. The structure of the chimeric antigen receptor is SP-Claudin 16scFv-TM-CD28-4-1BB-CD3zeta. The CAR-T cell with the carrier is obtained through a virus infection mode, and the CAR-T cell can recognize and target killing the ovarian cancer cells with high expression of the tumor-related antigen Claudin 16 through expression of the CAR structure.

Description

Technical Field

[0001] This invention belongs to the field of biomedical immune cell technology, specifically relating to a CAR vector that targets Claudin 16, a protein specifically highly expressed in ovarian cancer cells, its construction method, and its application. Background Technology

[0002] Ovarian cancer is a common malignant tumor in women, ranking third in incidence among female reproductive system malignancies, but with the highest mortality rate. Due to its insidious onset and lack of early symptoms, 70% of patients are diagnosed at an advanced stage, missing the opportunity for radical surgical treatment. Therefore, ovarian cancer is also known as the "silent killer." For decades, clinical treatment of ovarian cancer in my country has primarily relied on the three conventional therapies. 70% of patients relapse within 1-2 years after initial treatment, and some even experience multiple relapses. The overall 5-year survival rate has hovered around 30%, showing no significant improvement. In recent years, with the rise of immunotherapy, various immunotherapies have been applied to ovarian cancer, striving to control the progression of this "silent killer" and change the poor clinical treatment landscape for ovarian cancer. For more than half a century, cancer treatment has largely depended on radiotherapy, surgery, chemotherapy, and drugs targeting specific mutations in the tumor genome (which are increasingly being used). While these conventional therapies are effective for many patients, there is still an urgent need to develop new therapies. Utilizing the specific ability of the immune system to identify and destroy tumor cells is a new method for fighting cancer: immunotherapy. Ovarian cancer is a typical immunosuppressive tumor, so scientists believe that ovarian cancer patients can benefit from immunotherapy.

[0003] Chimeric antigen receptor (CAR) T-cell therapy is a type of cancer immunotherapy. This type of therapy involves extracting the patient's own T cells, equipping them with a receptor that targets cancer cells, mass-producing chimeric antigen receptor T cells in specialized equipment, and then injecting them into the patient to destroy tumor cells. Due to the high sensitivity of CAR-T therapy, its targeted / off-target toxicity can lead to more severe side effects compared to traditional antibody drugs. Therefore, the selection of targets for CAR-T therapy cannot follow the same criteria as traditional antibody therapy; instead, it requires finding more stringent tumor-specific antigens as targets.

[0004] Therefore, this invention is the first to use Claudin 16 as a target for CAR-T therapy. The CAR vector that targets Claudin 16 provided by this invention can both recognize ovarian cancer cells that are positive for Claudin 16 and enhance the killing ability of T lymphocytes, thereby improving the effect of CAR-T immunotherapy against ovarian cancer. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a specific CAR vector targeting Claudin 16, its construction method, and its applications. The CAR vector targeting Claudin 16 provided by this invention can both recognize Claudin 16-positive ovarian cancer cells and enhance the killing ability of T lymphocytes, thereby improving the efficacy of CAR-T immunotherapy against ovarian cancer.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0007] The first objective of this invention is to provide a CAR vector that targets Claudin 16, a protein specifically highly expressed in ovarian cancer cells, and which contains a gene encoding a chimeric antigen receptor.

[0008] The chimeric antigen receptor is used to target Caudin 16, a protein specifically highly expressed in ovarian cancer cells.

[0009] The structure of the chimeric antigen receptor is SP-Claudin 16 scFv-TM-CD28-4-1BB-CD3ζ, where SP is a signal peptide that guides the transmembrane transfer of newly synthesized proteins, Claudin 16scFv is a single-chain antibody that specifically recognizes Claudin 16 on the cell surface, TM is a transmembrane domain that connects the extracellular antigen-binding domain and the intracellular signaling domain, CD28-4-1BB is a co-stimulatory signaling molecule, and CD3ζ is a cytoplasmic signal transduction domain.

