PA-CAR-T cell as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of cell biology, and particularly relates to a PA-CAR-T cell as well as a preparation method and application thereof. The PA-CAR-T cell comprises a CAR-T cell and a capsule shell coating the CAR-T cell, the capsule shell is obtained by alternately overlapping polylysine layers and sodium alginate layers for n times from inside to outside, and n is larger than or equal to 1. The preparation method comprises the following steps: firstly, treating qualified T cells into a single-cell suspension, then sequentially adding a polylysine solution and an alginate solution into the cell suspension, and incubating, so that polylysine layers and sodium alginate layers are alternately formed on the surfaces of the T cells. The PA-CAR-T cell provided by the invention can delay the depletion of the CAR-T cell in CAR-T cell therapy, alleviate the cytokine release syndrome caused by infusion of the CAR-T cell, and maintain the tumor killing ability of the PA-CAR-T cell.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell biology, and specifically relates to a PA-CAR-T cell and a preparation method and application thereof. Background Art

[0002] Chimeric antigen receptor T cells (CAR-T) therapy is an individualized treatment method that uses the body's own immune system. It uses genetic engineering methods to introduce receptor genes (CARs) that can recognize tumor-specific antigens to achieve specific killing of tumor cells. The modified T cells are cultured and expanded in vitro and then returned to the patient's body. It can achieve complete remission in up to 70% to 90% of patients with relapsed and refractory B-cell blood tumors, and even achieve negative micro-residual lesions.

[0003] Although the remission rate of CAR-T cell therapy for blood tumors is high, most patients still cannot avoid relapse after CAR-T cell therapy, and the relapse rate will increase over time. Studies have found that CAR-T cell exhaustion is a key factor in the relapse of most patients. T cell exhaustion refers to the situation in which T cells that are exposed to antigen stimulation and inflammatory environment for a long time in the presence of factors such as chronic infection and tumors gradually lose their immune effector function and begin to lose memory T cell characteristics after being continuously stimulated during the immune response process.

[0004] In addition, cytokine release syndrome (CRS) is a serious adverse reaction in CAR-T cell therapy and an important cause of clinical failure of CAR-T cell therapy. A study published by the American Society of Hematology in 2024 showed that 57.5% of patients developed new CRS within the first seven days after CAR-T infusion. Between the 8th and 14th days after CAR-T infusion, 5.4% of patients developed new CRS. Among all patients, the incidence of grade ≥3 CRS was 5%.

[0005] Existing measures to intervene in CAR-T cell exhaustion mainly include using PD-1, CTLA-4 antibodies, or inducing CAR-T cells to secrete PD-1 antibodies, etc., to prevent CAR-T cell exhaustion. It is also possible to delay CAR-T cell exhaustion by knocking out genes such as PD-1, CTLA-4, TIM-3, and LAG-3 for CAR-T cell modification, etc. However, the effects of these above methods are not significant. Currently, the treatment for CRS is mainly symptomatic treatment, and the commonly used drugs are hormones, antipyretics, anti-allergy drugs, and cytokine receptor antagonists (Tocilizumab, Anakinra, Etanercept), etc. The US FDA has approved the IL-6 receptor blocker (Tocilizumab) for the treatment of CRS caused by CAR-T, but these monoclonal antibodies still face some serious problems such as systemic toxicity and short effector time.

[0006] It can be seen that in the existing technology, whether it is the treatment measures to prevent CAR-T cell exhaustion or the drugs to inhibit CRS, there are defects such as insufficient efficacy or adverse reactions. Therefore, finding an effective method to optimize CAR-T cells and thereby prevent CAR-T cell exhaustion and inhibit the occurrence of CRS is an urgent problem to be solved in improving CAR-T cell therapy. Summary of the Invention

[0007] In order to solve the above technical problems, one of the purposes of the present invention is to provide a PA-CAR-T cell and its preparation method and application.

[0008] The technical solution adopted by the present invention is as follows: A PA-CAR-T cell includes a CAR-T cell and a capsule shell coating the CAR-T cell. From the inside out, the capsule shell consists of a first polylysine layer, a second sodium alginate layer,..., an nth polylysine layer, an nth sodium alginate layer, where n≥1.

[0009] Preferably, the CAR-T cell is a CD19 CAR-T cell.

[0010] Preferably, n = 2.

