Chimeric antigen receptor T cell for expressing HPSE-pX-delta1-30 and application of chimeric antigen receptor T cell

Abstract

The invention belongs to the technical field of immune cell therapy, and particularly relates to a chimeric antigen receptor T cell for expressing HPSE-pX-delta 1-30 and application of the chimeric antigen receptor T cell. According to the invention, HPSE-pX-delta 1-30 is expressed through engineered CAR T cells, and the activity of HPSE is limited to TME by using an exosome, so that systematic off-target toxicity is avoided. The result of the embodiment shows that the CAR T cell has remarkable ECM infiltration capacity, tumor killing efficiency and lasting immune memory phenotype, has the characteristics of simplicity in preparation, high safety and remarkable curative effect, and can be used for preparing medicines for treating solid tumors rich in ECM.

Description

Technical Field

[0001] This invention belongs to the field of immunotherapy technology, specifically relating to a chimeric antigen receptor T cell expressing HPSE-pX-Δ1-30 and its application. Background Technology

[0002] Chimeric antigen receptor T-cell (CAR T-cell) therapy has demonstrated significant efficacy in the treatment of hematological malignancies; however, its application in solid tumors such as colorectal cancer faces major challenges. This is primarily attributed to the restriction of CAR T-cell infiltration and function by the dense extracellular matrix (ECM) within the tumor microenvironment (TME). The ECM forms a physical barrier, hindering T-cell infiltration and reducing contact with tumor cells, thus limiting efficacy. Among ECM components, heparan sulfate proteoglycan 2 (HSPG2) is highly expressed in tumor cells and the associated matrix, and its degrading enzyme, heparinase (HPSE), has been proposed to relax this barrier and promote T-cell entry into the tumor core. However, in current CAR T-cell manufacturing processes, HPSE expression declines sharply during T-cell expansion and effector differentiation, failing to maintain sufficient ECM degradation activity. Furthermore, constitutive overexpression of HPSE carries the risk of off-target effects, as HSPG2 is also widely present in vital organs such as the lung, kidney, and vascular basement membrane, potentially leading to systemic ECM damage.

[0003] Existing technologies attempt to improve T cell infiltration through direct overexpression of HPSE, such as engineered CAR T cells expressing HPSE to enhance ECM degradation and tumor invasion. However, these methods have not effectively limited HPSE activity at the tumor site and are prone to toxicity. Furthermore, CAR T cells in solid tumors often exhibit exhaustion phenotypes (e.g., high PD-1 and CD57 expression), insufficient memory differentiation, and cytokine imbalances, further weakening durable anti-tumor immunity. In recent years, CAR T-derived exosomes have been found to inherit tumor homing characteristics and carry CAR and apoptosis ligands (such as TRAIL and FasL), but engineered designs targeting ECM remodeling are lacking. Therefore, developing a novel chimeric antigen receptor and corresponding CAR T cells is crucial to overcoming the obstacles of CAR T therapy for solid tumors. Summary of the Invention

[0004] The purpose of this invention is to provide a chimeric antigen receptor T cell (CAR T cell) expressing HPSE-pX-Δ1-30. The CAR T cell provided by this invention can achieve local delivery of HPSE to simultaneously achieve ECM degradation, tumor killing and immune regulation.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a chimeric antigen receptor (CAR), the CAR comprising: a single-chain antibody (scFv, such as Anatumab) that specifically recognizes mesothelin (MSLN), a CD8α hinge and transmembrane region, a 4-1BB co-stimulatory signaling domain, a CD3ζ signaling domain, and an HPSE-pX-Δ1-30 fusion protein; The gene sequence of HPSE-pX-Δ1-30 is shown in SEQ ID NO:1.

[0006] HPSE is anchored to T-cell-derived exosomes via the pX-Δ1-30 protein. CAR T-derived exosomes naturally carry CAR box structures, thereby degrading ECM and promoting T-cell infiltration in the tumor microenvironment (TME).

[0007] Furthermore, the HPSE-pX-Δ1-30 fusion protein is linked to the CAR via a 2A peptide.

[0008] A nucleic acid molecule encoding the above-mentioned CAR, the sequence of which is shown in SEQ ID NO:2.

[0009] A CAR expression cassette, the expression cassette comprising the aforementioned nucleic acid molecule.

[0010] A CAR T cell, said CAR T cell being derived from the aforementioned CAR-modified T cells. This CAR T cell can express HPSE-pX-Δ1-30.

