A method for regulating CAR-T cells and its application

Abstract

The present invention discloses a method for regulating CAR-T cells and its application. The present invention regulates CAR-T cells through tyrosine kinase inhibitors, thereby alleviating and treating immune-related toxicities that may occur after CAR-T cells are used for treatment. The regulation method of the present invention can regulate the expression of various cytokines related to CAR-T cell immunotoxicity, perform early intervention on related toxicities, reduce the incidence and mortality of severe toxicities, and thus improve the safety of CAR-T cells. Dasatinib in the regulation method used in the present invention is an anti-tumor drug that has been approved for clinical treatment and can be applied in the treatment of various cancers, enabling the reuse of old drugs and having great economic value.

Description

Technical Field

[0001] The present invention belongs to the field of cellular immunotherapy, and particularly relates to a method for regulating CAR-T cells and its application. Background Art

[0002] Chimeric antigen receptor T-cell (CAR-T) is an immunotherapy technology that can achieve targeted killing of tumor cells by genetically modifying T cells. It shows great promise in cancer treatment, especially achieving remarkable efficacy in hematological malignancies, with a complete remission rate of up to 70%-90%. The success of CAR-T cells in the treatment of hematological tumors has inspired researchers to apply them to the treatment of solid tumors. More and more CAR-T cell therapies for solid tumors have been successively carried out and achieved certain effects. Currently, the FDA has approved 6 CAR-T cell products targeting CD19 or BCMA for the treatment of B-cell lymphoma. CAR-T is changing the treatment mode of malignant tumors and leading the tumor treatment into the era of cell therapy.

[0003] Due to the nature of CAR-T cells as living drugs, the cells are expected to persist for a long time, and the efficacy may last for decades. However, this may also be associated with severe immune-related toxicities, which thus become a challenge for the clinical application of CAR-T. Common immune-related toxicities of CAR-T treatment include cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). According to clinical CAR-T use findings, the incidence rates of CRS and ICANS are 57%-93% and 20%-70% respectively. In addition, the occurrence of CAR-T immune-related toxicities is mainly related to the acute cytokine production after CAR-T cell infusion. It has been found that CRS is characterized by acute systemic inflammation, elevated levels of circulating cytokines, and secondary organ dysfunction, involving a complex network of interactions among various cytokines. CAR-T immune-related toxicities can be controlled and treated by methods such as IL-6 receptor antagonists or corticosteroids, but severe cases may also be life-threatening. In addition, the use of corticosteroids will cause irreversible permanent damage to the activity of CAR-T cells, thus affecting the long-term treatment effect. Therefore, it is crucial to study strategies that can precisely control CAR-T cell immune-related toxicities.

[0004] Researchers optimize the structure of CAR to achieve more specific and safer targeting of tumor cells, thereby reducing the occurrence of immune-related toxicities. For example, suicide genes such as HSV-TK or iCasp9 are introduced to construct bispecific antigen CAR, inhibitory chimeric antigen receptors (iCAR), SynNotch CAR, and small molecule switch-regulated CAR. However, all of the above methods require complex genetic engineering and cannot directly control cytokine production.

[0005] Therefore, how to directly regulate the expression of cytokines in CAR-T cells by drugs, while not affecting the persistence of CAR-T cells, making it more precisely controllable has become the key to improving the safety of its clinical application. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the above prior art. For this purpose, the present invention provides a method and application for regulating CAR-T cells, which can regulate the expression of various cytokines in CAR-T cells, thereby alleviating and treating immune-related toxicities that may occur after CAR-T cells are used for treatment, including cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), and increasing the safety of CAR-T cells.

[0007] In the first aspect of the present invention, a method for regulating CAR-T cells is provided, and the method is to treat CAR-T cells with a tyrosine kinase inhibitor for 20-28 h.

[0008] In some embodiments of the present invention, the tyrosine kinase inhibitor includes dasatinib and / or its derivatives.

[0009] In some embodiments of the present invention, the derivatives include pharmaceutically acceptable salts, esters, hydrates, solvates, polymorphs, isomers or prodrugs of dasatinib.

[0010] In some embodiments of the present invention, the isomers include constitutional isomers, stereoisomers, optical isomers, regioisomers, geometric isomers.

[0011] In some embodiments of the present invention, the final concentration of the tyrosine kinase inhibitor in the system when treating CAR-T cells is 0.1-3 μM.