[0011] Another purpose of the present invention is to provide a preparation method of the above-mentioned PA-CAR-T cell, including the following steps: S1. Process the screened qualified T cells into a single-cell suspension. S2. Add a polylysine solution to the cell suspension and incubate to make the polylysine adsorb on the surface of the CAR-T cell to form a polylysine layer. S3. Add a sodium alginate solution and incubate to make the alginate wrap around the outside of the polylysine layer to form a sodium alginate layer. S4. Repeat the steps S2 and S3 once, twice or more times according to the requirements until the desired PA-CAR-T cells are obtained.

[0012] Preferably, the concentration of the single cell suspension is 1×10 5-7 cells / mL, preferably 1×10 6 cells / mL, the concentration of the polylysine solution is 0.01 - 1 mg / ml, preferably 0.1 mg / ml; the concentration of the sodium alginate solution is 0.1 - 1%, preferably 0.25%.

[0013] Preferably, in the steps S2 and S3, the incubation time is 2 - 60 min; after the incubation of the polylysine solution, the cell suspension is washed with PBS.

[0014] Preferably, the incubation time is 5 - 15 min, and after the incubation of the polylysine solution, the cell suspension is washed with PBS twice.

[0015] Preferably, in the step S1, the T cells are CD19 CAR-T cells, and the preparation method is: the primary T cells obtained by culturing human peripheral blood are amplified in vitro, and then the T cells obtained by amplified culture are mixed and cultured with lentivirus by the lentivirus transduction method to obtain T cells expressing the CD19 CAR receptor on the surface, which are CD19 CAR-T cells.

[0016] The third object of the present invention is to provide an application of the PA-CAR-T cells as described above in preparing a vector of a chimeric antigen receptor in CAR-T therapy.

[0017] The fourth object of the present invention is to provide an application of the PA-CAR-T cells as described above in preparing a CAR-T therapy reagent for delaying the in vivo exhaustion of CAR-T cells and / or inhibiting cytokine storm.

[0018] The beneficial effects of this application are as follows: Poly-L-lysine (PLL for short) is a positively charged amino acid polymer formed by polymerization of L-lysine monomers. Alginate is a natural polysaccharide widely present in the cell walls of brown algae such as kelp and giant kelp. It is considered a safe biomaterial that can be degraded in vivo and is non-toxic to human cells, and can be used as an outer layer anionic coating material for cells. Based on the layer-by-layer (LBL) self-assembly technology, this application enables the negatively charged anionic polymer and the positively charged cationic polymer to form a multi-layer thin film capsule shell outside the CAR-T cells through electrostatic interaction.

[0019] Through the screening of materials and the control of methods, the prepared PA-CAR-T cells have a small particle size, high mechanical strength, and the capsule shell is a semi-permeable membrane, allowing oxygen, nutrients, metabolites, and small molecule proteins to freely pass through, while macromolecular substances with immunological activity such as immune cells, dextran molecules with a molecular weight greater than 71KD, and immunoglobulin IgG (molecular weight 150KD) cannot pass through, thereby protecting the CAR-T cells inside the capsule shell from being attacked by the immune system.

[0020] After preparing the CAR-T cell single-cell suspension, polylysine solution is added, gently pipetted and mixed evenly, incubated at room temperature, then the polylysine is washed away with PBS. After that, sodium alginate solution is added, gently pipetted and mixed evenly, incubated at room temperature, and then the sodium alginate is washed away with PBS. The above steps are repeated to make the polylysine adsorbed on the surface of negatively charged T cells, and natural sodium alginate is tightly combined with polylysine through electrostatic deposition to form CAR-T cells coated with multi-layer materials.

[0021] The PA-CAR-T cells of the present invention form a relatively smooth capsule shell after coating (such as polylysine-sodium alginate-polylysine-sodium alginate), which can reduce the physical damage of external forces to cells, and at the same time does not affect the entry of small molecule substances such as oxygen and water into cells, ensuring the best cell viability and coating rate.

[0022] The PA-CAR-T cells provided by the present invention can delay the exhaustion of CAR-T cells in CAR-T cell therapy, reduce the cytokine release syndrome caused by the infusion of CAR-T cells, and at the same time retain their tumor killing ability. The method of coating CAR-T cells with the biocompatible material has extremely reduced operation difficulty and cost compared with other methods for delaying the exhaustion of CAR-T cells and reducing the cytokine release syndrome.

