CAR-T cell culture medium and application thereof

Abstract

The invention relates to the technical field of CAR-T cell culture, in particular to a CAR-T cell culture medium and application thereof. The CAR-T cell culture medium is one of CAR-T cell culture mediums in different culture stages: a T cell activation culture medium in a T cell activation stage, which comprises 2-Deoxyglucose; or a CAR-T cell culture medium in a continuous culture stage, wherein the CAR-T cell culture medium comprises 2-Deoxyglucose; or a CAR-T cell culture medium in a culture later stage, wherein the CAR-T cell culture medium comprises 2-Deoxyglucose. Compared with CAR-T cells cultured by a CAR-T cell culture medium without 2-Deoxyglucose, the CAR-T cell culture medium has the advantages that the proportion of the juvenile CAR-T cells is increased, and the anti-tumor effect is enhanced.

Description

[0001] This application is a divisional application with the application date of September 8, 2021, application number 202111049866.8, and the invention name is "CAR-T cell culture medium and its application". Technical Field

[0002] The present invention relates to the technical field of CAR-T cell culture, and in particular to a CAR-T cell culture medium and applications thereof. Background Art

[0003] Chimeric antigen receptor T-cell immunotherapy (CAR-T) involves introducing a chimeric antigen receptor (CAR) into T cells, enabling them to specifically recognize antigens on the surface of cancer cells and kill them. In recent years, this therapy has been considered one of the most promising treatments for cancer, offering numerous advantages unmatched by other therapies. However, as a novel cancer treatment, it still faces numerous limitations and shortcomings.

[0004] During CAR-T therapy, the engineered T cells are cultured in vitro to reach a therapeutic cell count before being infused back into the patient. Currently, the most established method for inducing and expanding CAR-T cells in vitro involves using anti-CD3 and anti-CD28 antibodies combined with IL-2 stimulation to induce differentiation. However, this method significantly accelerates the proliferation of differentiated CAR-T cells, significantly reducing the proportion of naive CAR-T cells and significantly increasing the number of terminal CAR-T cells. Terminal CAR-T cells have a short survival time in vivo and cannot provide long-term immune protection, making them difficult to meet clinical treatment needs. Naive CAR-T cells include CAR-TN cells, CAR-TSCM cells, and CAR-TCM cells, while terminal CAR-T cells include CAR-TEM cells and CAR-TE cells. CAR-TSCM cells and CAR-TCM cells tend to migrate to lymphoid tissues, while CAR-TEM cells and CAR-TE cells prefer peripheral tissues. Therefore, increasing the number of naive CAR-T cells is crucial for achieving optimal therapeutic effects in CAR-T therapy. In recent years, the proportion of naive CAR-T cells has been relatively increased by modifying cytokines such as IL-7 and IL-21. However, this approach still faces challenges, such as limited increase in the proportion of naive CAR-T cells and low cell proliferation. Therefore, obtaining CAR-T cells with a low differentiation state and strong anti-tumor ability remains an ongoing challenge in the field of adoptive cellular immunotherapy. Summary of the Invention

[0005] In view of this, the present invention provides a cell culture medium containing 2-Deoxyglucose or Simvastatin, which is a culture medium used in different stages of the CAR-T cell preparation process (T cell activation stage, CAR-T cell culture stage, and late CAR-T cell culture stage). The proportion of naive CAR-T cells in CAR-T cells cultured in this culture medium is significantly increased compared with CAR-T cell culture medium that does not contain 2-Deoxyglucose or Simvastatin, and the anti-tumor effect can be increased.

[0006] The cell culture medium is a CAR-T cell culture medium or a T cell culture medium, and the culture medium contains 2-Deoxyglucose or Simvastatin additives.

[0007] Preferably, the T cell culture medium is a T cell culture medium used in the T cell activation stage, which includes 2-Deoxyglucose or Simvastatin; or the CAR-T cell culture medium is one of the following CAR-T cell culture media for different culture stages: the CAR-T cell culture medium is a CAR-T cell culture medium a used in the culture stage, which includes 2-Deoxyglucose or Simvastatin; or the CAR-T cell culture medium is a CAR-T cell culture medium b used in the late culture stage, which includes 2-Deoxyglucose.