[0012] In some specific embodiments of the present invention, the final concentration of the tyrosine kinase inhibitor in the system when treating CAR-T cells is 1 μM.

[0013] In some specific embodiments of the present invention, the nucleotide sequence of the CAR-T cell is as shown in SEQ ID NO: 1.

[0014] In some embodiments of the present invention, the specific construction method of the CAR-T cell is derived from the existing publicly disclosed patent text: CN112940137A: A PD-1 gene knockout CAR-T cell targeting MUC1, its preparation method and application.

[0015] In some embodiments of the present invention, the chimeric antigen receptor (CAR) includes an antigen-binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain.

[0016] In some embodiments of the present invention, the antigen-binding domain is an antigen-binding fragment targeting a tumor antigen, and the scFv targeting MUC1.

[0017] In some embodiments of the present invention, the transmembrane domain is derived from CD8 and CD28.

[0018] In some embodiments of the present invention, the co-stimulatory domain is the intracellular domain of a co-stimulatory molecule.

[0019] In some embodiments of the present invention, the co-stimulatory molecule is 4-1BB.

[0020] In some embodiments of the present invention, the intracellular signaling domain is derived from CD3ζ.

[0021] In the second aspect of the present invention, there is provided the use of dasatinib and / or its derivatives as the sole active ingredient in the preparation of a preparation for inhibiting the release of cytokines by CAR-T cells.

[0022] The present invention discovers that dasatinib and / or its derivatives can be applied to the preparation of a preparation for inhibiting the release of cytokines by CAR-T cells, thereby treating the immune-related toxicities that may occur after CAR-T cells are used for treatment.

[0023] In some embodiments of the present invention, the immune-related toxicities include cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS).

[0024] In some embodiments of the present invention, the cytokines include IL-6, IL-10, IFN-γ, TNF-α, 4-1BB.

[0025] In some embodiments of the present invention, the preparation further comprises pharmaceutically acceptable excipients.

[0026] In some embodiments of the present invention, the pharmaceutically acceptable adjuvants include diluents, absorbents, wetting agents, binders, disintegrants, lubricants, colorants, coating materials, solvents, pH regulators, antibacterial agents, isotonicity regulators, chelating agents.

[0027] In some embodiments of the present invention, the preparation is an injection, and the administration methods include subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intravenous drip, intrathecal injection.

[0028] The beneficial effects of the present invention are as follows:

[0029] 1. The present invention provides a method for regulating CAR-T cells, which can directly down-regulate the expression of various CAR-T immune toxicity-related cytokines. Through this method, early intervention can be carried out on CAR-T cell immune-related toxicity, reducing the incidence and mortality of severe toxicity, thereby improving the safety of CAR-T cells.

[0030] 2. The regulatory substance used in the embodiments of the present invention is dasatinib, which is an anti-tumor drug approved for clinical treatment and can be used in the treatment of various cancers; the present invention further discovers its related symptoms of immune toxicity that occur during the process of regulating CAR-T cell therapy, which can achieve the new use of an old drug and has great economic value. Description of the Drawings

[0031] The following further describes the present invention in conjunction with the drawings and embodiments, wherein:

[0032] Figure 1 It is the positive rate of detecting the transfection of T cells into CAR-T cells by flow cytometry. Among them, Figure A is the T cells obtained by culturing and activating in Example 1, and Figure B is the CAR-T cells prepared in Example 1;

[0033] Figure 2 It is the influence of different concentrations of dasatinib on the cell viability and proliferation of CAR-T cells. Among them, Figure A is the cell survival rate of CAR-T cells, and Figure B is the number of viable cells of CAR-T cells;

[0034] Figure 3To investigate the effects of different dasatinib concentrations on cytokine expression in CAR-T cells, Figure A shows the relative mRNA expression levels of IL-6 in CAR-T cells, Figure B shows the relative mRNA expression levels of IL-10 in CAR-T cells, Figure C shows the relative mRNA expression levels of IFN-γ in CAR-T cells, and Figure D shows the relative mRNA expression levels of TNF-α in CAR-T cells;

[0035] Figure 4 To investigate the effects of different dasatinib concentrations on the relative mRNA expression levels of the T cell marker 4-1BB in CAR-T cells. Specific Embodiments

[0036] The following will clearly and completely describe the concept and technical effects of the present invention in combination with examples to fully understand the purpose, features, and effects of the present invention. Obviously, the described examples are only a part of the examples of the present invention, rather than all the examples. Based on the examples of the present invention, other examples obtained by those skilled in the art without creative efforts all fall within the scope of protection of the present invention.