[0023] According to the present invention, T lymphocytes of healthy donors can be prepared by large-scale coating and transformation, and then used for anti-tumor treatment of autologous tumor patients. By using the new concept of immune isolation and the new technology of coating immune cells with nanobiomaterials, the exhaustion and cytokine release syndrome occurring in CAR-T cell therapy for hematological malignancies can be delayed, so it has broad application prospects. Description of the Drawings

[0024] Figure 1 It is a flow cytometry diagram of the positive rate of CD19 CAR-T cells.

[0025] Figure 2 It is a flow cytometry diagram of the effect of lentiviral transduction on T cell apoptosis.

[0026] Figure 3 It is a flow cytometry diagram of the effect of lentiviral transduction on T cell differentiation.

[0027] Figure 4 It is a schematic diagram of the synthesis of PA-CAR-T cells.

[0028] Figure 5 It is a flow cytometry diagram of the encapsulation rate of biomaterial-coated CAR-T cells.

[0029] Figure 6 It is a flow cytometry diagram of the effect of biomaterial coating on cytokine release of CAR-T cells.

[0030] Figure 7 It is a confocal laser scanning microscopy image of biomaterial-coated CAR-T cells.

[0031] Figure 8 It is a flow cytometry diagram of cytokine release in the co-culture of PA-CAR-T cells and macrophages.

[0032] Figure 9 It is a flow cytometry diagram of exhaustion molecules of CAR-T cells in the co-culture of PA-CAR-T cells and tumor cells.

[0033] Figure 10 It is a flow cytometry diagram of the killing of tumor cells by PA-CAR-T cells. Detailed implementation manners

[0034] For the sake of easy understanding, the technical solutions of the present invention will be described in more detail below in conjunction with embodiments.

[0035] PA-CAR-T: P is poly-L-lysine, A is alginate, and CAR-T is chimeric antigen receptor T-cell.

[0036] Unless otherwise specified, various raw materials, reagents, instruments and equipment used herein can be purchased through the market or prepared by existing methods.

[0037] Example 1 1. Preparation of biomaterial-coated CD19 CAR-T cells S1. Aseptic isolation of human peripheral blood mononuclear cells (PBMC): Transfer 5 ml of peripheral blood evenly into a new 15 ml centrifuge tube with a pipette, pipette up and down to mix well, centrifuge at 500 g for 10 min. After centrifugation, pipette about 2 ml of the upper pale yellow plasma with a pipette and transfer it into a new 15 ml centrifuge tube. The remaining about 3 ml of liquid is the concentrated blood cells. Dilute the concentrated blood cells with physiological sodium chloride solution of the same volume as the concentrated blood cells. Keep a 15 ml centrifuge tube containing about 3 ml of Ficoll separation solution at a 45-degree angle to the horizontal plane. Pipette the diluted blood cells and slowly transfer the diluted blood cells to the upper layer of the separation solution at a position 1 cm above the inclined surface of the separation solution, making the interface between the blood cells and the separation solution obvious. Place the centrifuge tube with the transferred blood cells in a centrifuge and centrifuge at 700 g for 30 min at room temperature. After centrifugation, gently take out the centrifuge tube from the centrifuge and place it on a centrifuge tube rack. Open the cap of the centrifuge tube, discard the plasma dilution above the white film layer with a Pasteur pipette, aspirate the white film layer (mononuclear cells) in the centrifuge tube, transfer it into a new 15 ml centrifuge tube and add an appropriate amount of physiological sodium chloride solution to make the final volume 12 ml, and invert the tube up and down to mix well. Centrifuge at 500 g for 10 min. After centrifugation, disinfect the centrifuge tube with 75% alcohol and transfer it to a biosafety cabinet, discard the supernatant. Resuspend the cells with 5 ml of physiological sodium chloride solution. After resuspension, centrifuge at 500 g for 10 min. After centrifugation is completed, disinfect the centrifuge tube with 75% alcohol and transfer it to a biosafety cabinet.

[0038] S2. In vitro expansion and culture of human T cells: Seed the primary T cells obtained in step S1 at 1×10 6 ml into X-VIVO medium, add human T cell-activator CD3 / CD28 (3 μg / ml), add 30 U / ml IL-2, and culture in a CO2 incubator at 37°C. Change the medium approximately every two days and supplement with 30 U / ml IL-2.

