Large-scale multiplication culture method of CAR-T cells

Abstract

The invention provides a large-scale multiplication culture method of CAR-T cells, and belongs to the technical field of CAR-T cells. The preparation method comprises the following steps: coupling bevacizumab and heparinase, immobilizing the coupled bevacizumab and heparinase on anti-CD3 / CD28 magnetic beads to prepare modified magnetic beads, resuspending CAR-T cells by using an MACS T cell culture medium containing potassium chloride, decitabine liposome and 2-deoxy-D-glucose, adding the modified magnetic beads, uniformly mixing, and performing magnetic field stimulation culture to obtain the high-activity CAR-T cells. The high-activity CAR-T cells prepared by adopting the large-scale amplification culture method of the CAR-T cells not only have better wettability, can enter a tumor microenvironment, but also can improve recruitment of the CAR-T cells in the tumor microenvironment, and have better anti-tumor activity, meanwhile, the amplification rate is obviously improved by adopting the method, and the method is suitable for large-scale in-vitro amplification culture.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of CAR-T cell, in particular to a large-scale expansion culture method of CAR-T cell. BACKGROUND

[0002] The earliest research of CAR-T cell therapy started in 1989, which is Chimeric Antigen Receptor T-Cell Immunotherapy. It is a new precise targeted therapy for tumor treatment, and has achieved good results in clinical tumor treatment in recent years through optimization and modification. It is a very promising new tumor immunotherapy method which can precisely, quickly and efficiently cure cancer.

[0003] CAR-T cell is a T cell with stable CAR structure obtained by inserting the CAR gene sequence modified by genetic engineering technology into the T cell genome through retrovirus or lentivirus. The approved CAR-T cell is composed of single chain variable fragment (ScFv) antigen recognition domain, T cell activation domain (CD3z) derived from CD3 and co-stimulatory domain (CD28 or 4-1BB). CD19 CAR-T cell therapy has a significant effect on refractory / relapsed B-cell acute lymphoblastic leukemia. However, in the treatment of solid tumors such as lung cancer, liver cancer and colorectal cancer, the treatment targets include human epidermal growth factor receptor-2 (HER2), glypican3 (GPC3) and epidermal growth factor receptor (EGFR), but the presence of tumor microenvironment makes CAR-T cells unable to effectively infiltrate around the tumor, and long-term antigen stimulation causes functional exhaustion of CAR-T cells, which cannot effectively exert the killing function.

[0004] However, the low efficiency of CAR-T cell in vitro expansion greatly limits its application. SUMMARY

[0005] The purpose of the present application is to provide a large-scale expansion culture method of CAR-T cell. The high-activity CAR-T cell prepared by the method of the present application not only has good infiltrability and can enter the tumor microenvironment, but also can improve the recruitment of CAR-T cells in the tumor environment and has good anti-tumor activity. At the same time, the method of the present application significantly improves the expansion rate and is suitable for large-scale in vitro expansion culture.

[0006] The technical scheme of the present application is implemented as follows:

[0007] The present application provides a large-scale expansion culture method of CAR-T cells, wherein bevacizumab is coupled with heparinase, and then fixed on anti-CD3 / CD28 magnetic beads to prepare modified magnetic beads, and then the CAR-T cells are resuspended in MACS T cell culture medium containing potassium chloride, decitabine liposomes and 2-deoxy-D-glucose, mixed with the modified magnetic beads, and cultured under magnetic field stimulation to obtain high-activity CAR-T cells.

[0008] As a further improvement of the present application, the following steps are included:

[0009] S1. Preparation of decitabine liposomes: soybean phospholipid, cholesterol and pegylated phospholipid are added to an ethanol / dichloromethane solvent, heated and dissolved to form a uniform oil phase, the solvent is removed under reduced pressure, an acidic buffer solution is added, stirred and hydrated, homogenized to prepare a blank liposome aqueous dispersion, decitabine is added, an alkaline buffer solution is added to adjust the pH value, incubated, and freeze-dried to prepare decitabine liposomes;

[0010] S2. Preparation of bevacizumab-modified heparinase: bevacizumab is added to water, NHS and EDC are added, stirred and activated, heparinase is added, stirred and reacted, dialyzed, and freeze-dried to prepare bevacizumab-modified heparinase;

[0011] S3. Treatment of magnetic beads: bevacizumab-modified heparinase is added to water, NHS and EDC are added, stirred and activated, anti-CD3 / CD28 magnetic beads are added, stirred and reacted, and then separated by magnet to prepare modified magnetic beads;

[0012] S4. Preparation of culture medium: potassium chloride, decitabine liposomes, dasatinib and 2-deoxy-D-glucose are added to MACS T cell culture medium to prepare the culture medium;

[0013] S5. Large-scale expansion culture of CAR-T cells: CAR-T cells are resuspended in the culture medium, mixed with modified magnetic beads, and cultured under magnetic field stimulation to obtain high-activity CAR-T cells.