[0011] A method for preparing CAR T cells, the method comprising the following steps: (1) Peripheral blood mononuclear cells (PBMCs) and T cells were isolated and activated with anti-CD3 / CD28 antibody for 24-48 hours; (2) Use a lentiviral vector containing the above-mentioned CAR nucleic acid molecules to transfect activated T cells at MOI=8; (3) Expand the T cell population that stably expresses CAR in serum-free medium containing IL-2 for 10–14 days.

[0012] Furthermore, the method also includes: (4) detecting CAR expression by flow cytometry (FCM).

[0013] The present invention also provides the application of the HPSE-pX-Δ1-30 CAR T cells, the application including: (1) Application in enhancing the penetration and killing of tumor cells in vitro; for example, it showed better effects than standard CAR T in extracellular matrix (ECM) simulated invasion model and three-dimensional tumor spheroid experiment; (2) Its application in enhancing the invasion and clearance of solid tumors, such as showing stronger tumor suppression and prolonging survival in the HCT116 tumor-bearing mouse model; (3) Application in the preparation of drugs for the treatment of solid tumors.

[0014] Furthermore, the solid tumor is colorectal cancer, pancreatic cancer, or ovarian cancer.

[0015] The beneficial effects of this invention are: This invention utilizes engineered CAR T cells to express HPSE-pX-Δ1-30, and employs exosomes to limit HPSE activity in the tumor microenvironment (TME), thus avoiding systemic off-target toxicity. These cells exhibit significant ECM penetration, tumor-killing efficacy, and durable immune memory phenotype in both in vitro and in vivo models. They are characterized by simple preparation, high safety, and significant therapeutic effects, providing an innovative solution for immunotherapy of solid tumors. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 CAR box structure diagram of standard CAR T and HPSE-pX-Δ1-30 CAR T cells.

[0018] Figure 2 Expression rates of CAR in standard CAR T cells and HPSE-pX-Δ1-30 CAR T cells.

[0019] Figure 3 The results of the invasiveness assay assessing the ability of HPSE-pX-Δ1-30 CAR T cells to penetrate the matrix.

[0020] Figure 4 A statistical graph of the ability of HPSE-pX-Δ1-30 CAR T cells to penetrate the matrix in an invasive assay.

[0021] Figure 5 The result of assessing the anti-tumor efficiency of HPSE-pX-Δ1-30 CAR T cells using HCT116 tumor spheroids.

[0022] Figure 6 A statistical graph of the anti-tumor efficiency of HPSE-pX-Δ1-30 CAR T cells in assessing HCT116 tumor spheroids.

[0023] Figure 7 A graph showing the results of evaluating HPSE-pX-Δ1-30 CAR T cell subsets after co-culturing with tumor cells.

[0024] Figure 8 Statistical graph evaluating HPSE-pX-Δ1-30 CAR T cell subsets after co-culture with tumor cells

[0025] Figure 9 Statistical graph evaluating HPSE-pX-Δ1-30 CAR T cell subsets after co-culture with tumor cells

[0026] Figure 10 The results of evaluating the anti-tumor efficiency of HPSE-pX-Δ1-30 CAR T cells in NSG tumor-bearing mice are shown in the figure.

[0027] Figure 11 Statistical graph of NSG tumor-bearing mice evaluating the anti-tumor efficiency of HPSE-pX-Δ1-30 CAR T cells Detailed Implementation

[0028] This invention provides a chimeric antigen receptor T cell (CAR T cell) expressing HPSE-pX-Δ1-30. The CAR T cell contains an expression cassette encoding a CAR in its genetic design. The CAR includes an extracellular recognition domain targeting tumor-associated antigens, a CD8α hinge and transmembrane domain, and intracellular signaling domains of 4-1BB and CD3ζ. It further includes an expression cassette encoding a fusion protein of human heparanase (HPSE) and the exosome-loaded signal peptide pX-Δ1-30, thereby promoting the continuous secretion of HPSE-loaded exosomes by the CAR T cell, achieving local tumor extracellular matrix (ECM) degradation.

[0029] The CAR T cells in this invention can secrete exosomes carrying HPSE, which are used to degrade ECM and enhance tumor invasion. HPSE can target multivesicular bodies / exosomes via the ESCRT-associated protein ALIX, thereby achieving the secretion of HPSE exosomes to locally degrade ECM.

