Use of sodium bicarbonate in preparation of enhancer for MSLN-car-t treatment of solid tumors

Abstract

The use of sodium bicarbonate in the preparation of an enhancer for an MSLN-CAR-T treatment of solid tumors. The therapeutic effect of MSLN-CAR-T in solid tumors is improved by means of treating solid tumors with sodium bicarbonate and then performing an MSLN-CAR-T treatment.

Description

[Corrected according to Rule 26 24.10.2025] Use of sodium bicarbonate in the preparation of MSLN-CAR-T treatment of solid tumor enhancer TECHNICAL FIELD

[0001] The present application relates to the field of tumor treatment, in particular to the use of sodium bicarbonate in combination with MSLN-CAR-T in the preparation of a drug for treating solid tumors. BACKGROUND

[0002] Sodium bicarbonate is widely used in clinical practice for the treatment of metabolic acidosis, alkalization of urine, excessive gastric symptoms and non-specific drug poisoning. Some clinical trials use high-dose NaHCO3 to study the safety and pain relief of cancer patients. In the basic research of prostate cancer, colorectal cancer, liver cancer or spontaneous metastasis mouse models, oral NaHCO3 can increase the pH value of the tumor environment, significantly prevent metastasis, and does not affect the growth of the primary tumor. However, the role of sodium bicarbonate in CAR-T therapy has not been studied.

[0003] Chimeric antigen receptor T (CAR-T) cells use single-chain antibodies (scFvs) and T cell activation domains to modify T cells. When CAR is expressed on the T cell membrane, genetically modified CAR-T cells can recognize tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs) expressed on target cells and initiate killing functions, eliminating cancer cells without interacting with major histocompatibility complexes (MHCs). In recent years, CAR-T cells have achieved encouraging clinical results in the treatment of hematological tumors. However, solid tumors progress slowly, and CAR-T cell therapy for solid tumors has many limitations, such as antigen deficiency, antigen loss, and loss, antigen heterogeneity, T cell infiltration failure, T cell exhaustion and microenvironment immunosuppression, etc.

[0004] MSLN is a membrane glycoprotein expressed in normal mesothelial cells, but highly expressed in mesothelioma, breast cancer, ovarian cancer, lung cancer, pancreatic cancer and other cancers. In addition, some antibody-based therapeutic drugs, as well as vaccines and t cell therapies targeting MSLN are being clinically evaluated, but the results are not satisfactory. How to improve the efficacy of MSLN-CAR-T cell therapy for solid tumors needs further exploration. SUMMARY

[0005] Therefore, one of the purposes of the present application is to provide a use of sodium bicarbonate in the preparation of MSLN-CAR-T treatment of solid tumor enhancer; the second purpose of the present application is to provide a use of sodium bicarbonate in combination with MSLN-CAR-T in the preparation of a drug for treating solid tumors.

[0006] To achieve the above-mentioned purposes, the present application provides the following technical solutions:

[0007] 1. A pharmaceutical preparation for treating solid tumors, the pharmaceutical preparation comprising sodium bicarbonate and MSLN-CAR-T cells.

[0008] Preferably, in the pharmaceutical preparation, the sodium bicarbonate is orally administered, and the MSLN-CAR-T cells are injected.

[0009] Preferably, the concentration of the orally administered sodium bicarbonate is 200 mM.

[0010] 2. A method for treating solid tumors, wherein the pH is adjusted to 7.4-8.5 by sodium bicarbonate, the cells are treated at different pH values, and then MSLN-CAR-T cells are added at a target-to-effector ratio of 1:1-1:10.

[0011] Preferably, the solid tumors are triple-negative breast cancer, pancreatic cancer, ovarian cancer, lung cancer, or colorectal cancer.

[0012] The present application provides a method and application of sodium bicarbonate and MSLN-CAR-T for treating solid tumors, and further improves the therapeutic effect of MSLN-CAR-T on solid tumors, thereby providing a new idea for CAR-T treatment of solid tumors and laying a foundation for clinical treatment of solid tumors. BRIEF DESCRIPTION OF DRAWINGS

[0013] In order to make the objectives, technical solutions, and beneficial effects of the present application clearer, the present application is described below with reference to the following drawings:

[0014] FIG. 1 shows the positive rate of MSLN-CAR-T cells detected by flow cytometry.