[0014] As a further improvement of the present application, the mass ratio of the soybean phospholipid, cholesterol, pegylated phospholipid, and decitabine in step S1 is 10:3-5:1-2:0.5-1.5, the acidic buffer solution is Tris-HCl solution with pH=3-4, the basic buffer solution is Tris-HCl solution with pH=8-9, the pH value is adjusted to 7-7.4, the volume ratio of the ethanol / dichloromethane solvent is 3-5:7-10, the temperature of the incubation is 30-40℃, the CO2 concentration is 3-7v / v%, the rotation speed is 100-150r / min, and the time is 1-2h.

[0015] As a further improvement of the present application, the temperature of the heating in step S1 is 40-50℃, the time of the stirring hydration is 20-40min, and the rotation speed of the homogenization is 10000-15000r / min for 20-30min.

[0016] As a further improvement of the present application, the mass ratio of the bevacizumab, NHS, EDC, and heparinase in step S2 is 2-3:1-2:1-2:5-7, the time of the stirring activation is 20-30min, and the time of the stirring reaction is 10-12h.

[0017] As a further improvement of the present application, the mass ratio of the bevacizumab modified heparinase, NHS, EDC, and anti-CD3 / CD28 magnetic beads in step S3 is 10:2-3:2-3:13-15, the time of the stirring activation is 20-30min, and the time of the stirring reaction is 10-12h.

[0018] As a further improvement of the present application, in the medium in step S4, the content of potassium chloride is 50-60mmol / L, the content of decitabine liposome is 0.12-0.15g / L, the content of dasatinib is 10-15mg / L, and the content of 2-deoxy-D-glucose is 10-30mg / L.

[0019] As a further improvement of the present application, in step S5, the CAR-T cells are resuspended in the medium to a density of 10 5 -10 6 cells / mL, the modified magnetic beads are in an equal amount to the cells, and the conditions of the magnetic field stimulation culture are 30-38℃, a magnetic field strength of 10-50mT, a magnetic field frequency of 10-30Hz, and a culture time of 8-12 days.

[0020] The present application further protects a high-activity CAR-T cell obtained by a large-scale expansion culture method of the above-mentioned CAR-T cell.

[0021] The application further protects the use of the high-activity CAR-T cell in the preparation of an anti-tumor drug.

[0022] The application has the following beneficial effects:

[0023] In terms of solid tumors such as lung cancer, liver cancer, colorectal cancer, and the like, the treatment targets include human epidermal growth factor receptor 2 (HER2), glycosylphosphatidylinositol, protein glycan 3 (GPC3), epidermal growth factor receptor (EGFR), and the like, but due to the presence of a tumor microenvironment, CAR-T cells cannot effectively infiltrate the tumor periphery, and long-term antigen stimulation causes functional exhaustion of the CAR-T cells, which cannot effectively exert a killing function, and therefore, how to improve the infiltration of the CAR-T cells and increase the concentration of the CAR-T cells in the tumor periphery is an effective means to improve the anti-tumor effect.

[0024] Decitabine treatment of CAR-T cells can enhance the secretion of IFN-γ, thereby significantly improving the tumor inhibition rate, and has the effect of significantly enhancing the function of the CAR-T cells. However, decitabine has poor water solubility and is chemically unstable. Therefore, the application prepares a liposome, which reduces the decomposition rate in a suitable pH environment, and the prepared liposome has significantly improved dispersibility in water, thereby being able to significantly improve the function of the CAR-T cells and prolong the efficacy time.

[0025] The application uses bevacizumab to modify heparinase to modify anti-CD3 / CD28 magnetic beads, stimulates the overexpression of heparinase in CAR-T cells, and expresses heparinase in CAR-T cells to degrade heparan sulfate proteoglycan (HSPG), thereby enhancing tumor infiltration and anti-tumor activity. Bevacizumab can be coupled to the surface of CAR-T cells to increase the concentration of CXCL10 and CXCL11 in the tumor microenvironment, thereby promoting the recruitment of CAR-T cells in the tumor microenvironment and promoting the surface infiltration of CAR-T cells, and improving the killing power on solid tumors.