[0030] In the CAR T cells described in this invention, the extracellular recognition domain is a single-chain variable region (scFv, anetumab) targeting mesothelin (MSLN). In vitro, it exhibits at least three times the matrix penetration ability compared to control CAR T cells; in vivo, it significantly reduces tumor volume and prolongs survival in tumor-bearing animals; and it can be used to prepare drugs for treating ECM-rich solid tumors.

[0031] The sequence information involved in this invention is as follows: HPSE-pX-Δ1-30: Complete CAR sequence: Scfv(MSLN-Anetumab)-2ndCAR-FT2A-3FLAG-HPSE(36-543AA)-pX(del1-30aa): To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0032] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0033] The various instruments, equipment, raw materials, or reagents used in the embodiments of this invention are not subject to any special restrictions on their source; they are all conventional products that can be purchased through legitimate commercial channels and can be prepared according to conventional methods well known to those skilled in the art. The main reagents used in the embodiments of this invention are as follows: I. Cell Culture and Basic Culture Medium Reagents 1. Culture medium (1) DMEM high glucose medium: used for HCT116 cell culture; (2) RPMI 1640 medium: used for T cell culture; (3) X-VIVO15 medium: used for primary T cell and CAR T cell culture; (4) 10% fetal bovine serum: added to DMEM and RPMI 1640 medium; (5) 1% penicillin / streptomycin mixture: used for aseptic treatment of all cell culture media; (6) Penicillin / streptomycin / glutamine mixture (100×): Added to X-VIVO15 medium for primary T cell culture.

[0034] 2. Cell digestion and processing reagents

[0035] (1) Trypsin: used for passage of adherent cells; (2) 50 mmol / L β-mercaptoethanol: added to X-VIVO15 medium to maintain the activity of primary T cells; (3) Recombinant human IL-2: final concentration 10 ng / mL, used for T cell activation and CAR T cell culture.

[0036] II. Cell Isolation and Activation Reagents

[0037] 1. Primary cell separation reagents

[0038] (1) Human peripheral blood lymphocyte separation solution: used for PBMC separation; (2) EasySep Human T Cell Negative Selection Kit: Isolate high-purity primary T cells from PBMCs; (3) T cell activation reagent: ImmunoCult HuCD3 / CD28 / CD2 T cell activator: used for the activation of primary T cells, activation time 24-72 hours.

[0039] III. Functional Testing and Staining Reagents

[0040] 1. ECM Infiltration and 3D Tumor Spheroid Assay Reagents

[0041] (1) Matrigel: used for ECM-mimicking coating of Transwell chambers to detect CAR T cell infiltration ability; (2) 96-well U-shaped ultra-low adsorption (ULA) plate: used for the construction of 3D tumor spheres of HCT116 cells; (3) Breathe-Easy semipermeable membrane sealing film: used to prevent culture medium evaporation during 3D tumor spheroid culture.

[0042] (4) Red CellTracker: Used for marking T cells.

[0043] 2. Reagents for detecting cell viability and killing effect

[0044] CellTiter-Glo 3D Cell Viability Assay Reagent: Used to detect ATP levels in tumor cells in 3D tumor spheroid models and assess CAR T cell killing efficiency; V. FCM Detection Reagents Surface marker antibodies: Anti-human CD3 antibody, anti-human CD4 antibody, anti-human CD8 antibody, anti-human CD45RA antibody, anti-human PD-1 antibody, anti-human CD57 antibody, anti-human TRAIL antibody, anti-human FasL antibody.

[0045] Anti-human CAR antibodies (specific antibodies against MSLN scFv, such as anti-Anetumab scFv antibody): used to detect the expression rate of CAR on the surface of T cells.

[0046] Example 1

[0047] Construction of standardized CAR T and HPSE-pX-Δ1-30 CAR T

[0048] The CAR T lentiviral vector used in this embodiment was purchased from Suzhou Genewiz Biotechnology. The viral titer of the standard CAR T is 4.52 × 10⁻⁶. 8Tu / mL; the viral titer of HPSE-pX-Δ1-30 CAR T was 3.39 × 10⁻⁶. 9 Tu / mL ( Figure 1 ).

[0049] I. Separation of PBMCs by Density Gradient Centrifugation

[0050] 1. Collect fresh anticoagulated whole blood and infuse it into the anticoagulation tube using a disposable venous blood collection needle. Only 20 ml of blood is needed. Collect and use the blood immediately. Perform intercellular experiments directly after blood collection to avoid affecting cell viability.

[0051] 2. Transfer the 20 ml of whole blood obtained in the previous step into a 50 mL centrifuge tube, add 20 mL of PBS solution (1:1 ratio) to dilute the blood, and mix gently.