[0039] S3. Lentivirus preparation: One day (20 - 24 hours) before transfection, 0.4×10 6Seed the cells in a six-well plate so that the cell confluence can reach about 50% - 70% the next day. Gently mix the LipoFiter 3 liposome transfection reagent. For the cells in one well of the six-well plate to be transfected, take a clean and sterile centrifuge tube, add 4 μg of plasmid complex (second-generation CD19 CAR plasmid: PxpaX2: PMD2g = 6:3:1) to 250 μl of DMEM solution, and gently pipette to mix. Take another clean and sterile centrifuge tube, add 250 μl of DMEM solution, then add 10 μl of LipoFiter 3, and gently pipette to mix. Let it stand at room temperature for 5 minutes. Mix the DNA solution in the centrifuge tube with the LipoFiter 3 solution, and gently pipette to mix. Incubate at room temperature for 20 minutes. Add all 500 μl of the LipoFiter 3 - DNA mixture into one well of the six-well plate. When adding, pay attention to adding as evenly as possible to the whole well, then gently mix by making an "8" shape, and place it in the cell culture incubator for 6 - 12 h. Then remove the serum-free culture medium containing LipoFiter 3 - DNA, and add 2 ml of fresh serum-containing cell culture medium to each well and continue culturing. After 48 h, aspirate the supernatant and filter it through a 0.45 μm microporous filter membrane to obtain lentivirus.

[0040] S4. Lentivirus transduction: Mix 6 ml of PBS with 60 μl of RetroNectin, coat each well of a 12-well plate with 500 μl of the solution, and place it at 37°C for 1 h; discard the coating solution in the culture flask, wash twice with PBS, and wash once with T cell medium; dilute the virus supernatant with medium to 600 μl, add it to the 12-well plate, and centrifuge at 2000 g at 37°C for 2 h; Mix 300 μl of fresh T cell medium with 5×10 5 T cells, slowly add them to the virus plate, centrifuge at 1000 g at 37°C for 10 min, and let it stand and culture in an incubator at 37°C and 5% CO2; After 12 h of infection, replace with fresh medium, resuspend the T cells, and inoculate them into a six-well plate at a density of 1×10 6 / ml; Continue culturing for 60 h.

[0041] S5. Detection of the positive rate of CAR-T cells: Take the cells in the six-well plate, with the number of cells within 1×10 6 , wash the cells twice with PBS. Resuspend the cells with 1 ml of staining buffer, add 5 μl of IgG, F(ab')z fragment specific antibody, and use a BD flow cytometer to detect the proportion of positive expression of CAR-T cells in the FITC channel. The results are as Figure 1 shown. The positive rate of the CD19 CAR-T cells prepared in this example is (30 ± 7.9)%.

[0042] 2. Effects of Lentiviral Transduction on Apoptosis of CAR-T Cells Collect the CAR-T cells after lentiviral transduction. The number of cells is within 1×10 6 cells. Wash the cells twice with PBS. Resuspend the cells with 1 ml of staining buffer, add 5 μl of Hoechst33342 staining solution, gently mix, and incubate at 4°C in the dark for 15 min. Add 5 μl of PI staining solution and incubate at 4°C in the dark for 5 - 10 min after gently mixing. Wash and resuspend the cells with PBS. Detect the expression ratios of Hoechst and PI at the excitation wavelengths of 350 nm and 460 nm using a flow cytometer, and analyze the survival of CAR-T cells after lentiviral transduction. Conventional untransduced T cells are used as controls. The results are shown in Figure 2 . There is no significant difference in the apoptosis of T cells between the lentiviral transduction method in this example and the untransduced cells.

[0043] 3. Effects of Lentiviral Transduction on Differentiation of CAR-T Cells Take the cells in a six-well plate. The number of cells is within 1×10 6 cells. Wash the cells twice with PBS. Resuspend the cells with 1 ml of staining buffer, add 5 μl of CD4 and CD8 antibodies, and detect the positive expression ratios of CD4 and CD8 in the FITC channel and APC channel using a BD flow cytometer. The results are as shown in Figure 3 . After lentiviral transduction, the differentiation of CD4 and CD8 in CAR-T cells is significantly higher than that of untransduced normal T cells.