[0026] The addition of 2-deoxy-D-glucose in the culture medium can increase the content and expression of 2-deoxy-D-glucose in CAR-T cells, 2-deoxy-D-glucose enters the tumor microenvironment, destroys the tumor N-glycan barrier, and reduces the expression of CAR-T cell PD-1 / TIM-3, thereby improving the anti-tumor efficacy of CAR-T cells. The addition of the tyrosine kinase inhibitor dasatinib in the culture reduces the exhaustion induced by CAR continuous signals, and increases the proportion of memory T cells.

[0027] In the tumor microenvironment, tumor cells compete for limited resources, rapidly divide, leading to dense areas of apoptosis and necrosis, and resulting in poor patient prognosis. High extracellular potassium concentrations can trigger a starvation response in tumor-infiltrating T cells, thereby limiting T cell effector function and maintaining their stemness in vitro, and can also more effectively kill tumor cells. Therefore, this invention increases the potassium concentration in the culture medium to culture CAR-T cells without affecting the in vitro expansion efficiency of CAR-T cells, but significantly improves their anti-tumor activity.

[0028] The highly active CAR-T cells prepared by the large-scale expansion and culture method of the present invention not only have good infiltrative ability and can enter the tumor microenvironment, but also improve the recruitment of CAR-T cells in the tumor environment and have good anti-tumor activity. At the same time, the method of the present invention significantly improves the expansion rate and is suitable for large-scale in vitro expansion and culture. Detailed Implementation

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] NHS, N-hydroxysuccinimide; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0031] Example 1

[0032] This embodiment provides a method for large-scale expansion and culture of CAR-T cells, including the following steps:

[0033] S1. Preparation of decitabine liposomes: 1g soybean lecithin, 0.3g cholesterol, and 0.1g PEG2000-DSPE were added to 100mL ethanol / dichloromethane solvent (volume ratio 3:7), heated to 40℃, stirred and dissolved to form a homogeneous oil phase, the solvent was removed under reduced pressure, Tris-HCl solution with pH=3 was added, hydration was carried out by stirring for 20min, homogenization was carried out at 10000r / min for 20min to obtain a blank liposome aqueous dispersion, decitabine was added, Tris-HCl solution with pH=8 was added, the pH was adjusted to 7, the temperature was 30℃, the CO2 concentration was 3v / v%, the stirring speed was 100r / min, and the mixture was incubated for 2h, dialyzed, and freeze-dried to obtain decitabine liposomes;

[0034] S2. Preparation of bevacizumab-modified heparinase: 0.2g bevacizumab was added to 100mL of water, along with 0.1g NHS and 0.1g EDC. The mixture was stirred and activated for 20min. Then, 0.5g heparinase was added, and the mixture was stirred and reacted for 10h. The mixture was dialyzed in a 5000Da dialysis bag for 12h and then freeze-dried to obtain bevacizumab-modified heparinase.

[0035] S3. Treatment of magnetic beads: 1g of bevacizumab-modified heparinase was added to 150mL of water, along with 0.2g of NHS and 0.2g of EDC. The mixture was stirred and activated for 20min. Then, 1.3g of anti-CD3 / CD28 magnetic beads were added and the mixture was stirred and reacted for 10h. The magnets were separated, washed, and freeze-dried to obtain the modified magnetic beads.

[0036] S4. Preparation of culture medium: Potassium chloride, decitabine liposomes, dasatinib, and 2-deoxy-D-glucose were added to the MACS T cell culture medium to prepare the culture medium, wherein the content of potassium chloride was 50 mmol / L, the content of decitabine liposomes was 0.12 g / L, the content of dasatinib was 10 mg / L, and the content of 2-deoxy-D-glucose was 10 mg / L;

[0037] S5. Large-scale expansion and culture of CAR-T cells: Resuspend CAR-T cells in culture medium and adjust the cell density to 102. 5 Cells were cultured at a concentration of 10 mT / mL with an equal number of modified magnetic beads. The mixture was then incubated at 30°C with a magnetic field strength of 10 mT and a magnetic field frequency of 10 Hz for 12 days to obtain highly active CAR-T cells.