[0052] 3. Take two 50 mL centrifuge tubes and add 20 mL of human peripheral blood lymphocyte separation medium. Then, gently add 20 mL of diluted blood to the top layer of the human peripheral blood lymphocyte separation medium in each centrifuge tube. Be sure to be gentle to avoid mixing the two solutions and keep the interface between the two liquids clear. Use a 5 mL Pasteur tube to slowly add the blood to the tube wall. Now, add 40 mL of diluted blood to each centrifuge tube.

[0053] 4. Place the diluted blood in a centrifuge at 2000 rpm for 20 minutes. Note: The centrifuge can accelerate normally, but the deceleration should be slow. You can set it to no break or only 10% braking.

[0054] 5. After centrifugation, the liquid levels in the tube, from top to bottom, are: diluted plasma layer, PBMC layer (i.e., white film layer), separation liquid layer, and red blood cell layer. After centrifugation, discard the plasma layer, carefully aspirate the PBMC layer using a Pasteur tube, and transfer it to a 15 mL centrifuge tube.

[0055] 6. Add 10 mL of PBS washing buffer to the centrifuge tube to resuspend the cells. Centrifuge at 250 g for 10 min at room temperature, discard the supernatant, and repeat this step 1-2 times. Add 1 mL of RPMI 1640 medium to resuspend the cells, count (calculate the total number of cells) and assess viability. Adjust the volume to 5 × 10⁻⁶ cells / mL. 7 Cell count per ml. Perform the following separation and activation.

[0056] II. Isolation of Pan T cells

[0057] 1. Transfer 300 μL of PBMC to a 5 mL flow cytometry tube and add 15 μL of isolation cocktail. Mix and incubate at room temperature for 5 minutes.

[0058] 2. Vortex RapidSpheres™ 30s. (Note: The particles should appear to be uniformly dispersed.)

[0059] 3. Add 12ul RapidSpheres™ for sampling and mixing.

[0060] 4. Add RPMI 1640 medium to fill the sample to 2.5 ml. Gently pipette up and down 2-3 times to mix.

[0061] 5. Place the tube (without the cap) into the magnet and incubate at room temperature for 3-5 minutes.

[0062] 6. Pick up the magnet and pour the enriched cell suspension into a new test tube. (Note: Invert the magnet and tube for 2-3 seconds, then return them to an upright position.)

[0063] III. T-cell activation

[0064] 1. The separated T cells were divided into 1×10⁻⁶ cells. 6 / ml was inoculated into well plates.

[0065] 2. Add CD3 / CD28 T cell activator at a concentration of 25 μL / mL. Use for CAR T cell construction 24-48 hours later.

[0066] IV. Construction of CAR T

[0067] 1. After approximately 48 hours of T cell activation, count the T cell concentration, obtaining 200,000-800,000 T cells. Centrifuge each sample and add 100 μL of IL-2-free, double-antibody X-VIVO15 medium. Select MOI=8 and add 4.2 μL of virus infection enhancement medium. Follow the instructions for V... 病毒 =(8×N cell ) / V 病毒滴度

[0068] Calculate the volume of virus added, then select a centrifugal force of 1000g and centrifuge at 37℃ for 10 minutes.

[0069] 2. After 12 hours, add 100 μL of IL-2-free, double-antibody X-VIVO15 medium.

[0070] 3. After 24 hours, the culture was transferred to a 6-well plate for further culture, with a culture volume of 2 ml.

[0071] 4.2 days later, the culture was expanded. CAR expression levels were detected by flow cytometry 1-2 weeks later. Figure 2 ).

[0072] Example 2

[0073] Evaluate the efficacy of HPSE-pX-Δ1-30 CAR T cells in in vitro tumor killing.

[0074] I. Transwell Invasiveness Tests: 1. Four groups were set up: primary T cells, CAR T cells, HPSE-pX-Δ1-30 CAR T cells, and HPSE-pX-Δ1-30 CART-NSMi cells (NSMi is an exosome inhibitor) (T cells were first labeled with Celltrack).

[0075] 1.2 Experimental Procedure: 1.2.1 The matrix collagen concentration is 8-12 mg / ml, which needs to be diluted to 0.2-0.3 mg / ml with serum-free culture medium. Note: The entire matrix gel process must be performed on ice.

[0076] 1.2.2 24 hours before the experiment, lay 7×10⁻⁶ m² of concrete in the bottom chamber. 4 HCT116 cells were used. Experimental T cells were starved for 12-24 hours using serum-free medium (RPMI 1640).