[0044] Example 2. Preparation of PA-CAR-T Cells by Coating with Biomaterials Refer to Figure 4 . In a 1 ml single-cell suspension of 1×10 6 cells, add 1 ml of polylysine solution with a concentration of 0.1 mg / ml and a molecular weight distribution range of 70000 - 150000. Gently pipette 20 times with a pipette gun to allow full contact between CAR-T cells and polylysine. Incubate at room temperature for 10 min, add 2 ml of PBS, and centrifuge at 1500 rpm for 5 min for one wash. Resuspend with 1 ml of PBS to obtain a single-cell suspension, add 1 ml of 0.25% sodium alginate solution, gently pipette 20 times with a pipette gun to allow full contact between sodium alginate and the polylysine layer, incubate at room temperature for 10 min, and centrifuge at 1500 rpm for 5 min for one wash. Coat, centrifuge, and wash in the same manner as above to obtain CAR-T cells coated with biomaterials. Preliminary studies have shown that the coating efficiency of 2 layers of biomaterials is low, and the coating of 6 layers of biomaterials has a significant impact on cell viability. Therefore, 4 layers of biomaterials are preferably used to coat CAR-T cells for subsequent experiments.

[0045] 1. Coating rate Collect 1×10 6 CAR-T cells transduced with lentivirus, and wash the cells twice with PBS. Use fluorescently labeled sodium alginate to encapsulate CAR-T cells by the above method. Detect the efficiency of biomaterial encapsulation of CAR-T with a BD flow cytometer in the FITC channel. The results are shown in Figure 5 as shown, showing that the biomaterial encapsulation rate can reach 98.1%, forming an effective encapsulation.

[0046] 2. Effect of biomaterial-encapsulated CAR-T cells on cytokine secretion of CAR-T cells Collect CAR-T cells transduced with lentivirus, encapsulate CAR-T cells by the above method, and then culture them in a six-well plate. Collect 1×10 6 cells, and wash the cells twice with PBS. Resuspend the cells with 1 ml of staining buffer, add 5 μl of TNF-α and IFN-γ antibodies, and detect the proportions of positive expressions of TNF-α and IFN-γ in CAR-T cells with a BD flow cytometer in the V450 channel and the APC channel. The results are as Figure 6 shown, showing that the biomaterial in the present invention does not affect the cytokine expression of CAR-T cells.

[0047] 3. Laser confocal microscopy of biomaterial-coated CAR-T cells Collect 1×10 6 CAR-T cells transduced with lentivirus, and wash the cells twice with PBS. First, stain with 15 μl of IgG, F(ab')z fragment specific antibody. Encapsulate CAR-T cells by the steps of Example 1 ⑥, and fluorescently label the first layer of sodium alginate and the second layer of polylysine from the outside to the inside. Centrifuge (2000 rpm x 5 min), discard the supernatant, add 4% paraformaldehyde fixative and fix at room temperature for 15 min, centrifuge and wash to discard the supernatant, then add 100 μl of ready-to-use DAPI staining working solution, and incubate at room temperature in the dark for 20 min. Wash with washing buffer (600 μl / tube); after resuspension and centrifugation, aspirate and discard the supernatant, leave 20 - 30 μl of supernatant in each tube of sample, blow the cells evenly, drop them on a glass slide, smear evenly, and air dry naturally; then drop a small drop of anti-quenching mounting medium, carefully take out the coverslip, with the cells facing down, gently buckle it on the mounting medium. Observe the surface fluorescence of the cells under a laser confocal microscope. The results are as Figure 7 shown, showing that the biomaterial can effectively encapsulate CAR-T cells.

[0048] Example 3. Co-culture experiment 1. Add the PA-CAR-T cells and CAR-T cells prepared in Example 2 and macrophages to each well of a six-well plate at a ratio of 10:1, and keep the total number of cells in each well at 1×10 6 . Co-culture for 6 days. Collect the cells in the well plate at different time points. The number of cells is within 1×10 6 . Wash the cells twice with PBS. Add 5 μl of CD3 and TNF-α flow antibodies to each sample, and detect the proportion of TNF-α positive expression of CAR-T cells by a BD flow cytometer in the PE and APC channels. The results are shown in Figure 8. The TNF-α positive expression of PA-CAR-T cells is lower than that of CAR-T cells at different time points, indicating that the biomaterial coating can effectively prevent the up-regulation of TNF-α caused by the contact between CAR-T cells and macrophages.

[0049] 2. Add the PA-CAR-T cells and CAR-T cells prepared in Example 2 and RAMOS cells to each well of a six-well plate at a ratio of 1:1, and keep the total number of cells in each well at 1×10 6 . Add fresh RAMOS cells at a ratio of 1:1 on the 3rd, 6th, and 9th days. Co-culture for 9 days. Collect the cells in the well plate at different time points. The number of cells is within 1×10 6 . Wash the cells twice with PBS and resuspend the cells with 1 ml of staining buffer. Add 5 μl of CD3 and PD-1 antibodies to each sample, and detect the proportion of PD-1 positive expression of CAR-T cells by a BD flow cytometer in the PE and APC channels. The results are shown in Figure 9 . As the co-culture time progresses, the PD-1 expression of CAR-T cells is higher on the 10th day than on the 2nd day. The expression of PD-1 of the biomaterial-coated CAR-T cells is lower than that of the uncoated CAR-T cells, indicating that PA-CAR-T can effectively delay the exhaustion of CAR-T cells.