[0015] FIG. 2 shows the cell killing efficiency detected by LDH after MDA-MB-231 and HCC38 cells were pretreated with different pH values of medium for 48 h, and then co-cultured with T cells or MSLN-CAR-T cells at an E:T ratio of 1:0.5, 1:1, 1:5, and 1:10 for 6 h (A: MDA-MB-231; B: HCC38 cells; all experiments were performed at least three times, and representative data from one experiment are shown. Quantitative data are expressed as mean ± SD, and P values were calculated using unpaired two-tailed t tests).

[0016] FIG. 3 shows the changes in transplanted tumors in MDA-MB-231 tumor-bearing mice at 10, 14, 18, 20, and 22 days after inoculation, and the changes in transplanted tumors in HCC38 tumor-bearing mice at 10, 14, 18, 20, and 22 days after inoculation.

[0017] FIG. 4 shows the positive rate of MSLN-CAR-T cells detected by flow cytometry.

[0018] Figure 5 shows the cell killing efficiency detected by LDH after PANC-1, AsPC-1 cells were pretreated with different pH value of medium for 48h, and then co-cultured with T cells or MSLN-CAR-T cells at E:T ratio of 1:1, 1:5, 1:10 for 6h. All experiments were performed at least three times, and representative data from one experiment were shown. Quantitative data were presented as mean ± SD, and P value was calculated using unpaired two-tailed t test.

[0019] Figure 6 shows the changes of transplanted tumors in AsPC-1 tumor-bearing mice at 6, 11, 18, 21, 26 days after inoculation, and the changes of transplanted tumors in PANC-1 tumor-bearing mice at 6, 9, 13, 16, 20 days after inoculation.

[0020] Figure 7 shows the positive rate of MSLN-CAR-T cells detected by flow cytometry.

[0021] Figure 8 shows the cell killing efficiency detected by LDH after SK-OV-3 cells were pretreated with different pH value of medium for 48h, and then co-cultured with T cells or MSLN-CAR-T cells at E:T ratio of 1:1, 1:5, 1:10 for 6h. All experiments were performed at least three times, and representative data from one experiment were shown. Quantitative data were presented as mean ± SD, and P value was calculated using unpaired two-tailed t test.

[0022] Figure 9 shows the changes of transplanted tumors in SK-OV-3 tumor-bearing mice at 6, 9, 13, 16, 20 days after inoculation. DETAILED DESCRIPTION

[0023] The present application will be further described below in conjunction with the accompanying drawings and specific examples, so that those skilled in the art can have a better understanding.

[0024] The reagents used in the present application and their numbers are as follows:

[0025] Ficoll (GE, 17144003); fetal bovine serum (Gibco / Life Technologies, 10099-141); interleukin-2 (IL-2; Proteintech, HZ-1015); CD3 / CD28 beads (1:3; B&L Biotech, GMP-TL601); polybrene (8 μg / mL; Beyotime, ST1380-10g); MSLN-CAR vector purchased from Suzhou ICARTAB Company (LIC012A); Lipo3000 (Invitrogen, L3000015); Lenti-X (TaKaRa, 2109257A); HIV ELISA kit (PerkinElmer, NEK050001KT); MSLN pe conjugated antibody (RD, FAB32652P); Protein L (Pierce, 2997)-streptavidin APC (Invitrogen, S21374); EGFR-FITC (ICARTAB, 21092702); sodium bicarbonate (Solebao); lactate dehydrogenase (LDH) assay kit (Beyotime, C0017); Matrigel (Biocoat, 356231); NOG mice (CIEA, Charles River); fluorescein (2 μg / 20 g; Promega, P1043).

[0026] Example 1

[0027] The application of sodium bicarbonate and MSLN-CAR-T combined therapy for triple negative breast cancer, the specific steps are as follows:

[0028] (1) MSLN-CAR-T cell preparation

[0029] A, collect fresh blood of healthy volunteers, all subjects are informed consent, use Ficoll to separate normal peripheral blood mononuclear cells, and culture the primary cells in medium containing 10% fetal bovine serum and 100 U / ml interleukin-2.

[0030] B, activate T cells in mononuclear cells: use anti-human CD3 and CD28 antibodies to activate T cells, and culture in medium containing 10% fetal bovine serum and 100 U / ml interleukin-2.

