A culture system, method and application thereof for enhancing memory phenotype and anti-tumor activity of CAR-T cells

Abstract

The application provides a culture system, method and application thereof for enhancing CAR-T cell memory phenotype and anti-tumor activity, relates to the technical field of biological medicine, and comprises a basic culture medium, cytokines and a synergistic agent, wherein the synergistic agent comprises IWR-1-endo and an aromatic hydrocarbon receptor antagonist. The system is used for in-vitro expansion of CAR-T cells by adding a plurality of cytokines and synergistic agents in the basic culture medium, can significantly improve the in-vitro expansion capacity of CAR-T cells, and can prolong the expansion culture time to 20 days, still has a high proportion of T stem cell memory cells (Tscm) and central memory T cells (Tcm), improves the maintenance of Tscm and Tcm phenotypes and the improvement of in-vivo and in-vitro persistent anti-tumor activity. The technical problem that the culture system in the prior art cannot efficiently expand CAR-T cells in-vitro while specifically promoting and maintaining the memory phenotype of the CAR-T cells is solved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a culture system, method, and application for enhancing the memory phenotype and antitumor activity of CAR-T cells. Background Technology

[0002] CAR-T cell therapy has achieved success in the treatment of hematologic malignancies. One of the key steps in this therapy is the large-scale expansion of genetically modified T cells in vitro to obtain a sufficient number of therapeutic cells. Currently, conventional CAR-T cell preparation procedures heavily rely on cytokine stimulation to activate and proliferate T cells.

[0003] However, existing technologies have significant limitations: First, while long-term or high-dose use of IL-2 can promote proliferation, it easily induces terminal differentiation of T cells, leading to T stem cell memory cells (Tscm) with long-term memory function and stronger reproliferative potential (CD62L). + CD45RA + T cells), and central memory T cells (Tcm), (CD62L) + CD45RA - The proportion of CAR-T cells decreases, while the proportion of short-acting effector T cells increases. Secondly, this directly affects the persistence of CAR-T cells in the patient after infusion, potentially leading to disease relapse. Furthermore, the expansion efficiency sometimes still fails to meet clinical needs. Therefore, developing a novel strategy that can efficiently expand CAR-T cells in vitro while specifically promoting and maintaining their memory phenotype is crucial for improving the long-term efficacy of CAR-T therapy.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] One of the objectives of this invention is to provide a culture system for enhancing the memory phenotype and anti-tumor activity of CAR-T cells, thereby solving the technical problem that existing culture systems cannot efficiently expand CAR-T cells in vitro while specifically promoting and maintaining their memory phenotype.

[0006] A second objective of this invention is to provide the application of the above-mentioned culture system in the preparation of CAR-T cells or in the preparation of products for the preparation of CAR-T cells.

[0007] The third objective of this invention is to provide a method for preparing CAR-T cells.

[0008] The fourth objective of this invention is to provide a CAR-T cell.

[0009] The fifth objective of this invention is to provide the application of CAR-T cells prepared by the above-described method in the preparation of drugs for treating tumors.

[0010] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a culture system for enhancing the memory phenotype and anti-tumor activity of CAR-T cells, comprising a basal culture medium, cytokines, and synergists; The synergist comprises IWR-1-endo at a final concentration of 0.5–5 µM and an aryl hydrocarbon receptor antagonist at 1 µM. The cytokines include at least one of the following: stem cell growth factor at a final concentration of 50-100 ng / ml, FMS-like tyrosine kinase 3 ligand at 50-100 ng / ml, thrombopoietin at 20-100 ng / ml, interleukin-3 at 10-20 ng / ml, interleukin-6 at 10-20 ng / ml, and interleukin-2 at 50-200 U / ml.

[0011] Furthermore, the basal culture medium is a hematopoietic cell culture medium.

[0012] Furthermore, the aromatic hydrocarbon receptor antagonist includes StemRegenin 1.