[0038] Example 2

[0039] This embodiment provides a method for large-scale expansion and culture of CAR-T cells, including the following steps:

[0040] S1. Preparation of decitabine liposomes: 1g soybean lecithin, 0.5g cholesterol, and 0.2g PEG2000-DSPE were added to 100mL of ethanol / dichloromethane solvent (volume ratio 5:10), heated to 50℃, stirred and dissolved to form a homogeneous oil phase, the solvent was removed under reduced pressure, Tris-HCl solution with pH=4 was added, hydration was carried out by stirring for 40min, homogenization was carried out at 15000r / min for 30min to obtain a blank liposome aqueous dispersion, decitabine was added, Tris-HCl solution with pH=9 was added, the pH was adjusted to 7.4, the temperature was 40℃, the CO2 concentration was 7v / v%, the stirring speed was 150r / min, and incubation was carried out for 1h, dialyzed, and freeze-dried to obtain decitabine liposomes;

[0041] S2. Preparation of bevacizumab-modified heparinase: 0.3g bevacizumab was added to 100mL of water, along with 0.2g NHS and 0.2g EDC. The mixture was stirred and activated for 30min. Then, 0.7g heparinase was added, and the mixture was stirred and reacted for 12h. The mixture was dialyzed in a 5000Da dialysis bag for 12h and then freeze-dried to obtain bevacizumab-modified heparinase.

[0042] S3. Treatment of magnetic beads: 1g of bevacizumab-modified heparinase was added to 150mL of water, along with 0.3g of NHS and 0.3g of EDC. The mixture was stirred and activated for 30min. Then, 1.5g of anti-CD3 / CD28 magnetic beads were added and the mixture was stirred and reacted for 12h. The magnets were separated, washed, and freeze-dried to obtain the modified magnetic beads.

[0043] S4. Preparation of culture medium: Potassium chloride, decitabine liposomes, dasatinib, and 2-deoxy-D-glucose were added to the MACS T cell culture medium to prepare the culture medium, wherein the content of potassium chloride was 60 mmol / L, the content of decitabine liposomes was 0.15 g / L, the content of dasatinib was 15 mg / L, and the content of 2-deoxy-D-glucose was 30 mg / L;

[0044] S5. Large-scale expansion and culture of CAR-T cells: Resuspend CAR-T cells in culture medium and adjust the cell density to 102. 6 Cells were cultured at a concentration of 38°C, with an equal number of modified magnetic beads added and mixed thoroughly. The magnetic field strength was 50 mT and the magnetic field frequency was 30 Hz. The cells were then stimulated for 8 days to obtain highly active CAR-T cells.

[0045] Example 3

[0046] This embodiment provides a method for large-scale expansion and culture of CAR-T cells, including the following steps:

[0047] S1. Preparation of decitabine liposomes: 1g soybean lecithin, 0.4g cholesterol, and 0.15g PEG2000-DSPE were added to 100mL of ethanol / dichloromethane solvent (volume ratio 4:8), heated to 45℃, stirred and dissolved to form a homogeneous oil phase, the solvent was removed under reduced pressure, Tris-HCl solution with pH=3.5 was added, and the mixture was stirred and hydrated for 30min. The mixture was homogenized at 12000r / min for 25min to obtain a blank liposome aqueous dispersion. Decitabine was added, and Tris-HCl solution with pH=8.5 was added to adjust the pH to 7.2. The temperature was 35℃, the CO2 concentration was 5v / v%, the stirring speed was 120r / min, and the mixture was incubated for 1.5h. The mixture was dialyzed and freeze-dried to obtain decitabine liposomes.

[0048] S2. Preparation of bevacizumab-modified heparinase: 0.25 g bevacizumab was added to 100 mL of water, along with 0.15 g NHS and 0.15 g EDC. The mixture was stirred and activated for 25 min. Then, 0.6 g heparinase was added, and the mixture was stirred and reacted for 11 h. The mixture was dialyzed through a 5000 Da dialysis bag for 12 h and then freeze-dried to obtain bevacizumab-modified heparinase.

[0049] S3. Treatment of magnetic beads: 1g of bevacizumab-modified heparinase was added to 150mL of water, along with 0.25g of NHS and 0.25g of EDC. The mixture was stirred and activated for 25min. Then, 1.4g of anti-CD3 / CD28 magnetic beads were added and the mixture was stirred and reacted for 11h. The magnets were separated, washed, and freeze-dried to obtain the modified magnetic beads.