[0031] C. Preparation of MSLN-CAR-T cells: MSLN-CAR vector (the second generation of MSLN-CAR vector contains msln-specific target single-chain antibody, with a CD8 signal sequence, two intracellular costimulatory signal regions (CD3 zeta and 4-1BB), a CD8 hinge region and a transmembrane sequence) and 3 helper packaging plasmids (psPAX2, pRRE and pRSV-Rev) were transfected with Lipo3000, and the filtered 48 or 72 h HEK293T cell culture supernatant was collected. The supernatant was added to the virus supernatant with virus concentration reagent Lenti-X at a ratio of 3:1, centrifuged at 4°C, 4000g for 30 minutes, resuspended, and the virus was obtained for standby. The virus titer was determined using the HIV ELISA kit. Part of the T cells in the activated mononuclear cells were inoculated into retronectin-coated culture plates, and added with virus and polybrene, centrifuged at 32°C, 900g for 90 minutes, and incubated at 37°C for 24 hours; finally, the MSLN-CAR-T cells were cultured in normal culture medium. Another part of the T cells in the activated mononuclear cells were used as control group T cells for continuous culture.

[0032] D. Flow cytometry detection of MSLN-CAR-T positive rate: centrifugal collection of cells, PBS washing 2 times; next, the cells were stained, precipitated and washed according to the antibody instructions; finally, the cells were incubated at 4°C in PBS containing 2% FBS for flow cytometry (BD, FACSAria II). The expression of MSLN on cancer cells was detected using human MSLN pe conjugated antibody (RD, FAB32652P), the expression of CAR on CAR-T cells was detected using EGFR-FITC and protein L-streptavidin APC, and the data analysis was performed using Flow Jo 7.6.1, and the results are shown in Figure 1. The results showed that the percentage of CAR-T cells expressing MSLN-scFv was about 33%.

[0033] (2) Preparation of triple-negative breast cancer cells

[0034] Take out the MDA-MB-231, HCC38 cell (human triple negative breast cancer cell line) freeze tube from the liquid nitrogen tank, immediately put it into the 37°C water bath, shake it from time to time, and melt it within 1 minute; wipe the surface of the freeze tube clean, sterilize it with alcohol, and then perform subsequent operations on the clean bench; transfer the cells in the freeze tube into a 10ml centrifuge tube containing 5ml DMEM medium, mix with a glass dropper; place the 10ml centrifuge tube containing MDA-MB-231, HCC38 cells in a horizontal centrifuge, 1200rpm / minute, centrifuge for 10 minutes; discard the supernatant, add 5ml DMEM high-sugar (containing 10% fetal bovine serum by volume fraction) complete medium, mix with a glass dropper, and transfer the cells into a 50ml cell culture bottle; 37°C, 5% CO2 incubator for 12 hours.

[0035] (3) Effect of combination therapy of sodium bicarbonate and MSLN-CAR-T

[0036] A, sodium bicarbonate treatment of tumor cells, using sodium bicarbonate to adjust the pH of the culture medium, so that the pH is 6.5, 7.4 and 8.5, and the concentration of 2×10 4 MDA-MB-231 and HCC38 cells are inoculated in 96-well plates and cultured in different pH value culture medium for 48 hours.

[0037] B, in vitro killing: collect control T cells and MSLN-CAR-T cells, add them to a 96-well plate at a ratio of 1:0.5, 1:1, 1:5 and 1:10 (target cells: effector cells). The target cells refer to tumor cells, and the effector cells refer to T cells or MSLN-CAR-T cells. The lactate dehydrogenase (LDH) level is determined by using a kit. Add 10μl of 10×lysis buffer and react for 1h, then centrifuge at 400×g for 5min, transfer 60μl of supernatant to a new plate, and add 60μl of LDH working solution. Incubate the plate in the dark for 30min, and detect the absorbance (OD) values at 490nm and 620nm. Each sample uses 4-5 replicates. Calculate the cytotoxicity (%) by the OD values of the sample and the control. The results are shown in Figure 2. The results show that under the same pH culture conditions, the killing effect of untreated T cells on TNBC cells has no significant difference. The killing effect of MSLN-CAR-T cells on TNBC cells at pH 8.5 and pH 7.4 is higher than that at pH 6.5. Under alkaline conditions, the killing efficiency of MSLN-CAR-T cells on HCC38 cells is greatly improved to 80%.