[0013] Secondly, the present invention provides the application of the above-mentioned culture system in the in vitro expansion of CAR-T cells or in the preparation of products for the in vitro expansion of CAR-T cells.

[0014] Furthermore, the product includes the aforementioned culture system; Preferably, the product includes reagents or kits.

[0015] Thirdly, the present invention provides a method for culturing CAR-T cells, including culturing CAR-T cells using the above-described culture system.

[0016] Furthermore, the cultivation time is 7-20 days; Preferably, the culture system is replaced with a new one every 2 to 3 days, preferably every 2 days.

[0017] Fourthly, the present invention provides a CAR-T cell obtained by the above-described culture method.

[0018] Furthermore, the proportion of T stem cell memory cells in the CAR-T cells is ≥40%.

[0019] Fifthly, the present invention provides the application of CAR-T cells obtained by the culture method described above in the preparation of drugs for treating tumors; Preferably, the drug comprises CAR-T cells cultured using the above-described culture method; Preferably, the drug further includes a pharmaceutically acceptable carrier.

[0020] This invention provides a culture system for enhancing the memory phenotype and antitumor activity of CAR-T cells. This system adds multiple cytokines and potentiators to the basal culture medium for in vitro expansion of CAR-T cells, significantly improving their in vitro expansion capacity. The expansion culture time can be extended to 20 days, while still maintaining a high proportion of T stem cell memory cells (Tscm) and central memory T cells (Tcm), thus enhancing the maintenance of the Tscm and Tcm phenotypes and improving their sustained in vitro and in vivo antitumor activity. This solves the technical problem in existing technologies where culture systems cannot efficiently expand CAR-T cells in vitro while specifically promoting and maintaining their memory phenotype. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 The flow cytometry dynamic analysis diagrams of CAR-T cell subpopulations obtained by amplification in different culture systems provided in the embodiments of the present invention are shown, where Q1 is terminal effector cells (Te), Q2 is T stem cell memory cells (Tscm), Q3 is central memory cells (Tcm), and Q4 is effector memory cells (Tem). Figure 2 The graphs showing the change in the proportion of long-term memory T cells in different culture systems during expansion culture according to embodiments of the present invention are shown in the figure, where **** indicates p<0.001; Figure 3 This is a statistical chart showing the fold expansion of long-term memory T cells in different culture systems provided in this embodiment of the invention. Figure 4 The graph shows the proportion and expansion fold of central memory T cells in different culture systems provided in the embodiments of the present invention. In this graph, A represents the proportion of central memory T cells and B represents the expansion fold of central memory T cells. Figure 5The results of cytotoxicity detection of tumor cells by CAR-T cells amplified in different culture systems provided in the embodiments of the present invention under co-culture with different effector-target ratios are shown. Among them, A is the result after 24 hours and B is the result after 48 hours. Figure 6 The above is a statistical chart showing the dynamic secretion level of inflammatory factors during in vitro killing of CAR-T cells obtained by expansion in different culture systems provided in the embodiments of the present invention under co-culture with different effector-target ratios. In the chart, A is IENγ, B is granzyme B, C is tumor necrosis factor α (TNF-α), and D is interleukin 2 (IL-2). Figure 7 The following is an evaluation chart of the in vitro tumor suppression effect of CAR-T cells obtained by expansion in different culture systems under different effector-target ratios in the embodiments of the present invention. In this chart, A represents the in vitro tumor killing efficiency, and B represents the remaining proportion of CAR-T cells after tumor killing. Figure 8 The results of anti-tumor detection of CAR-T cells obtained by amplification in different culture systems provided in the embodiments of the present invention in mice. Detailed Implementation

[0023] In this document, "prepared from" and "comprising" are synonymous. The terms "comprising," "including," "having," "containing," or any other variations thereof as used herein are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0024] In this document, the conjunction "composed of" excludes any unspecified elements, steps, or components. If used in a claim, this phrase will make the claim closed, meaning it does not contain any materials other than those described, except for conventional impurities associated with them.