[0050] S4. Preparation of culture medium: Potassium chloride, decitabine liposomes, dasatinib, and 2-deoxy-D-glucose were added to the MACS T cell culture medium to prepare the culture medium, wherein the content of potassium chloride was 55 mmol / L, the content of decitabine liposomes was 0.135 g / L, the content of dasatinib was 12 mg / L, and the content of 2-deoxy-D-glucose was 20 mg / L;

[0051] S5. Large-scale expansion and culture of CAR-T cells: Resuspend CAR-T cells in culture medium and adjust the cell density to 102. 6 Cells were cultured at a concentration of 35°C, with an magnetic field strength of 30 mT and a magnetic field frequency of 20 Hz for 10 days to obtain highly active CAR-T cells.

[0052] Comparative Example 1

[0053] The difference from Example 3 is that step S1 was not performed.

[0054] Specifically as follows:

[0055] S1. Preparation of bevacizumab-modified heparinase: 0.25g bevacizumab was added to 100mL of water, along with 0.15g NHS and 0.15g EDC. The mixture was stirred and activated for 25min. Then, 0.6g heparinase was added, and the mixture was stirred and reacted for 11h. The mixture was dialyzed through a 5000Da dialysis bag for 12h and then freeze-dried to obtain bevacizumab-modified heparinase.

[0056] S2. Treatment of magnetic beads: 1g of bevacizumab-modified heparinase was added to 150mL of water, along with 0.25g of NHS and 0.25g of EDC. The mixture was stirred and activated for 25min. Then, 1.4g of anti-CD3 / CD28 magnetic beads were added and the mixture was stirred and reacted for 11h. The magnets were then separated to obtain the modified magnetic beads.

[0057] S3. Preparation of culture medium: Potassium chloride, decitabine, dasatinib, and 2-deoxy-D-glucose were added to the MACS T cell culture medium to prepare the culture medium, wherein the content of potassium chloride was 55 mmol / L, the content of decitabine was 0.135 g / L, the content of dasatinib was 12 mg / L, and the content of 2-deoxy-D-glucose was 20 mg / L.

[0058] S4. Large-scale expansion and culture of CAR-T cells: Resuspend CAR-T cells in culture medium and adjust the cell density to 102. 6 Cells were cultured at a concentration of 35°C, with an magnetic field strength of 30 mT and a magnetic field frequency of 20 Hz for 10 days to obtain highly active CAR-T cells.

[0059] Comparative Example 2

[0060] The difference from Example 3 is that steps S2 and S3 were not performed.

[0061] Specifically as follows:

[0062] S1. Preparation of decitabine liposomes: 1g soybean lecithin, 0.4g cholesterol, and 0.15g PEG2000-DSPE were added to 100mL of ethanol / dichloromethane solvent (volume ratio 4:8), heated to 45℃, stirred and dissolved to form a homogeneous oil phase, the solvent was removed under reduced pressure, Tris-HCl solution with pH=3.5 was added, and the mixture was stirred and hydrated for 30min. The mixture was homogenized at 12000r / min for 25min to obtain a blank liposome aqueous dispersion. Decitabine was added, and Tris-HCl solution with pH=8.5 was added to adjust the pH to 7.2. The temperature was 35℃, the CO2 concentration was 5v / v%, the stirring speed was 120r / min, and the mixture was incubated for 1.5h. The mixture was then freeze-dried to obtain decitabine liposomes.

[0063] S2. Preparation of culture medium: Potassium chloride, decitabine liposomes, dasatinib, and 2-deoxy-D-glucose were added to the MACS T cell culture medium to prepare the culture medium, wherein the content of potassium chloride was 55 mmol / L, the content of decitabine liposomes was 0.135 g / L, the content of dasatinib was 12 mg / L, and the content of 2-deoxy-D-glucose was 20 mg / L;

[0064] S3. Large-scale expansion and culture of CAR-T cells: Resuspend CAR-T cells in culture medium and adjust the cell density to 102. 6Add an equal number of anti-CD3 / CD28 magnetic beads to the cells at a concentration of 35°C, a magnetic field strength of 30 mT, and a magnetic field frequency of 20 Hz. Stimulate the cells with the magnetic field for 10 days to obtain highly active CAR-T cells.

[0065] Comparative Example 3

[0066] The difference from Example 3 is that decitabine liposomes were not added in step S4.