[0037] The experimental materials and reagents used, unless otherwise specified, are all conventional consumables and reagents that can be obtained commercially.

[0038] Example 1 Preparation of CAR-T Cells

[0039] The CAR-T cells used in the examples of the present invention are CAR-T cells targeting MUC1. The specific preparation method can refer to the Chinese patent document CN112940137A (A PD-1 gene knockout CAR-T cell targeting MUC1, its preparation method and application). The specific operation steps are as follows:

[0040] 1. Construction of the lentiviral expression vector pLVX-EF1α-CAR 5E5

[0041] The CAR structure is composed of an antigen-binding domain, a transmembrane domain, a co-stimulatory signal transduction region, and a signal transduction domain in series. In this example, the nucleotide sequence of CAR is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. The antigen-binding domain is an antigen-binding fragment targeting a tumor antigen, specifically an scFv targeting MUC1. The transmembrane domain is derived from CD8 and CD28. The co-stimulatory domain is the intracellular domain 4-1BB of a co-stimulatory molecule. The intracellular signal transduction domain is derived from CD3ζ.

[0042] It was constructed by cloning the sequence shown in SEQ ID NO: 1 between the EcoRI and MluI restriction sites of the lentiviral vector pLVX-EF1α-IRES-Puro (purchased from Shanghai Linyuan Biotechnology Co., Ltd.) to obtain a recombinant lentiviral expression vector (named pLVX-EF1α-CAR 5E5), and then the plasmid was extracted using an Endo-free Plasmid MaxiKit (purchased from Omega). At the same time, a large amount of the helper vector plasmids (pSPAX2 and pMD2.G) for lentiviral packaging was extracted. The concentration and purity of the extracted plasmids were detected using a UV spectrophotometer, and then they were stored in a -20°C refrigerator for subsequent lentiviral packaging.

[0043] 2. Lentiviral Packaging and Purification

[0044] The cryopreserved HEK293T cells (purchased from ATCC) were thawed and passaged using DMEM complete medium (DMEM medium (Gibco) + 10% FBS). The HEK293T cells were seeded into a 10-layer cell factory at a density of 3×10 6 / mL, and 1 L of DMEM complete medium was added. After overnight culture, the cells could reach 80 - 90% confluence for plasmid transfection.

[0045] One T75 culture flask (flask A) was prepared and added with the lentiviral expression plasmid pLVX-EF1α-CAR 5E5 (840 μg), the lentiviral packaging plasmid pSPAX2 (840 μg), and the lentiviral envelope plasmid pMD2.G (420 μg), and supplemented with serum-free DMEM to 60 mL. Another T75 culture flask (flask B) was prepared and added with 5.25 mL of 1 mg / mL PEI MAX40K (polysciences), and supplemented with serum-free DMEM to 60 mL. The liquids in flasks A and B were mixed well and allowed to stand for 5 min. The solution in flask B was added to the solution in flask A, mixed well, and allowed to stand for 20 min to form a DNA-PEI complex.

[0046] The DNA-PEI complex was added to 1 L of DMEM medium containing 5% FBS, mixed well, and used to replace the culture medium in the 10-layer cell factory. The culture supernatant (about 1 L) was collected 48 h after transfection and stored in a 2 - 8°C refrigerator. At the same time, 1 L of fresh DMEM medium containing 5% FBS was added to the 10-layer cell factory, and the culture supernatant (about 1 L) was collected 24 h later and stored in a 2 - 8°C refrigerator. This process was repeated once. The approximately 3 L of culture supernatant collected three times was mixed, and a capsule filter (Sartorius) was used to remove cells and cell debris.

[0047] The clarified and filtered lentiviral supernatant was passed through a tangential flow filtration system (Sartorius The KR2I was concentrated to 200 - 300 mL. After filtration through a 0.45 μm filter membrane, chromatography purification was carried out. The purified lentivirus was sterilized by filtration through a 0.22 μm filter (Sartorius), aliquoted, and stored in a -80 °C refrigerator.

[0048] 3. Preparation of CAR-T cells

[0049] 50 mL of peripheral blood from tumor patients or healthy volunteers was collected, anticoagulated with heparin, and the serum obtained after centrifugation was inactivated at 56 °C for standby.