[0050] 3. Add the PA-CAR-T cells and CAR-T cells prepared in Example 2 and RAMOS cells to each well of a six-well plate at a ratio of 5:1, and keep the total number of cells in each well at 1×10 6 . Add fresh RAMOS cells at a ratio of 5:1 on the 3rd, 6th, and 9th days. Co-culture for 9 days. Collect the cells in the well plate at different time points. The number of cells is within 1×10 6Wash the cells twice with PBS within [quantity], resuspend the cells with 400 μl of staining buffer, add 5 μl of Annexin V staining solution, gently mix and incubate at 4°C in the dark for 15 min. Add 5 μl of PI staining solution and incubate at 4°C in the dark for 15 min after gently mixing, then wash and resuspend the cells with PBS. Observe with a fluorescence microscope, and use a flow cytometer to detect the expression ratio of Annexin V and PI at excitation wavelengths of 350 nm and 460 nm, and analyze the survival of CAR-T cells after coating. Conventional uncoated CAR-T cells are used as controls. The results are shown in Figure 10. For PA-CAR-T cells, the apoptosis rate of tumor cells was lower than that of CAR-T cells at the beginning of co-culture. As time extended and the material gradually detached, there was no significant difference in the tumor-killing efficacy of CAR-T cells and uncoated CAR-T cells.

[0051] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the present invention; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention should be included within the protection scope of the present invention.

Claims

1. A PA-CAR-T cell, characterized in that: The invention comprises CAR-T cells and a capsule encapsulating the CAR-T cells. From the inside to the outside, the capsule is obtained by alternately stacking a poly-lysine layer and a sodium alginate layer n times, n≥1, each layer is 50-100 nm thick, and the diameter of the PA-CAR-T cells ranges from 7.4 to 7.8 μm.

2. The PA-CAR-T cell according to claim 1, characterized in that The CAR-T cells are CD19 CAR-T cells.

3. The PA-CAR-T cell according to claim 1, characterized in that The n=2, and the molecular weight distribution range of the poly-lysine is 70000-150000.

4. A method for preparing PA-CAR-T cells according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1. Process the qualified T cells into single cell suspension; S2. adding a poly-lysine solution to the cell suspension and incubating the cell suspension to allow the poly-lysine to be adsorbed on the surface of the CAR-T cells to form a poly-lysine layer; S3. Add sodium alginate solution and incubate to wrap the sodium alginate around the outside of the poly-lysine layer to form a sodium alginate layer; Optionally, S4. Repeat steps S2 and S3 once, twice or more, until the desired PA-CAR-T cells are obtained.

5. The preparation method according to claim 4, characterized in that The single cell suspension concentration was 1×10 5-7 / mL, preferably 1×10 6 The concentration of the poly-lysine solution is 0.01-1 mg / ml, preferably 0.1 mg / ml; the concentration of the sodium alginate solution is 0.1-1%, preferably 0.25%.

6. The preparation method according to claim 5, characterized in that In the steps S2 and S3, 0.1-5 ml of poly-lysine solution and 0.1-5 ml of sodium alginate solution are added respectively, and the mixture is gently blown 10-50 times to allow poly-lysine to be adsorbed on the surface of negatively charged T cells and sodium alginate to be adsorbed on the surface of the poly-lysine layer.

7. The preparation method according to claim 6, characterized in that The incubation time is 5 to 15 min. After the incubation with the poly-lysine solution is completed, the cell suspension is washed twice with PBS.

8. Use of the PA-CAR-T cell according to any one of claims 1 to 3 in preparing a vector of a chimeric antigen receptor in CAR-T therapy.

9. Use of the PA-CAR-T cell as claimed in any one of claims 1 to 3 as a carrier of chimeric antigen receptor in CAR-T therapy.

10. Use of the PA-CAR-T cells according to any one of claims 1 to 3 in the preparation of a reagent for CAR-T therapy that delays CAR-T cell exhaustion in vivo and / or inhibits cytokine storm.

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