[0010] Preferably, the Claudin 16scFv includes a heavy chain variable region VH, a light chain variable region VL, and a flexible linker strand connecting the heavy chain variable region VH and the light chain variable region VL. The nucleotide sequence of the heavy chain variable region VH is shown in SEQ ID NO.2, the nucleotide sequence of the light chain variable region VL is shown in SEQ ID NO.3, and the nucleotide sequence of the flexible linker strand is shown in SEQ ID NO.4.

[0011] Preferably, the recombinant vector is a recombinant vector containing a gene encoding a chimeric antigen receptor, a lentiviral packaging plasmid, and an envelope plasmid.

[0012] Preferably, the recombinant vector is GV230-Claudin 16scFv.

[0013] A second object of the present invention is to provide a host cell containing, as described above, a vector or chromosome in which exogenous nucleic acid molecules as described above are integrated.

[0014] A third objective of this invention is to provide a method for obtaining CAR-T cells that specifically overexpress the protein Claudin 16 in ovarian cancer cells. The method involves packaging the recombinant vector as described above with lentivirus to obtain a high-titer lentivirus, and then infecting T cells with the lentivirus to obtain chimeric antigen receptor-T cells.

[0015] Preferably, the lentivirus packaging uses 293T cells as the packaging cells and employs a three-plasmid packaging system, which includes a PSPAX2 plasmid, a pMD2G plasmid, and a recombinant vector, with a mass ratio of 5:3:3 for the PSPAX2 plasmid, pMD2G plasmid, and recombinant vector.

[0016] A fourth object of the present invention is to provide the use of the chimeric antigen receptor, the nucleic acid molecule, the carrier, or the cell as described above for the preparation of a medicament or preparation for the treatment of ovarian cancer.

[0017] A fifth objective of this invention is to provide a method for constructing a CAR vector that targets Claudin 16, a protein specifically highly expressed in ovarian cancer cells.

[0018] The steps are as follows:

[0019] (1) The chimeric antigen receptor was stored on a PEE6.4 plasmid;

[0020] (2) The PEE6.4 plasmid and lentiviral vector contained in step (1) were double-digested. The digestion products were separated by agarose gel electrophoresis to obtain the target fragment containing the chimeric antigen receptor and the double-digested lentiviral vector.

[0021] (3) The target fragment containing the chimeric antigen receptor in step (2) is ligated with the lentiviral vector in step (2), the ligation product is transformed, the plasmid is extracted, and a recombinant plasmid containing the chimeric antigen receptor structure is obtained. The recombinant plasmid is a CAR vector that targets the ovarian cancer cell-specific high-expression protein Claudin 16.

[0022] Preferably, the double enzyme digestion in step (2) is XoHI and BamHI, and the mass ratio of the target fragment containing the chimeric antigen receptor and the lentiviral vector conjugated in step (3) is 10:1.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This invention provides the structure and construction method of a CAR vector that targets the ovarian cancer cell-specific high-expression protein Claudin 16, and its structure contains a unique claudin 16 single-chain antibody;

[0025] The Claudin 16 single-chain antibody is a specific structure determined based on the high expression of Claudin 16 in ovarian cancer tissues. On the one hand, it can target and bind to ovarian cancer cells; on the other hand, it endows T cells with new antigen specificity, effectively avoiding the immune escape mechanism of tumor cell MHC downregulation. When T cells obtain this vector through viral infection and express this CAR structure, they can target and kill ovarian cancer cells. This invention is the first to use Claudin 16 as a target for CAR-T therapy. The CAR vector targeting Claudin 16 provided by this invention can both recognize Claudin 16-positive ovarian cancer cells and enhance the killing ability of T lymphocytes, thereby improving the efficacy of CAR-T immunotherapy against ovarian cancer. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the structure of the lentiviral expression vector GV230 of the present invention;

[0027] Figure 2 This is a schematic diagram of the CAR structure of the present invention;

[0028] Figure 3 Electrophoresis diagram of nucleic acid for identification of lentiviral recombinant vector by double digestion with XoHI and BamHI;

[0029] Figure 4 The graph shows the flow cytometry results of the positive rate of Claudin 16-CAR-T cells;

[0030] Figure 5 A schematic diagram illustrating the high expression of Claudin 16 in the ovarian cancer cell line OVCA-R3;

[0031] Figure 6 The image shows the killing effect of Claudin 16-CAR-T cells on the ovarian cancer cell line OVCA-R3.