[0050] The precipitated cells were diluted with physiological saline and added to a centrifuge tube containing Ficoll solution (purchased from GE). Peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation, washed twice with physiological saline, and counted for standby.

[0051] PBMC was resuspended with lymphocyte medium KBM 581 serum-free cell medium (Corning), and the cell density was adjusted to 1 - 2×10 6 / mL. It was inoculated into a T75 cell culture flask, and anti-human CD3 monoclonal antibody (OKT-3) was added to activate PBMC. At the same time, 500 IU / mL of recombinant human interleukin-2 (rhIL-2) and 5 - 10% plasma were supplemented, and cultured in a 37 °C, 5% CO2 incubator to obtain activated T cells.

[0052] Take the T cells activated overnight, add the purified lentivirus (MOI = 5), centrifuge for infection, and place it in a 37 °C, 5% CO2 incubator for culture.

[0053] After 24 h of lentivirus infection, centrifuge to change the medium, resuspend it in KBM581 medium, add 5 - 10% plasma and 500 IU / mL rhIL-2 to continue amplification.

[0054] After PBMC was stimulated and cultured overnight, it was counted, centrifuged, and then resuspended with lymphocyte medium KBM 581 serum-free cell medium containing recombinant human interleukin-2 (rhIL-2) to make the density of T lymphocytes reach 2 - 5×10 6 / mL. The T lymphocytes were aliquoted into 6-well plates, and purified lentivirus solution (MOI = 5) and polybrene (final concentration 6 μg / mL) were added. Centrifuge for infection, 700 g, 1.5 h, and place it in a 37 °C, 5% CO2 incubator for culture.

[0055] After centrifugation and medium change 24 hours after lentiviral infection, the cells were resuspended in KBM581 medium, supplemented with 5-10% plasma and 500 IU / mL rhIL-2, and further amplified in a 37°C, 5% CO2 incubator. The medium was changed in a timely manner and the cells were gradually transferred to culture flasks or bags with a larger volume.

[0056] On the 3rd day after lentiviral infection, the positive rate of T cells transfected into CAR-T cells was detected by flow cytometry. The results were as Figure 1 shown. The positive rate of the CAR-T cells prepared in this example was approximately 85.9%.

[0057] Example 2: Regulation of gene expression of CAR-T cells by dasatinib

[0058] 1. Treatment of CAR-T cells with dasatinib

[0059] The CAR-T cells prepared in Example 1 were taken, centrifuged at 12,000 g for 10 min at 4°C, the supernatant was discarded, and the cells were resuspended in KBM581 medium, and the cell density was adjusted to 1×10 6 cells / mL. 2 mL of the CAR-T cell suspension was inoculated into a 12-well plate to obtain 2×10 6 cells / well. The 12 wells were divided into 4 groups, with 3 wells in each group. One group was added with dimethyl sulfoxide (DMSO) as the control group, and the other 3 groups were respectively added with equal volumes of dasatinib at different concentrations, so that the final concentrations of dasatinib in the system were 1 μM, 5 μM, and 10 μM), and cultured in a 37°C, 5% CO2 incubator. After 24 hours, 10 mL of the solution was taken from each group for cell counting and calculation of cell viability.

[0060] The formula for calculating the cell viability in this example was cell viability = number of live cells / total number of cells × 100%.

[0061] The results were as Figure 2As shown, the cell viability of CAR-T cells in the DMSO treatment group was 96.20% ± 0.44%, and the cell viabilities of CAR-T cells in the dasatinib treatment groups at 1 μM, 5 μM, and 10 μM were 97.7% ± 0.40%, 95.23% ± 0.15%, and 85.10 ± 2.56% respectively. Dasatinib at 1 μM and 5 μM had no significant effect on the viability of CAR-T cells, while dasatinib at 10 μM significantly reduced the cell viability of CAR-T cells. In addition, in the statistical graph of the number of live cells, dasatinib at 1 μM also had no significant effect on the number of live CAR-T cells, while the number of live CAR-T cells decreased significantly under the treatment of 5 μM and 10 μM dasatinib, being only 57.84% ± 5.29% and 40.17 ± 2.34% of the control group respectively. The above results indicate that dasatinib at 1 μM has no cytotoxicity to CAR-T cells and does not affect their proliferation.