[0025] In this document, when a quantity, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1~5” is disclosed, the described range should be interpreted as including the ranges “1~4”, “1~3”, “1~2”, “1~2 and 4~5”, “1~3 and 5”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0026] In this document, “and / or” is used to indicate that one or both of the situations described may occur, for example, A and / or B includes (A and B) and (A or B).

[0027] In this document, unless otherwise stated, arbitrary numbering is used to distinguish one entity or behavior from another, and is not required to require or imply any actual relationship, order or importance between these entities or behaviors, such as numbering I...V; A1, A2...A7; first, second...fifth, etc.

[0028] In this document, unless otherwise stated, “optional,” “optional,” “optional,” or “optional” means that the event or situation described below may, but does not have to, occur, including the circumstances in which the event or situation may or may not occur.

[0029] In this article, “each…independently selected”, “…independently selected respectively”, and “…independently selected” are interchangeable and should all be interpreted broadly. They refer to the range or options that each member of a set of variables or components can choose independently, that is, the choice of each variable or component is independent and is not affected by the choice of other variables or components.

[0030] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. 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.

[0031] This invention provides a culture system for enhancing the memory phenotype and anti-tumor activity of CAR-T cells, comprising a basal culture medium, cytokines, and synergists; The synergist comprises IWR-1-endo at a final concentration of 0.5–5 µM and an aryl hydrocarbon receptor antagonist at 1 µM. The cytokines include at least one of the following: stem cell growth factor at a final concentration of 50-100 ng / ml, FMS-like tyrosine kinase 3 ligand at 50-100 ng / ml, thrombopoietin at 20-100 ng / ml, interleukin-3 at 10-20 ng / ml, interleukin-6 at 10-20 ng / ml, and interleukin-2 at 50-200 U / ml.

[0032] This system, by adding multiple cytokines and potentiators to the basal culture medium for the in vitro expansion of CAR-T cells, significantly enhances the in vitro expansion capacity of CAR-T cells. The expansion culture time can be extended to 20 days, while still maintaining a high proportion of T stem cell memory cells (Tscm) and central memory T cells (Tcm), thus improving the maintenance of the Tscm and Tcm phenotype and enhancing the sustained anti-tumor activity in vitro and in vivo. This solves the technical problem in existing technologies where culture systems cannot efficiently expand CAR-T cells in vitro while specifically promoting and maintaining their memory phenotype.

[0033] The final concentration of IWR-1-endo can be, but is not limited to, 0.5µM, 1µM, 1.5µM, 2µM, 2.5µM, 3µM, 3.5µM, 4µM, 4.5µM or 5µM, or any value between 0.5 and 5µM.

[0034] The final concentration of stem cell growth factor (SCF) can be, but is not limited to, 50 ng / ml, 55 ng / ml, 60 ng / ml, 65 ng / ml, 70 ng / ml, 75 ng / ml, 80 ng / ml, 85 ng / ml, 90 ng / ml, 95 ng / ml or 100 ng / ml, or any value between 50 and 100 ng / ml.

[0035] The final concentration of FMS-like tyrosine kinase 3 ligand (FLT3L) can be, but is not limited to, 50 ng / ml, 55 ng / ml, 60 ng / ml, 65 ng / ml, 70 ng / ml, 75 ng / ml, 80 ng / ml, 85 ng / ml, 90 ng / ml, 95 ng / ml or 100 ng / ml, or any value between 50 and 100 ng / ml.

[0036] The final concentration of thrombopoietin (TPO) can be, but is not limited to, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml or 100 ng / ml, or any value between 20 and 100 ng / ml.

[0037] The final concentration of interleukin-3 (IL3) can be, but is not limited to, 10 ng / ml, 11 ng / ml, 12 ng / ml, 13 ng / ml, 14 ng / ml, 15 ng / ml, 16 ng / ml, 17 ng / ml, 18 ng / ml, 19 ng / ml or 20 ng / ml, or any value between 10 and 20 ng / ml.