[0032] Figure 7 ELISA detection of IFN-gamma factor in CAR-T cells 24 h after OVCAR3 killing. Detailed Implementation

[0033] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0034] This invention provides a CAR vector targeting Claudin 16, a protein specifically highly expressed in ovarian cancer cells. The specific scheme is as follows: it includes a chimeric antigen receptor and a vector. The chimeric antigen receptor is linked to the vector and includes a Claudin 16 single-chain antibody. The Claudin 16 single-chain antibody is composed of nucleotide sequences as shown in SEQ ID NO.2 and SEQ ID NO.3, and a flexible linker as shown in SEQ ID NO.4. The antibody Anti-claudin 16, which expresses the tumor-associated antigen Claudin 16 on the surface of ovarian cancer cells, is responsible for recognizing the highly expressed Claudin 16 in ovarian cancer tissue, greatly increasing the targeting of CAR-T cells and reducing tumor immune escape.

[0035] The structural composition of the chimeric antigen receptor is SP-Claudin 16 scFv-TM-CD28-4-1BB-CD3ζ (e.g., Figure 2 (As shown).

[0036] SP expresses a signal peptide that guides the transmembrane transfer of newly synthesized proteins; the nucleotide sequence of SP is shown in SEQ ID NO. 1. Claudin 16scFv represents a Claudin 16 single-chain antibody. TM is the transmembrane region, connecting the extracellular antigen-binding domain and the intracellular signaling domain, anchoring the CAR structure to the T cell membrane; the nucleotide sequence of TM is shown in SEQ ID NO. 5. CD28-4-1BB is the co-stimulatory domain, transducing proliferation signals and inducing cytokine production and stimulating T cell activation; the CD28 nucleotide sequence is shown in SEQ ID NO. 6, and the 4-1BB nucleotide sequence is shown in SEQ ID NO. 7. CD3ζ is the signal transduction domain; when the extracellular region binds to the target antigen, it transduces a TCR-like signal into the cell, activating the T cell; the CD3ζ nucleotide sequence is shown in SEQ ID NO. 8. The CAR structure mentioned in this invention endows T cells with stronger proliferative capacity and longer viability, resulting in stronger tumor cell killing ability. The vector includes the PEE6.4 plasmid and a lentiviral vector. The lentiviral vector is GV230, and its structure is shown in SEQ ID NO. 8. Figure 1 As shown in the figure. By analyzing the restriction site map of this vector, the insert fragment was selected for double digestion with Xho I and BamHI.

[0037] I. Experimental Materials

[0038] 1. Lentiviral expression plasmid GV230, lentiviral packaging plasmid pMD2G, and vector plasmid PSPAX2 were purchased from Jikai Gene; the structure of lentiviral expression plasmid GV230 is as follows. Figure 1 As shown;

[0039] 2. The CAR structural sequence was synthesized by Nanjing Genscript Biotech Co., Ltd. and preserved in the form of PEE6.4 plasmid;

[0040] 3. Restriction endonucleases XhoI and BamHI were purchased from NEB;

[0041] 4.T4 DNA ligase and dd H2O were purchased from Takara;

[0042] 5. Competent cells, gel extraction kit, and plasmid miniprep kit were purchased from Novizan Biotechnology Co., Ltd.

[0043] 6.293T cells and OVCA-R3 cells were purchased from the Chinese Academy of Sciences Cell Bank;

[0044] 8. FBS, DMEM, 1640 medium, PBS, and Opti-MEM were purchased from Gibco;

[0045] 9. CD3 monoclonal antibody, CD28 monoclonal antibody, CH38 protein, and IL-2 were purchased from Suzhou Nearshore Protein Technology Co., Ltd.