[0077] 1.2.3 Two hours in advance, spread the diluted gel on the upper surface of the membrane at the bottom of the chamber (using a 12µm pore size), and place it in a 37°C incubator for 60 minutes to allow Matrigel to polymerize into a gel (100µl for 24-well plates and 50µl for 48-well plates).

[0078] 1.2.4 Wash once with PBS, resuspend the cells in serum-free medium, and adjust the cell density to 2.5 x 10⁻⁴. 5 / ml (48-well plate).

[0079] 1.2.5 Add 100 μL of cell suspension to the small chamber and 250-300 μL of RPMI 1640 complete culture medium containing 20% ​​FBS and hIL-2 to the lower chamber (be careful to remove air bubbles between the small chamber and the lower culture medium).

[0080] 1.2.6 After 24 hours of routine culture, photographs were taken of the upper and lower chambers, and the number of T cells was counted using a Boehringer-Bauer counting chamber.

[0081] The results showed that HPSE-ΔpX1-30 CAR T could penetrate the streptocele from the upper ventricle to the lower ventricle more significantly, and this effect could be weakened by exosome blockers. Figure 3 Its penetration capability is nearly four times greater than that of standardized CAR T. Figure 4 ).

[0082] II. 3D tumor spheroid assessment of the anti-tumor effect of HPSE-ΔpX1-30 CAR T: 1. Four groups were set up: control group, primary T, CAR T, and HPSE-ΔpX1-30 CAR T.

[0083] 2. Six wells for each condition, with an initial seeding cell count of 1 × 10⁶ cells. 4 After 5 days of culture, the tumor spheroids can be considered to be 3 × 10⁻⁶. 4 One cell, then add 3 × 10 5 The corresponding T cells were co-cultured for 72 hours.

[0084] 3. After aspirating 150 μL of supernatant, add 100 μL of culture medium to flush out the T cells and immediately photograph the tumor spheroids. Wash the tumor spheroids twice in PBS to remove T cells. Place in a new 96-well plate and photograph. Figure 5 Then, the ATP experiment was conducted.

[0085] 4. ATP assay (CellTiter-Glo® 3D cell viability assay)

[0086] Incubate the CellTiter-Glo® 3D reagent at 37°C to room temperature (do not place in an incubator, as this will affect the fluorescence signal). Use a pipette to transfer individual spheroids from the culture medium to a white 96-well microplate (using a white plate avoids interference from the fluorescence signal in other wells). Confirm that the spheroids have been successfully transferred (record the volume of culture medium transferred; an equal volume of reagent will be added in the next step; the volume of culture medium transferred in this step is 60 µl).

[0087] Add 60 µl of reagent to each well using a multichannel pipette. This procedure must be performed in a biosafety cabinet to prevent contamination of the CellTiter-Glo® 3D reagent.

[0088] Wrap the culture plate in aluminum foil to protect it from light, place it on a shaker, and shake at 120 rpm for 5 minutes to thoroughly mix the culture medium and reagents; then incubate at room temperature for 25 minutes. Detect its luminescence (…). Figure 6 The results showed that HPSE-ΔpX1-30 CART significantly reduced tumor size and viability.

[0089] 5. The collected T cells were used for phenotypic determination. The results show that ( Figure 7-9 HPSE-ΔpX1-30 CAR T cells showed an increased proportion of memory-like T cells and decreased exhaustion markers such as PD-1 and CD57.

[0090] Example 3.

[0091] Evaluate the efficacy of HPSE-pX-Δ1-30 CAR T cells in killing tumors in vivo.

[0092] To evaluate the therapeutic efficacy of CAR T cells, we further designed a low-target-ratio in vivo treatment experiment. NSG mice were implanted with tumors subcutaneously. Seven days later, they were treated with CAR-T cells via tail vein injection. Tumor growth was then monitored, and mouse survival was recorded. Simultaneously, FCM was used to analyze CD3 levels in the mouse tumor tissue. Figure 10 ).

[0093] 1. After expanding MSLN+-Fluc+-HCT116 cells to the required number, digest the cells, count and aspirate the cells, centrifuge at 950 rpm for 5 min, wash the cells twice with 1×PBS, and discard the supernatant. Then, use 1×PBS at a concentration of 2.5×10⁻⁶ cells / mL. 6 Cells were resuspended at a density of cells / mL for later use (Matrix gel concentration was 5.5 mg / mL).