[0038] The final concentration of interleukin-6 (IL6) can be, but is not limited to, 10 ng / ml, 11 ng / ml, 12 ng / ml, 13 ng / ml, 14 ng / ml, 15 ng / ml, 16 ng / ml, 17 ng / ml, 18 ng / ml, 19 ng / ml or 20 ng / ml, or any value between 10 and 20 ng / ml.

[0039] The final concentration of interleukin-2 (IL2) can be, but is not limited to, 50 U / ml, 100 U / ml, 120 U / ml, 140 U / ml, 160 U / ml, 180 U / ml or 200 U / ml, or any value between 50 and 200 U / ml.

[0040] In some specific embodiments, the basal culture medium is a hematopoietic cell culture medium, specifically SFEM II hematopoietic cell culture medium.

[0041] In some specific embodiments, the aromatic hydrocarbon receptor antagonist includes StemRegenin 1 (SR1).

[0042] According to another aspect of the invention, the above-described culture system is also provided for use in in vitro expansion of CAR-T cells or in the preparation of products for in vitro expansion of CAR-T cells.

[0043] In some specific embodiments, the product includes the culture system described above. In some specific embodiments, the product includes reagents or kits.

[0044] According to another aspect of the present invention, a method for culturing CAR-T cells is also provided, comprising culturing CAR-T cells using the culture system described above.

[0045] In some specific implementations, the culture time is 7 to 20 days; in some specific implementations, a new culture system is replaced every 2 to 3 days, preferably every 2 days.

[0046] According to another aspect of the present invention, a CAR-T cell is also provided, which is obtained by the culture method described above.

[0047] Data shows that CAR-T cells cultured using this medium are significantly superior to those cultured using conventional culture systems in terms of long-term memory phenotype ratio, cytotoxicity, and secretion of inflammatory factors, making it suitable for clinical translation of tumor immunotherapy.

[0048] In some specific implementations, the proportion of T stem cell memory cells in the CAR-T cells is ≥40%.

[0049] According to another aspect of the present invention, the application of CAR-T cells obtained by the culture method described above in the preparation of drugs for treating tumors is also provided; In some specific embodiments, the drug includes CAR-T cells cultured using the above-described culture method.

[0050] In some specific embodiments, the drug further includes a pharmaceutically acceptable carrier. This pharmaceutically acceptable carrier includes, but is not limited to, saline, buffer solutions, glucose, and combinations thereof.

[0051] The present invention will be further illustrated below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0052] Reagents: Hematopoietic cell culture medium SFEM II: purchased from STEMCELL Technologies (Canada), StemSpan.

[0053] IWR-1-endo: Purchased from MedChemExpress (MCE), lot number HY-12238.

[0054] Example 1 A culture system for enhancing the memory phenotype and antitumor activity of CAR-T cells includes hematopoietic cell culture medium SFEM II, 100 ng / ml SCF, 100 ng / ml FLT3L, 100 ng / ml TPO, 20 ng / ml IL3, 20 ng / ml IL6, 1 µM SR1, 200 U / ml IL2, and 1 µM IWR-1-endo.

[0055] Example 2: Preparation and In Vitro Expansion of CAR-T Cells 1. Materials: Peripheral blood from healthy donors, CD3 / CD28 activated magnetic beads, CD19-41BBz CAR lentivirus, IWR-1-endo, recombinant human IL-2, RPMI 1640 medium, hematopoietic cell medium SFEM II, fetal bovine serum, SR1, IL3, IL6, SCF, FLT3L, TPO.

[0056] 2. Grouping Experimental group: The culture system of Example 1 was used as the dry conditioned medium.

[0057] Control group: Standard culture system (RPMI 1640 + 10% FBS + 200 U / mL IL-2) was used only.

[0058] 3. T cell isolation or activation (1) Mononuclear cell acquisition: Peripheral blood from the donor is taken and diluted with an equal volume of PBS solution in an anticoagulated blood collection tube and mixed well; mononuclear cells are separated using Ficoll lymphocyte separation solution at a speed of 400g for 20-25 minutes (25 minutes in this example), with the centrifuge acceleration being 2 up and 0 down, and the white membrane layer is collected as mononuclear cells; the collected blood sample can also be directly treated with red blood cell lysis buffer to remove red blood cells and obtain red blood cell-free mononuclear cells.