[0038] C, in vivo killing: MDA-MB-231, HCC38 cells (1×10 6) with Matrigel and PBS at a ratio of 50:50 subcutaneously injected into the right subcutaneous of female NOG mice. NaHCO3(200 mM) was added to the drinking water, and CAR-T cells (5 x 10 6 ) were injected via the tail vein 3 days after tumor implantation. One day before treatment, the mice were imaged, divided into groups with approximately equal tumor burden, randomly assigned to treatment groups, and kept blind until the end of the study. Imaging after intraperitoneal injection of luciferin (2 μg / 20 g), the effect of tumor treatment was evaluated by live mouse imaging (BLT, AniView100). The results are shown in Figure 3, and the results show that the treatment group treated with sodium bicarbonate has reduced fluorescence intensity. It is shown that sodium bicarbonate can improve the killing ability of MSLN-CAR-T on breast cancer cells.

[0039] This example is the best embodiment of the present application.

[0040] Example 2

[0041] The application of sodium bicarbonate and MSLN-CAR-T combined treatment of pancreatic cancer, the specific steps are as follows:

[0042] (1) Preparation of MSLN-CAR-T cells

[0043] A, collect fresh blood of healthy volunteers, all subjects are informed consent, use Ficoll to separate normal human peripheral blood mononuclear cells, and culture the primary cells in medium containing 10% fetal bovine serum and 100 U / ml interleukin-2.

[0044] B, activate T cells in mononuclear cells. Use microspheres coated with anti-human CD3 and CD28 antibodies, culture in medium containing 10% fetal bovine serum and 100 U / ml interleukin-2, and activate T cells.

[0045] C, obtain MSLN-CAR-T cells by lentiviral transduction of T cells

[0046] MSLN-CAR vector (the second generation MSLN-CAR vector contains msln specific target single-chain antibody, with a CD8 signal sequence, two intracellular costimulatory signal regions (CD3 zeta and 4-1BB), a CD8 hinge region and a transmembrane sequence) and 3 helper packaging plasmids (psPAX2, pRRE and pRSV-Rev) were transfected with Lipo3000, and the filtered 48 or 72 h HEK293T cell culture supernatant was collected. The supernatant was added to the virus supernatant with virus concentration reagent Lenti-X at a ratio of 3:1, centrifuged at 4°C, 4000g for 30 minutes, resuspended, and the virus was obtained for standby. The virus titer was determined using the HIV ELISA kit. Part of the T cells in the activated mononuclear cells were inoculated into retronectin-coated culture plates, and the virus and polybrene were added, centrifuged at 32°C, 900g for 90 minutes, and incubated at 37°C for 24 hours. Finally, the MSLN-CAR-T cells were cultured in normal culture medium. Another part of the T cells in the activated mononuclear cells were used as control T cells for continuous culture.

[0047] D, flow cytometry detection of MSLN-CAR-T positive rate, centrifugal collection of cells, PBS washing 2 times. Next, according to the antibody instructions, the cells were stained, precipitated and washed. Finally, the cells were incubated at 4°C in PBS containing 2% FBS for flow cytometry (BD, FACSAria II). Human MSLN pe conjugated antibody (RD, FAB32652P) was used to detect the expression of MSLN on cancer cells. EGFR-FITC and protein L-streptavidin APC were used to detect CAR expression on CAR-t cells. Data analysis was performed using Flow Jo 7.6.1, and the results are shown in Figure 4.

[0048] (2) Preparation of pancreatic cancer cells

[0049] The PANC-1, AsPC-1 cell (human pancreatic cancer cell line) cryopreservation tube was taken out from the liquid nitrogen tank and immediately placed in a 37°C water bath, shaken from time to time, and melted within 1 minute. The surface of the cryopreservation tube was wiped clean, alcohol disinfected, and subsequent operations were performed on the clean bench. The cells in the cryopreservation tube were transferred to a 10 ml centrifuge tube containing 5 ml of DMEM medium, and mixed with a glass dropper. The 10 ml centrifuge tube containing PANC-1, AsPC-1 cells was placed in a horizontal centrifuge at 1200 rpm / min for 10 minutes. The supernatant was discarded, 5 ml of DMEM high glucose (containing 10% fetal bovine serum by volume fraction) complete culture medium was added, and the cells were mixed with a glass dropper and transferred to a 50 ml cell culture bottle. The cells were cultured at 37°C in a 5% CO2 incubator for 12 hours.