[0067] Specifically as follows:

[0068] S4. Preparation of culture medium: Potassium chloride, dasatinib, and 2-deoxy-D-glucose were added to the MACS T cell culture medium to prepare the culture medium, wherein the content of potassium chloride was 55 mmol / L, dasatinib was 12 mg / L, and the content of 2-deoxy-D-glucose was 20 mg / L.

[0069] Comparative Example 4

[0070] The difference from Example 3 is that potassium chloride was not added in step S4.

[0071] Specifically as follows:

[0072] S4. Preparation of culture medium: Decitabine liposomes, dasatinib, and 2-deoxy-D-glucose were added to the MACS T cell culture medium to prepare the culture medium, wherein the content of decitabine liposomes was 0.135 g / L, dasatinib was 12 mg / L, and the content of 2-deoxy-D-glucose was 20 mg / L.

[0073] Comparative Example 5

[0074] The difference from Example 3 is that dasatinib was not added in step S4.

[0075] Specifically as follows:

[0076] S4. Preparation of culture medium: Potassium chloride, decitabine liposomes, and 2-deoxy-D-glucose were added to the MACS T cell culture medium to prepare the culture medium, wherein the content of potassium chloride was 55 mmol / L, the content of decitabine liposomes was 0.135 g / L, and the content of 2-deoxy-D-glucose was 20 mg / L.

[0077] Comparative Example 6

[0078] The difference from Example 3 is that 2-deoxy-D-glucose was not added in step S4.

[0079] Specifically as follows:

[0080] S4. Preparation of culture medium: Potassium chloride, decitabine liposomes, and dasatinib were added to the MACS T cell culture medium to prepare the culture medium, wherein the content of potassium chloride was 55 mmol / L, the content of decitabine liposomes was 0.135 g / L, and the content of dasatinib was 12 mg / L.

[0081] Test Example 1

[0082] Highly active CAR-T cells cultured in Examples 1-3 or Comparative Examples 1-6 were seeded into K562 cell lines at an effector-to-target ratio of 40:1 in 96-well plates, while another group was seeded into BT549 cell lines at an effector-to-target ratio of 5:1 in 96-well plates. After co-culturing for 5 hours, the cells were centrifuged, the supernatant was discarded, and 100 ml of DMSO was added to each well for shaking and dissolution for 10 min. The absorbance (A value) was measured at 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Blank control, target cell control, and effector cell control were also included. The absorbance of each well was subtracted from the value of the blank control well, and the average A value of the three replicates was calculated. The cytotoxic activity of the effector cells was calculated based on the killing rate. The results are shown in Table 1.

[0083] Kill rate (%) = [Target cell control A value - (Experimental well A value - Effector cell control A value)] / Target cell control A value × 100%

[0084] Table 1

[0085]

[0086] As shown in the table above, the highly active CAR-T cells obtained by the methods in Examples 1-3 of this invention have a high killing rate against both the K562 cell line and the BT549 cell line.

[0087] Test Example 2

[0088] An appropriate amount of target cells A-673 were placed in 96-well plates as the positive target cell group. Highly active CAR-T cells cultured in Examples 1-3 or Comparative Examples 1-6 were added to the corresponding wells at an effector cell to target cell ratio of 25:1. Each sample was tested in triplicate. DMEM-Basic medium containing 10% fetal bovine serum was used as the co-culture system. After incubation at 37°C for 24 h, the 96-well plates were centrifuged at 300g for 15 min at room temperature, and 120 µL of supernatant was collected. The IFN-γ content (pg / mL) was detected at room temperature according to the instructions of the enzyme-linked immunosorbent assay kit. The results are shown in Table 2.

[0089] Table 2

[0090]

[0091] As can be seen from the table above, the content of IFN-γ secreted by the highly active CAR-T cells obtained by the methods in Examples 1-3 of this invention is significantly increased.

[0092] Test Example 3

[0093] The in vitro expansion efficiency and cell viability of highly active CAR-T cells cultured in Examples 1-3 and Comparative Examples 1-6 were calculated. The results are shown in Table 3.

[0094] Expansion efficiency = (Total number of cells after culture × Percentage of CAR-T cells in the total number of cells) / (Total number of cells before culture × Percentage of CAR-T cells in the total number of cells)

[0095]

[0096] As can be seen from the table above, the highly active CAR-T cells obtained by the methods in Examples 1-3 of this invention have high expansion efficiency and high cell survival rate.