[0059] (2) T cell purification: CD3 T cell negative selection was performed using the EasySep™ Human T Cell Negative Selection Kit (STEM CELL, Canada) according to the kit instructions. Specifically, the appropriate amount of negative selection antibody mixture was added according to the cell quantity, and incubated for 5 minutes; rapid spheres were vortexed for 30 seconds to homogenize the cells, and the appropriate amount of rapid spheres were added according to the instructions to bring the total volume to 2.5 ml. The mixture was then mixed and allowed to stand for 3 minutes. The test tube containing the cells was placed in a magnetic rack and allowed to stand for 3 minutes. The cells that were not bound by the magnetic beads were poured out, and the cells were centrifuged at 300g for 5 minutes. The collected cells were the T cells.

[0060] (3) T cell activation: Anti-CD3 / CD28 magnetic beads activate T cells at a ratio of 3:1, meaning 3 beads correspond to one T cell. Before use, the protective agent inside the anti-CD3 / CD28 beads needs to be removed by washing them three times with PBS. The T cells mixed with anti-CD3 / CD28 were seeded in 24-well plates at a cell density of 1~1.5×10⁻⁶ cells / wells. 6 Cells / well (Cell density in this example is 1×10⁻⁶) 6 (cells / pores).

[0061] 4. CAR-T cell expansion and culture (1) Viral transfection to prepare CAR-T cells: The centrifuge was preheated to 32℃~37℃ (in this example, it was preheated to 32℃), and the high-speed preheating was 3500rpm for about 0.5 hours; after T cells were activated for 24 hours, the T cells were centrifuged to remove the supernatant, and 500ul of viral culture medium and polybrene (10 mg / ml, 1:1000) were added; the centrifuge was set at 2000rpm, the rate of increase was 2, the rate of decrease was 0, and the temperature was 32℃~37℃ (in this example, it was 32℃) for 1 hour; then it was placed in a 37℃ carbon dioxide incubator for 6 hours, and 500ul of complete culture medium was gently added; after 24 hours of culture, the culture medium containing the virus was removed by centrifugation, the control group was replaced with standard culture medium (RPMI 1640 + 10% FBS + 200 U / mL IL-2), and other groups were replaced with the corresponding dry conditioned medium for amplification culture.

[0062] (2) CAR-T cell sorting and expansion culture: Three days after virus transfection, i.e., the fourth day of T cell culture, the virus infection efficiency was detected by flow cytometry. CAR-T cells were sorted by flow cytometry and cultured in the later stage. The medium was changed every 2-3 days thereafter (in this example, the medium change interval was 2 days) to maintain the concentration of cytokines and IWR-1-endo.

[0063] 5. Phenotypic and functional analysis of CAR-T cells Samples were taken and counted on days 4, 7, 10, 14 and 20 of culture, and CAR-T cell phenotype and function were analyzed.

[0064] 1) Flow cytometry was used to detect phenotypic markers (CD62L, CD45RA). CAR-T cell phenotypic results are as follows: Figure 1 As shown, the proportions of Q1 and Q4 in the experimental group were significantly lower than those in the control group, while the proportions of Q2 and Q3 were significantly higher than those in the control group. This demonstrates that the dry culture medium in the experimental group can maintain a high proportion of dryness in the expanded CAR-T cells, improve their self-renewal capacity, enable them to survive for a long time, and have a lasting anti-tumor effect.

[0065] 2) Amplification efficiency, such as Figure 2 and Figure 3 As shown, at the start of CAR-T cell sorting, the Tscm ratio was the same in the control group and the experimental group. After day 7, the Tscm ratio and expansion fold of the experimental group were significantly higher than those in the control group. As the culture time increased, the Tscm ratio and expansion fold of the control group gradually decreased, while the Tscm ratio of the experimental group gradually increased and remained at a high level. This indicates that the dry culture medium in the experimental group significantly promoted the proliferation of dry CAR-T cells.