[0050] (3) Effect of sodium bicarbonate and MSLN-CAR-T combination therapy

[0051] A, bicarbonate treatment of tumor cells, using sodium bicarbonate to adjust the pH of the culture medium, so that the pH is 6.5, 7.4 and 8.5, 2×10 4 PANC-1, AsPC-1 cells were seeded in 96-well plates and cultured in different pH value of the medium for 24 hours.

[0052] B, in vitro killing, T cells and MSLN-CAR-T cells were collected, and added to 96-well plates at a ratio of 1:1, 1:5 and 1:10 (target cells:effector cells) for 6h. The lactate dehydrogenase (LDH) level was determined by using the kit. 10μl of 10×lysis buffer was added for 1h, and after centrifugation at 400×g for 5min, 60μl of supernatant was transferred to a new plate, and 60μl of LDH working solution was added. The plate was incubated in the dark for 30min, and the absorbance (OD) values at 490nm and 620nm were detected. Each sample used 4-5 replicates. The cytotoxicity (%) was calculated by the OD values of the sample and the control. The results are shown in Figure 5. Under alkaline conditions, the killing efficiency of MSLN-CAR-T cells to AsPC-1 cells was greatly improved to 80%.

[0053] C, in vivo killing. PANC-1, AsPC-1 cells carrying luciferase (1×10 6 ) were injected subcutaneously into the right subcutaneous of female NOG mice at a ratio of 50:50 of Matrigel and PBS. NaHCO3(200mM) was added to the drinking water, and CAR-T cells (5×10 6 ) were injected into the tail vein 3 days after tumor implantation. One day before treatment, the mice were imaged, and they were divided into groups with approximately equal tumor load, randomly assigned to treatment groups, and kept blind until the end of the study. Imaging was performed after intraperitoneal injection of luciferin (2μg / 20g), and the effect of tumor treatment was evaluated by live mouse imaging (BLT, AniView100). The results are shown in Figure 6. The results show that the treatment group treated with sodium bicarbonate has a reduced fluorescence intensity, indicating that sodium bicarbonate can improve the killing ability of MSLN-CAR-T to breast cancer cells.

[0054] The detection results of this embodiment are the same as those of Example 1.

[0055] Example 3

[0056] The application of sodium bicarbonate and MSLN-CAR-T combined treatment of ovarian cancer, the specific steps are as follows:

[0057] (1) MSLN-CAR-T cell preparation

[0058] A, Fresh blood was collected from healthy volunteers with informed consent. Peripheral blood mononuclear cells were isolated by Ficoll and primary cells were cultured in medium containing 10% fetal bovine serum and 100 U / ml interleukin-2.

[0059] B, T cells in mononuclear cells were activated. Microspheres coated with anti-human CD3 and CD28 antibodies were used to culture T cells in medium containing 10% fetal bovine serum and 100 U / ml interleukin-2.

[0060] C, MSLN-CAR-T cells were obtained by lentiviral transduction of T cells. The second-generation MSLN-CAR vector contains a single-chain antibody specific for msln, with a CD8 signal sequence, two intracellular costimulatory signal regions (CD3 zeta and 4-1BB), a CD8 hinge region, and a transmembrane sequence. Virus was filtered and collected from the supernatant of HEK293T cells transfected with MSLN-CAR vector and three helper packaging plasmids (psPAX2, pRRE, and pRSV-Rev) for 48 or 72 h using Lipo3000. Lenti-X was added to the virus supernatant at a ratio of 3:1. Cells were resuspended in fresh medium at a ratio of 1:3 for standby. Virus titer was determined using an HIV ELISA kit.

[0061] D, Flow cytometry was used to detect the positive rate of MSLN-CAR-T. Cells were collected by centrifugation and washed twice with PBS. Next, cells were stained, precipitated, and washed according to the antibody instructions. Finally, cells were incubated in PBS containing 2% FBS at 4°C for flow cytometry (BD, FACSAria II). Human MSLN pe conjugated antibody (RD, FAB32652P) was used to detect the expression of MSLN on cancer cells. EGFR-FITC and protein L-streptavidin APC were used to detect CAR expression on CAR-T cells. Flow Jo 7.6.1 was used for data analysis, and the results are shown in Figure 7.