[0097] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. 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 method for large-scale expansion culture of CAR-T cells, characterized by, After coupling bevacizumab with heparinase, fixing on anti-CD3 / CD28 magnetic beads, modified magnetic beads are prepared, and then CAR-T cells are resuspended with MACS T cell medium containing potassium chloride, decitabine liposomes and 2-deoxy-D-glucose, mixed with modified magnetic beads, and cultured under magnetic field stimulation to obtain high-activity CAR-T cells.

2. The method for large-scale expansion and culture of CAR-T cells according to claim 1, characterized in that, It comprises the following steps: S1. Preparation of decitabine liposomes: soybean phospholipid, cholesterol and pegylated phospholipid are added to ethanol / dichloromethane solvent, heated to dissolve, form a uniform oil phase, remove the solvent under reduced pressure, add an acidic buffer solution, stir to hydrate, homogenize, prepare a blank liposome aqueous dispersion, add decitabine, add a basic buffer solution, adjust the pH value, incubate, freeze-dry to prepare decitabine liposomes; S2. Preparation of bevacizumab-modified heparinase: bevacizumab is added to water, NHS and EDC are added, stirred to activate, heparinase is added, stirred to react, dialyzed, freeze-dried to prepare bevacizumab-modified heparinase; S3. Magnetic bead treatment: Bevacizumab-modified heparinase is added to water, NHS and EDC are added, stirred to activate, anti-CD3 / CD28 magnetic beads are added, stirred to react, and the modified magnetic beads are prepared by magnetic separation; S4. Preparation of medium: potassium chloride, decitabine liposomes, dasatinib and 2-deoxy-D-glucose are added to MACS T cell medium to prepare the medium; S5. Large-scale expansion culture of CAR-T cells: CAR-T cells are resuspended with the medium, mixed with modified magnetic beads, and cultured under magnetic field stimulation to obtain high-activity CAR-T cells.

3. The large-scale expansion culture method of the CAR-T cell according to claim 2, characterized by, In step S1, the mass ratio of soybean phospholipid, cholesterol, pegylated phospholipid and decitabine is 10:3-5:1-2:0.5-1.5, the acidic buffer solution is Tris-HCl solution with pH=3-4, the basic buffer solution is Tris-HCl solution with pH=8-9, the pH value is adjusted to 7-7.4, the volume ratio of ethanol / dichloromethane solvent is 3-5:7-10, the incubation temperature is 30-40℃, the CO2 concentration is 3-7v / v%, the rotation speed is 100-150r / min, and the time is 1-2h.

4. The large-scale expansion culture method of the CAR-T cell according to claim 2, characterized by, In step S1, the heating temperature is 40-50℃, the stirring hydration time is 20-40min, and the homogenization rotation speed is 10000-15000r / min for 20-30min.

5. The large-scale expansion culture method of the CAR-T cell according to claim 2, characterized by, In step S2, the mass ratio of bevacizumab, NHS, EDC and heparinase is 2-3:1-2:1-2:5-7, the stirring activation time is 20-30min, and the stirring reaction time is 10-12h.

6. The method of claim 2, wherein the CAR-T cells are expanded in a large scale culture. In step S3, the mass ratio of bevacizumab-modified heparinase, NHS, EDC and anti-CD3 / CD28 magnetic beads is 10:2-3:2-3:13-15, the stirring activation time is 20-30min, and the stirring reaction time is 10-12h.

7. The method of claim 2, wherein the CAR-T cells are expanded in a large scale culture.

8. The method of claim 2, wherein the CAR-T cells are expanded in a large scale culture. The content of potassium chloride in the medium in step S4 is 50-60 mmol / L, the content of decitabine liposome is 0.12-0.15 g / L, the content of dasatinib is 10-15 mg / L, and the content of 2-deoxy-D-glucose is 10-30 mg / L.

8. The method for large-scale expansion and culture of CAR-T cells according to claim 2, characterized in that, The CAR-T cells in step S5 are resuspended in medium to a density of 10 5 -10 6 cells / mL, the modified magnetic beads are in equal number to the cells, the culture conditions of the magnetic field stimulation are 30-38°C, the magnetic field strength is 10-50 mT, the magnetic field frequency is 10-30 Hz, and the culture time is 8-12 days.

9. A high-activity CAR-T cell obtained by the large-scale expansion culture method of the CAR-T cell according to any one of claims 1-8.

10. Use of the high-activity CAR-T cell according to claim 9 in the preparation of an antitumor drug.

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