[0094] 2. After feeding NSG mice for one week, 200 μL (5 × 10⁻⁶) of cell suspension was aspirated using a syringe. 5 One tumor cell was slowly inoculated subcutaneously in the right hypochondrium of the back.

[0095] 3. 7-15 days after tumor injection, wait until the tumor tissue grows to 100-200mm. 3 At approximately 10:00 PM, the CAR T cell positivity rate was detected by flow cytometry. CAR T cells were counted and aspirated, centrifuged at 950 rpm for 5 min, washed twice with 1×PBS, and the supernatant was discarded. Cells were then washed with 1×PBS at a concentration of 2.5 × 10⁻⁶ cells / mL. 7 Resuspend cells at 5 million cells / mL for later use (5 million cells / 200 μL per mouse). Draw 200 μL of CAR T cell suspension into a syringe and slowly inject it into the mouse via tail vein injection.

[0096] 4. Endpoint assessment: On day 28 after CAR T injection, major organs (heart, liver, spleen, lung, kidney and brain) of mice in each treatment group were collected for H&E and CD3 staining to assess the safety of CAR T cell therapy, and T cell infiltration was measured in tumor tissue.

[0097] The results showed that HPSE-ΔpX1-30 CAR T cells significantly reduced tumor volume and tumor size. Figure 10 It significantly prolonged the survival time of mice, and at the same time, up to 9.5% of HPSE-ΔpX1-30 CAR T cells infiltrated the tumor. Finally, HPSE-ΔpX1-30 CAR T cells also maintained good safety. Figure 11 ).

[0098] The CAR T cells of this invention exhibited significantly enhanced killing activity against 3D tumor spheroids of HCT116 colorectal cancer cells in vitro. Simultaneously, the proportion of memory-like T cells increased, while exhaustion markers such as PD-1 and CD57 decreased. In vivo experiments showed that HPSE-pX-Δ1-30 CAR T cells significantly inhibited tumor growth in tumor-bearing mice, prolonged survival, and promoted intratumoral CD3+. + T-cell infiltration. Regarding safety, no significant abnormalities were observed in body weight, liver index, or major organ pathology in mice treated with this CAR T therapy.

[0099] The CAR T cells of the present invention and their application in the preparation of drugs for the treatment of solid tumors (including colorectal cancer, breast cancer, ovarian cancer and other ECM-rich solid tumors) can significantly improve T cell tumor infiltration, anti-tumor activity and persistence, and reduce systemic toxicity, making them suitable for clinical immunotherapy.

[0100] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A chimeric antigen receptor (CAR), characterized in that, The CAR comprises: a single-chain antibody that specifically recognizes mesothelin, a CD8α hinge and transmembrane region, a 4-1BB co-stimulatory signaling domain, a CD3ζ signaling domain, and an HPSE-pX-Δ1-30 fusion protein; The gene sequence of HPSE-pX-Δ1-30 is shown in SEQ ID NO:

1.

2. The CAR according to claim 1, characterized in that, The HPSE-pX-Δ1-30 fusion protein is linked to the CAR via a 2A peptide.

3. A nucleic acid molecule encoding the CAR of claim 1, characterized in that, The sequence of the nucleic acid molecule is shown in SEQ ID NO:

2.

4. A CAR expression cassette, characterized in that, The expression cassette comprises the nucleic acid molecule of claim 3.

5. A CAR T cell, characterized in that, The CAR T cells are obtained from the CAR-modified T cells according to claim 1.

6. A method for preparing CAR T cells, characterized in that, The method includes the following steps: (1) Isolate peripheral blood mononuclear cells and T cells, and activate them with anti-CD3 / CD28 antibody for 24-48 hours; (2) Using a lentiviral vector containing the nucleic acid molecule of the CAR as described in claim 3, activated T cells are transfected at MOI = 8; (3) Expand CAR T cells in serum-free medium containing IL-2 for 10–14 days to obtain CAR T cells.

7. The method according to claim 6, characterized in that, The method further includes: (4) detecting CAR expression by flow cytometry.

8. The application of the CAR T cells of claim 5 or the CAR T cells prepared by the method of claim 6 in in vitro enhancement of tumor cell penetration and killing.

9. The use of the CAR T cells of claim 5 or the CAR T cells prepared by the method of claim 6 in enhancing the invasion and clearance of solid tumors.

10. The use of the CAR T cells of claim 5 or the CAR T cells prepared by the method of claim 6 in the preparation of a medicament for treating solid tumors.

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