[0046] II. Construction method of CAR vector targeting Claudin 16, a protein specifically highly expressed in ovarian cancer cells.

[0047] The lentiviral expression plasmid GV230 and the PEE6.4 plasmid targeting the CAR structure (Claudin 16 single-chain antibody) were simultaneously digested with XhoI and BamHI. The digestion products were subjected to agarose gel electrophoresis, and the target bands were recovered. The recovered fragments were ligated at a CAR structure:GV230 ratio of 10:1, and then transformed into competent cells. The transformed competent cells were plated on LB agar and incubated overnight at 37°C. A portion of the colonies were picked and cultured in 3 mL of LB liquid medium on a shaker for enrichment and plasmid extraction. The extracted products were verified by XhoI and BamHI digestion. Plasmids with the correct band size were sent to Sangon Biotech for sequencing. Figure 3 The plasmids and bacterial cultures with correct sequencing results were preserved.

[0048] III. Viral Packaging Targeting Claudin 16, a Protein Highly Expressed Specifically in Ovarian Cancer Cells

[0049] (I) Virus preparation

[0050] Lentiviral packaging was performed using a three-plasmid packaging system. The three plasmids were the lentiviral expression plasmid GV230 containing the CAR structure, the lentiviral packaging plasmid pMD2G, and the envelope plasmid PSPAX2. 293T cells were used.

[0051] The specific implementation steps are as follows:

[0052] (1) Plating within 24 hours before transfection: Select 293T cells in logarithmic growth phase and plating them when the growth density reaches 90%.

[0053] (2) Once the growth density reaches 80-90% and the cells are in good condition, virus packaging can be carried out;

[0054] (3) The virus was packaged according to the ratio of PSPAX2 plasmid, pMD2G plasmid and CAR vector (recombinant expression plasmid) targeting the ovarian cancer cell-specific high-expression protein Claudin 16 in a ratio of 5:3:3.

[0055] (4) PEI was selected as the transfection reagent and the amount added was 6 μL / μg plasmid;

[0056] (5) Mix the plasmid mixture in step (3) and the transfection reagent mixture in step (4) into a tube, let it stand at room temperature for 5 minutes, then add it to the cells in the medium and continue culturing.

[0057] (6) Collect the culture supernatant after 48 hours and filter it through a 0.45 μm filter membrane;

[0058] (7) The collected virus solution was concentrated using the PEG8000 concentration method, and the virus titer was determined. The solution was then aliquoted and stored at -80℃ for later use.

[0059] (II) Viral fluid infecting T cells

[0060] 1. PBMC separation

[0061] 1) Collect approximately 10 mL of fresh human peripheral blood using a vacuum blood collection tube containing an anticoagulant;

[0062] 2) Dilution: Add an equal volume of PBS at room temperature and gently mix by pipetting.

[0063] 3) Sample addition: Take two 50mL centrifuge tubes, and add 10mL of lymphocyte separation solution (Ficoll) into the centrifuge tubes (the volume ratio of Ficoll to the blood before dilution is 1:1). Slowly add the diluted blood to the Ficoll tube about 1cm above the Ficoll surface along the tube wall.

[0064] 4) Centrifugation: at room temperature, 800 rpm, 30 min. After centrifugation, the liquid will separate into four layers from the bottom of the tube to the surface: red blood cells and granulocytes, layered liquid, mononuclear cells, and plasma.

[0065] 5) Recovery: Insert the pipette directly into the cloud layer (or first aspirate the upper layer of plasma), gently aspirate the cloud layer, and place it into a new centrifuge tube;

[0066] 6) Washing: Add at least 3 times the volume of PBMCs (peripheral blood mononuclear cells) of PBS, incubate at room temperature, 1000 rpm, for 10 min, twice;

[0067] 7) Cell counting: Discard the supernatant, add 1 mL of lymphocyte culture medium, mix well by pipetting, and prepare a PBMC cell suspension. Count the cells using a hemocytometer: Mix one drop of PBMC suspension with one drop of 2% trypan blue staining solution and add to the hemocytometer. Count the total number of cells under a microscope.