[0066] 3) Memory phenotype analysis, such as Figure 4 As shown, on day 7 before culture, CD62L in the control group + CD45RA + T stem cell memory cells (Tscm), CD62L + CD45RA - The proportion of central memory T cells (Tcm) increased rapidly. On day 7, the proportion of Tcm in the control group was higher than that in the experimental group. However, after day 7, the proportion of Tcm in the control group decreased rapidly, while the experimental group maintained a higher proportion of Tcm. On day 20 of culture, CD62L in the experimental group... + CD45RA - The proportion of Tcm was as high as 35%, while that of the control group was only about 10%. Figure 4 Long-term in vitro culture data further showed that the high proportion of Tscm and Tcm in the experimental group could be maintained for a longer period of time.

[0067] Example 2 In vitro killing experiment With CD19 + Raji lymphoma cells were used as target cells (E). CAR-T cells (T) and target cells (E) were co-cultured at different effector-to-target ratios. The results were as follows: Figures 5-7 As shown in the figure. Compared with the control group, with the same number of effector cells, the experimental group of CAR-T cells showed significantly improved cytotoxicity and in vitro killing efficiency against target cells, and the remaining proportion of CAR-T cells was significantly increased, achieving sustained anti-tumor activity.

[0068] Example 3: In vivo antitumor experiment A Raji lymphoma model was established in NOD-SCID mice, and equal numbers of CAR-T cells were infused into either the experimental or control groups. Results are as follows: Figure 8 As shown, tumor growth in the experimental group mice was inhibited, survival was significantly prolonged, and high levels of CAR-T cells could still be detected in peripheral blood at longer time points after infusion, demonstrating superior in vivo persistence.

[0069] In summary, the culture system provided by this invention can improve the cell expansion efficiency, maintain the cell memory phenotype, and enhance anti-tumor activity and persistence.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A culture system for enhancing the memory phenotype and antitumor activity of CAR-T cells, characterized in that, Includes basal culture medium, cytokines, and synergists; The synergist comprises IWR-1-endo at a final concentration of 0.5–5 µM and an aryl hydrocarbon receptor antagonist at 1 µM. The cytokines include at least one of the following: stem cell growth factor at a final concentration of 50-100 ng / ml, FMS-like tyrosine kinase 3 ligand at 50-100 ng / ml, thrombopoietin at 20-100 ng / ml, interleukin-3 at 10-20 ng / ml, interleukin-6 at 10-20 ng / ml, and interleukin-2 at 50-200 U / ml.

2. The culture system according to claim 1, characterized in that, The basal culture medium is a hematopoietic cell culture medium.

3. The culture system according to claim 1, characterized in that, The aromatic hydrocarbon receptor antagonist includes StemRegenin 1.

4. The use of the culture system according to any one of claims 1 to 3 in the in vitro expansion of CAR-T cells or in the preparation of products for in vitro expansion of CAR-T cells.

5. The application according to claim 4, characterized in that, The product includes the culture system described in any one of claims 1 to 3.

6. A method for culturing CAR-T cells, characterized in that, This includes culturing CAR-T cells using the culture system described in any one of claims 1 to 3.

7. The cultivation method according to claim 6, characterized in that, The cultivation period is 7-20 days; Change to a new culture system every 2 to 3 days.

8. A CAR-T cell, characterized in that, It is obtained by the culture method described in claim 6 or 7.

9. The CAR-T cells according to claim 8, characterized in that, The proportion of T stem cell memory cells in the CAR-T cells is ≥40%.

10. The use of CAR-T cells obtained by the culture method according to claim 6 or 7 in the preparation of drugs for treating tumors; The drug comprises CAR-T cells cultured using the culture method described in claim 6 or 7; The drug also includes a pharmaceutically acceptable carrier.

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