[0062] 2. Extract total cellular RNA

[0063] (1) Take the CAR-T cell suspensions in the above-mentioned groups respectively, centrifuge at 500 g for 5 min at 4°C, discard the supernatant, add 0.5 mL of total RNA extraction reagent (TRIzol reagent, purchased from Nanjing Novoprotein Scientific Inc.) to resuspend the cell pellet, and mix well.

[0064] (2) Add 0.1 mL of chloroform, shake vigorously for 15 s, and let stand for 3 min. Centrifuge at 12000 g for 10 min at 4°C.

[0065] (3) Transfer the top layer liquid to a new 1.5 mL centrifuge tube, add 1 mL of isopropanol, and let stand in a -20°C refrigerator for more than 20 min.

[0066] (4) Centrifuge at 12000 g for 10 min at 4°C, discard the supernatant, and add 1 mL of 70% ethanol to wash the pellet in the centrifuge tube. Centrifuge at 7500 g for 10 min at 4°C, discard the supernatant, and repeat this step once.

[0067] (6) Place the centrifuge tube with the pellet in a laminar flow hood to dry, resuspend the pellet with 1 mL of ddH2O, and detect the concentration of the extracted RNA using a NanoDrop nucleic acid analyzer.

[0068] 3. cDNA synthesis

[0069] Use a cDNA synthesis kit (RevertAid First Strand cDNA Synthesis Kit, purchased from Thermo Fisher Scientific (China) Co., Ltd.) to reverse transcribe the total RNA extracted in the above steps into cDNA. The steps are as follows:

[0070] (1) In a new RNase-free PCR tube, prepare the reaction system shown in Table 1:

[0071] Table 1

[0072] Name Dosage Total RNA extracted in the above steps 1 μg <![CDATA[10× Reaction Buffer (containing MgCl2)]]> 1 μL Deoxyribonuclease I (EN0521) 1 μL Nuclease-free water 10 μL

[0073] (2) Vortex to mix well, centrifuge briefly, and incubate at 37 °C for 30 min;

[0074] (3) Add 1 μL of 50 mM EDTA to the mixture, vortex to mix well, centrifuge briefly, and incubate at 65 °C for 10 min.

[0075] (4) In a sterile PCR tube, prepare the reaction system shown in Table 2:

[0076] Table 2

[0077] Name Volume (μL) RNA 10 Oligo(dT)18 (from cDNA synthesis kit) 1 Nuclease-free water 1

[0078] (5) Vortex the liquid in the tube to mix well, centrifuge briefly, incubate at 65 °C for 5 min, and then immediately cool on ice;

[0079] (6) Add the components shown in Table 3 to the tube:

[0080] Table 3

[0081] Name Volume (μL) 5× Reaction Buffer 4 RiboLock RNase inhibitor (20 U / μL) 1 10 mM Deoxynucleoside Triphosphate Mix 2 RevertAid M-MuLV RT (200 U / μL) 1 Total volume 20

[0082] (7) Mix well and centrifuge briefly. Incubate the reaction solution in the tube at 42 °C for 60 min and at 70 °C for 5 min to complete the synthesis of cDNA.

[0083] 4. Detection of the relative expression levels of cytokines by real-time fluorescence quantitative PCR

[0084] Use the qPCR kit TB Premix Ex TaqTM (Tli RNaseH Plus, purchased from Takara Biotechnology (Beijing) Co., Ltd. (TAKARA)), and operate according to the instructions to detect the relative expression levels of cytokines (IL-6, IL-10, IFN-γ, TNF-α, 4-1BB) in 4 groups of CAR-T cells. The steps are as follows:

[0085] (1) Design and synthesis of qPCR primers:

[0086] The detection primers for IL-6 are IL6-F / R. The sequence of the upstream primer IL6-F is as shown in SEQ ID NO: 3; the sequence of the downstream primer IL6-R is as shown in SEQ ID NO: 4.

[0087] The detection primers for IL-10 are IL10-F / R. The sequence of the upstream primer IL10-F is as shown in SEQ ID NO: 5; the sequence of the downstream primer IL10-R is as shown in SEQ ID NO: 6.

[0088] The detection primers for IFN-γ are IFN-γ-F / R. The sequence of the upstream primer IFN-γ-F is as shown in SEQ ID NO: 7; the sequence of the downstream primer IFN-γ-R is as shown in SEQ ID NO: 8.