[0062] (2) Preparation of ovarian cancer cells

[0063] Take out the SK-OV3 cell (human ovarian cancer cell line) frozen tube from the liquid nitrogen tank, immediately put it into the 37°C water bath, shake it from time to time, and melt it within 1 minute; wipe the surface of the frozen tube clean, sterilize it with alcohol, and then perform the subsequent operation on the clean bench; transfer the cells in the frozen tube into a 10ml centrifuge tube containing 5ml DMEM medium, mix with a glass dropper; place the 10ml centrifuge tube containing SK-OV3 cells in a horizontal centrifuge, 1200rpm / minute, centrifuge for 10 minutes; discard the supernatant, add 5ml DMEM high-sugar (containing 10% fetal bovine serum by volume fraction) complete medium, mix with a glass dropper, and transfer the cells into a 50ml cell culture bottle; 37°C, 5% CO2 incubator for 12 hours.

[0064] (3) Effect of sodium bicarbonate and MSLN-CAR-T combination therapy

[0065] A, sodium bicarbonate treatment of tumor cells. Adjust the pH of the culture medium with sodium bicarbonate to 6.5, 7.4 and 8.5, and add 2x10 4 SK-OV3 cells were seeded in 96-well plates and cultured in medium with different pH values for 24 hours.

[0066] B, in vitro killing. T cells and MSLN-CAR-T cells were collected and added to 96-well plates at a ratio of 1:1, 1:5 and 1:10 (target cells: effector cells) for 6h. The lactate dehydrogenase (LDH) level was determined using a kit. Add 10μl of 10x lysis buffer for 1h, centrifuge at 400xg for 5min, then transfer 60μl of supernatant to a new plate, add 60μl of LDH working solution. Incubate the plate in the dark for 30min, and detect the absorbance (OD) values at 490nm and 620nm. Each sample uses 4-5 replicates. Calculate the cytotoxicity (%) by the OD values of the sample and the control, and the results are shown in Figure 8. The results show that the killing efficiency of MSLN-CAR-T cells on SK-OV3 cells is greatly improved.

[0067] C, in vivo killing. SK-OV3 cells carrying luciferase (1x10 6 ) were injected subcutaneously into the right flank of female NOG mice at a ratio of 50:50 with Matrigel and PBS. Add NaHCO3 (200mM) to the drinking water, and inject CAR-T cells (5x10 6). One day before treatment, the mice were imaged, divided into groups with approximately equal tumor burden, randomly assigned to treatment groups, and kept blind until the end of the study. After intraperitoneal injection of luciferin (2 μg / 20 g), the tumor treatment effect was evaluated by live mouse imaging (BLT, AniView100), and the results are shown in Figure 9. The results show that the treatment group treated with sodium bicarbonate has reduced fluorescence intensity, indicating that sodium bicarbonate can improve the killing ability of MSLN-CAR-T on ovarian cancer cells.

[0068] The detection results obtained in this example are the same as those in Example 1.

[0069] Since MSLN is highly expressed in other solid tumors such as breast cancer, lung cancer, colorectal cancer, and since the MSLN target is improved after pH treatment in colorectal cancer and lung cancer, it is speculated that the combination of sodium bicarbonate and MSLN-CAR-T can be used for the treatment of colorectal cancer and lung cancer.

[0070] The above examples are only preferred examples for fully illustrating the present application, and the protection scope of the present application is not limited thereto. Any equivalent replacement or transformation made by those skilled in the art on the basis of the present application is within the protection scope of the present application. The protection scope of the present application is subject to the claims.

Claims

1. A pharmaceutical preparation for the treatment of solid tumors, characterized in that: The pharmaceutical preparation comprises sodium bicarbonate and MSLN-CAR-T cells.

2. The pharmaceutical preparation according to claim 1, characterized in that: The sodium bicarbonate in the pharmaceutical preparation is orally taken and the MSLN-CAR-T cells are injected.

3. The pharmaceutical preparation according to claim 1, characterized in that: The oral concentration of sodium bicarbonate is 200 mM.

4. A method of treating a solid tumor, comprising: The cells are treated at different pH values by adjusting the pH to 7.4-8.5 with sodium bicarbonate, and then the MSLN-CAR-T cells are added at a target-effector ratio of 1:1-1:

10.

5. Use according to claim 3, characterized in that: The solid tumor is triple-negative breast cancer, pancreatic cancer, ovarian cancer, lung cancer or colorectal cancer.

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