[0068] 2. T cell activation and lentiviral infection

[0069] (1) Experimental procedure:

[0070] Day 0: 24-well plate coating: Take a 24-well plate and add 175 μL (10 μg / mL) of CD3 monoclonal antibody solution, 175 μL (10 μg / mL) of CD28 monoclonal antibody solution, and 175 μL (50 μg / mL) of CH-38 protein solution to each well. After adding, gently shake to mix, seal the plate with sealing film, and place it in a 4°C refrigerator overnight.

[0071] Day 1: Cleaning the coated plates: Take out the 24-well plates coated yesterday, discard the supernatant, wash twice with sterile PBS, and then add PBS for later use;

[0072] PBMC plating: Collect PBMC cells, count them, and finally adjust the concentration to 0.5 × 10⁻⁶. 6 Add 500 μL of cell suspension to each well, i.e., add 2.5 × 10⁶ cells / mL. 5 One cell;

[0073] Viral infection: Infection was performed with an MOI of 30. 1 mL of virus culture medium suspension was prepared, added to a 24-well plate, and centrifuged at 1000 g for 30 min at room temperature.

[0074] Day 1-Day 2: Observe cell status;

[0075] Day 3: Transfer all cells from the 24-well plate to a 60mm culture dish containing 3mL of culture medium and observe the cell status;

[0076] Day 4-Day 7: Observe the cell status and cell number. If the cells begin to proliferate significantly and the cell density is high in a local area, add 3 mL of culture medium.

[0077] Day 8: At this point, the cells in the 60mm culture dish have reached confluence, and are transferred to a 100mm culture dish with 15mL of culture medium added for continued culture.

[0078] Day 9-Day 10: Observe the cell status. When the cells are fully filled in the 100mm culture dish, stop further growth, enrich the cells and calculate the expansion ratio, detect the infection efficiency, and perform subsequent experiments such as cell killing detection.

[0079] IV. Identification and Detection of CAR-T Cells

[0080] (I) Flow cytometry detection of CAR structural positive expression rate

[0081] 1) The obtained NC group cells (uninfected with virus) and sample group cells (infected with virus) were gently washed twice with PBS at 1000 rpm / 3 min, and the waste liquid was discarded.

[0082] 2) Add 50 μL of biotin-labeled Claudin 16 protein to each group of cells and incubate at 4°C for 30 min. The final concentration of biotin-labeled Claudin 16 protein is 5 μg / ml.

[0083] 3) After 30 min, centrifuge and discard the supernatant. Wash the cells twice with PBS buffer containing 2% BSA.

[0084] 4) Add 50 μL of PE-labeled streptavidin to each group of cells and incubate at 4°C for 30 min. The dilution ratio of PE-labeled streptavidin is 1:50.

[0085] 5) After 30 min, centrifuge and discard the supernatant, then wash the cells twice with PBS buffer containing 2% BSA;

[0086] 6) Resuspend the cells in 400 μL of PBS in each tube and analyze them using a flow cytometer. Figure 4 ).

[0087] (II) Detection of CAR-T cell killing level

[0088] Ovarian cancer OVCA R3 cell line highly expresses Claudin16 ( Figure 5 Taking ovarian cancer OVCA R3 cells as an example, the lactate dehydrogenase (LDH) release assay was used to detect the killing efficiency of effector T cells against target cells.

[0089] 1) Collect suspensions of target cells and effector cells separately, centrifuge at 1500 rpm for 5 min, discard the supernatant, resuspend in 3 mL of sterile PBS, and centrifuge at 1200 rpm for 5 min. Resuspend effector cells and target cells separately in 1 mL of AIM-V complete culture medium, mix well, and count using a hemocytometer.