[0089] The detection primers for TNF-α are TNF-α-F / R. The sequence of the upstream primer TNF-α-F is as shown in SEQ ID NO: 9; the sequence of the downstream primer TNF-α-R is as shown in SEQ ID NO: 10.

[0090] The detection primers for 4-1BB are 4-1BB-F / R. The sequence of the upstream primer 4-1BB-F is as shown in SEQ ID NO: 11; the sequence of the downstream primer 4-1BB-R is as shown in SEQ ID NO: 12.

[0091] (2) Prepare the reaction system as shown in Table 4:

[0092] Table 4

[0093]

[0094] (3) Mix well and centrifuge briefly.

[0095] (4) Perform amplification according to the following PCR reaction program:

[0096] Pre-denature at 95°C for 30 s; denature at 95°C for 5 s, anneal at 60°C for 32 s, read the plate, for a total of 40 cycles.

[0097] (5) After the qPCR reaction, export the Ct values of each sample and use 2 -△△Ct to analyze the relative expression levels of cytokines in CAR-T cells treated with different concentrations of dasatinib.

[0098] The results are as Figure 3As shown, dasatinib at final concentrations of 1 μM, 5 μM, and 10 μM can all significantly reduce the mRNA expression levels of IL-6, IL-10, IFN-γ, and TNF-α in CAR-T cells. In particular, low-concentration dasatinib (1 μM) is the most effective in downregulating IL-6. The occurrence of CRS in CAR-T clinical practice is highly correlated with IL-6, IL-10, IFN-γ, TNF-α, etc., and tocilizumab (IL-6 receptor monoclonal antibody) is commonly used to block IL-6 / IL-6R to treat CRS. In addition, the expression of the activated T cell marker 4-1BB was also detected by qPCR, and the results are as Figure 4 shown. Both 1 μM and 5 μM dasatinib can significantly downregulate the mRNA expression level of 4-1BB, and the inhibitory effect of 1 μM is stronger, indicating that 1 μM dasatinib can also effectively inhibit the activation of CAR-T cells.

[0099] In summary, 1 μM dasatinib can not only significantly downregulate the expression of various cytokines related to the occurrence of immunotoxicity in CAR-T cells (related to IL-6, IL-10, IFN-γ, TNF-α), but also significantly downregulate the expression of the T cell marker 4-1BB, inhibit the activation of CAR-T cells, and 1 μM dasatinib has no cytotoxicity to CAR-T cells. Therefore, low-concentration dasatinib can be used to treat immune-related toxicities that occur during CAR-T cell therapy.

[0100] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge scope of those of ordinary skill in the art. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for regulating CAR-T cells, characterized in that, The method is to treat CAR-T cells with a tyrosine kinase inhibitor for 20 to 28 hours. The regulated CAR-T cells refer to inhibiting the expression of cytokines and CAR-T cell activation markers in CAR-T cells. The cytokines include IL-6, IL-10, IFN-γ, and TNF-α. The CAR-T cell activation marker is 4-1BB. The tyrosine kinase inhibitor is dasatinib. The CAR-T cells are PD-1 gene knockout CAR-T cells targeting MUC1. When treating CAR-T cells with the tyrosine kinase inhibitor, the final concentration in the system is 1 μM. The nucleotide sequence of the CAR of the CAR-T cells is as shown in SEQ ID NO:

1. The method is a non-disease treatment method.

2. Use of dasatinib as the sole active ingredient in the preparation of a preparation for inhibiting the release of cytokines and CAR-T cell activation markers by CAR-T cells. The cytokines include IL-6, IL-10, IFN-γ, and TNF-α. The CAR-T cell activation marker is 4-1BB. The CAR-T cells are PD-1 gene knockout CAR-T cells targeting MUC1. The nucleotide sequence of the CAR of the CAR-T cells is as shown in SEQ ID NO:

1.

3. The application according to claim 2, wherein The preparation also includes pharmaceutically acceptable excipients.

4. The application according to claim 3, wherein The pharmaceutically acceptable excipients include diluents, absorbents, wetting agents, binders, disintegrants, lubricants, colorants, coating materials, solvents, pH regulators, antibacterial agents, isotonicity regulators, chelating agents.

5. The application according to claim 2, characterized in that The preparation is an injection, and the administration methods include subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intravenous drip, and intrathecal injection.

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