[0090] 2) Setting effector cells: When the target cell (E:T) ratio is 2.5:1, add 1 x 10-1 target cells to each well of a 96-well plate.4 Add 2.5 x 10⁻⁶ effector cells to each well. 4 The volume is 50 μL. When the E:T ratio is set to 5:1, 1 x 10⁶ target cells are added to each well of the 96-well plate. 4 Add 5 x 10-1 effector cells to each well 4 The volume is 50 μL. When the E:T ratio is set to 10:1, add 1 x 10⁶ target cells to each well of the 96-well plate. 4 Add 1x10 effector cells to each well 5 The volume is 50 μL. The LDH killing assay requires triplet wells. After preparing the effector cells and target cells, seed them into 96-well plates, seal the 96-well plates with sealing film, and centrifuge at 250 g for 5 min. Then, incubate them in a 37°C incubator for 24 h.

[0091] 3) Add 10 μL of lysis buffer to each well of the largest lysis group 45 min before detection, incubate in a 37°C incubator for 45 min, add LDH substrate and incubate for 10 min, and read the absorbance at 490 nm using a microplate reader.

[0092] 4) Results: LDH killing results showed that Claudin16 CAR-T cells had high specific killing effect on the Claudin16-expressing ovarian cancer cell line OVCA R3, and this effect was significantly dose-dependent; the higher the amount of CAR-T cells, the higher the killing effect. However, no killing effect was observed on the Claudin16-negative ovarian cancer cell line OVCA429 at any effector-to-target cell ratio (10:1, 5:1, 2.5:1). Figure 6 Claudin16-mediated CART cells can specifically kill Claudin16-positive tumor cells in a dose-dependent manner.

[0093] (III) ELISA detection of cytokine secretion

[0094] 1) Prepare 5ug / ml capture antibody using CBS coating buffer according to the specified ratio;

[0095] 2) Add 100 μL / well of the coating solution prepared in step 1) to the ELISA high affinity 96-well plate, seal and place in a 4°C refrigerator overnight;

[0096] 3) Clean the coated 96-well plate three times with PBST (0.05% Tween 20);

[0097] 4) Add 200 μL of PBS blocking buffer containing 3% BSA to each well and block at room temperature for 2 h;

[0098] 5) The standard was serially diluted 7 times, and the sample (the supernatant of CAR-T cells after 24 hours of killing by OVCAR3 in the cell killing assay) was serially diluted 5 times.

[0099] 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;

[0100] 7) Wash with PBST 4 times;

[0101] 8) Dilute the HRP-labeled detection antibody to a concentration of 0.5 ug / ml using PBS containing 0.1% BSA, add 100 μL to each well, and incubate at room temperature for 1 h;

[0102] 9) Wash 5 times with PBST, add 100 μL of TMB reagent to each well, and incubate at room temperature for 15 min;

[0103] 10) Add 50 μL / well stop solution to stop color development;

[0104] 11) Detect OD value using an enzyme-linked immunosorbent assay (ELISA) reader at 450nm.

[0105] The results are as follows Figure 7 As shown in the figure, 10:1 represents effector cell:target cell ratio of 10:1; 5:1 represents effector cell:target cell ratio of 5:1; 2.5:1 represents effector cell:target cell ratio of 2.5:1; NC represents the blank control. After co-culturing CAR-T cells and OVCAR3 cells for 24 hours, there was a significant release of the cytokine IFN-gamma, with the highest IFN-gamma release observed at an effector cell:target cell ratio of 10:1.

[0106] In summary, the CAR vector targeting the ovarian cancer cell-specific high-expression protein Claudin 16 provided by this invention is applied to infect T cells to obtain CAR-T cells expressing the antibody Anti-claudin 16 against the tumor-associated antigen Claudin 16 on the surface of ovarian cancer cells, enabling it to more accurately recognize and kill ovarian cancer cells expressing the aforementioned tumor-associated antigen.

[0107] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A CAR vector targeting Claudin 16, a protein specifically highly expressed in ovarian cancer cells, comprising a gene encoding a chimeric antigen receptor, characterized in that, The chimeric antigen receptor is used to target Caudin 16, a protein specifically highly expressed in ovarian cancer cells. The structure of the chimeric antigen receptor is SP-Claudin 16 scFv-TM-CD28-4-1BB-CD3ζ, where SP is a signal peptide that guides the transmembrane transfer of newly synthesized proteins, Claudin 16scFv is a single-chain antibody that specifically recognizes Claudin 16 on the cell surface, TM is a transmembrane domain that connects the extracellular antigen-binding domain and the intracellular signaling domain, CD28-4-1BB is a co-stimulatory signaling molecule, and CD3ζ is a cytoplasmic signal transduction domain. The Claudin 16scFv includes a heavy chain variable region VH, a light chain variable region VL, and a flexible linker strand connecting the heavy chain variable region VH and the light chain variable region VL. The nucleotide sequence of the heavy chain variable region VH is shown in SEQ ID NO.2, the nucleotide sequence of the light chain variable region VL is shown in SEQ ID NO.3, and the nucleotide sequence of the flexible linker strand is shown in SEQ ID NO.

4. SP expresses a signal peptide that guides the transmembrane transfer of newly synthesized proteins. The nucleotide sequence of SP is shown in SEQ ID NO.

1. Claudin 16scFv represents a Claudin 16 single-chain antibody. TM is the transmembrane region, connecting the extracellular antigen-binding domain and the intracellular signaling domain, anchoring the CAR structure to the T cell membrane. The nucleotide sequence of TM is shown in SEQ ID NO.

5. CD28-4-1BB is the co-stimulatory domain, transducing proliferation signals and inducing cytokine production and stimulating T cell activation. The nucleotide sequence of CD28 is shown in SEQ ID NO.6, and the nucleotide sequence of 4-1BB is shown in SEQ ID NO.

7. CD3ζ is the signal transduction domain. When the extracellular region binds to the target antigen, it transduces a TCR-like signal into the cell, activating the T cell. The nucleotide sequence of CD3ζ is shown in SEQ ID NO.

8.

2. The CAR vector targeting the ovarian cancer cell-specific high-expression protein Claudin 16 according to claim 1, characterized in that, The vector is GV230-Claudin 16scFv, which is constructed using the lentiviral expression plasmid GV230 and the chimeric antigen receptor of claim 1.

3. A host cell, characterized in that, The host cell contains the vector as described in any one of claims 1-2.

4. A method for obtaining CAR-T cells that specifically highly express the protein Claudin 16 in ovarian cancer cells, characterized in that, The vector described in any one of claims 1-2 is packaged with lentivirus to obtain a high-titer lentivirus, and the lentivirus is used to infect T cells to obtain chimeric antigen receptor-T cells.

5. The method for obtaining CAR-T cells that specifically highly express the protein Claudin 16 in ovarian cancer cells according to claim 4, characterized in that, The lentivirus packaging uses 293T cells as the packaging cells and employs a three-plasmid packaging system, which includes a PSPAX2 plasmid, a pMD2G plasmid, and a vector, with a mass ratio of 5:3:3 for the PSPAX2 plasmid, pMD2G plasmid, and vector.

6. The use of a carrier as described in any one of claims 1-2, or a cell as described in claim 3, characterized in that, Used to prepare drugs or preparations for the treatment of ovarian cancer.

7. A method for constructing a CAR vector targeting Claudin 16, a protein specifically highly expressed in ovarian cancer cells, as described in any one of claims 1-2, characterized in that, The construction method includes the following steps: (1) The chimeric antigen receptor was stored on a PEE6.4 plasmid; (2) The PEE6.4 plasmid and lentiviral vector contained in step (1) were double-digested, and the digestion products were separated by agarose gel electrophoresis to obtain the target fragment containing the chimeric antigen receptor and the double-digested lentiviral vector. (3) The target fragment containing the chimeric antigen receptor in step (2) is ligated with the lentiviral vector in step (2), the ligation product is transformed, the plasmid is extracted, and a recombinant plasmid containing the chimeric antigen receptor structure is obtained. The recombinant plasmid is a CAR vector that targets the ovarian cancer cell-specific high-expression protein Claudin 16.

8. The construction method according to claim 7, characterized in that, The double digestion in step (2) is XoHI and BamHI, and the mass ratio of the chimeric antigen receptor target fragment and the lentiviral vector conjugate in step (3) is 10:1.

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