[0008] Preferably, the T cell culture medium includes 2-Deoxyglucose at a concentration of 1-3 mM.

[0009] Preferably, the T cell culture medium includes Simvastatin at a concentration of 1-3 μM.

[0010] Preferably, the CAR-T cell culture medium a includes 2-Deoxyglucose at a concentration of 1-3 mM.

[0011] Preferably, the CAR-T cell culture medium a includes Simvastatin at a concentration of 1-3 μM.

[0012] Preferably, the CAR-T cell culture medium b includes 2-Deoxyglucose at a concentration of 1-3 mM.

[0013] Furthermore, in the T cell culture medium, CAR-T cell culture medium a, or CAR-T cell culture medium b, except for 2-Deoxyglucose or Simvastatin involved in the present invention, the other components are conventional components of the culture medium used in the T cell activation stage conventionally used in the art. In certain specific embodiments, 2-Deoxyglucose or Simvastatin is added to the 1640 culture medium at different stages.

[0014] The present invention also aims to provide a method for improving the anti-tumor effect of CAR-T cells / CAR-T cell products. The method comprises adding different active substances to the culture medium used in different culture stages of CAR-T cells for cultivation. The proportion of naive CAR-T cells in the cultured CAR-T cells / CAR-T cell products is greatly increased, thereby improving the anti-tumor effect. The naive CAR-T cells include CAR-TN cells, CAR-TSCM cells, and CAR-TCM cells.

[0015] The method includes: using the aforementioned T cell culture medium for activation in the T cell activation stage; and / or using the aforementioned CAR-T cell culture medium a for culture in the CAR-T cell culture stage; and / or using the aforementioned CAR-T cell culture medium b for culture in the late CAR-T cell culture stage.

[0016] Preferably, in the T cell activation stage, the activation is performed for 24-48 hours; and / or in the CAR-T cell culture stage, the culture is performed for 1-14 days; and / or in the late CAR-T cell culture stage, the culture is performed for 36-48 hours.

[0017] Specifically, one of 2-Deoxyglucose and Simvastatin can be added to the culture medium in all three stages of CAR-T cell preparation, or one of 2-Deoxyglucose and Simvastatin can be added separately in each stage.

[0018] The present invention also provides a method for preparing CAR-T cells or CAR-T cell products using 2-deoxyglucose or simvastatin. In addition to adding 2-deoxyglucose or simvastatin to the culture medium, it is not ruled out that adding 2-deoxyglucose and / or simvastatin at other stages can also achieve the corresponding effect.

[0019] Preferably, 2-Deoxyglucose or Simvastatin is used as a culture medium additive in the preparation of CAR-T cells / CAR-T cell products.

[0020] Preferably, when preparing CAR-T cells, different culture media are used according to different preparation stages of CAR-T cells, and the 2-Deoxyglucose or Simvastatin is used as a culture medium additive in the culture media at different stages.

[0021] More preferably, the 2-Deoxyglucose is used as a culture medium additive in the culture medium used in the T cell activation stage and / or the CAR-T cell culture stage and / or the late stage of CAR-T cell culture; more preferably, the added amount of the 2-Deoxyglucose is 1-3 mM;

[0022] More preferably, the Simvastatin is used as a culture medium additive in the culture medium used in the T cell activation stage and / or the CAR-T cell culture stage. More preferably, the added amount of the Simvastatin is 1-3 μM.

[0023] The present invention also aims to provide a CAR-T cell / CAR-T cell product prepared using the aforementioned CAR-T cell culture medium. Compared to CAR-T cells / CAR-T cell products cultured in conventional culture media, these CAR-T cells / CAR-T cell products contain a higher proportion of naive CAR-Ts, effectively expanding the cell population and enhancing anti-tumor effects.

[0024] The present invention also provides a method for preparing CAR-T cells or CAR-T cell products that can enhance anti-tumor efficacy. 2-Deoxyglucose or Simvastatin can be used as an additive in the preparation of CAR-T cells or CAR-T cell products to increase the proportion of naive CAR-T cells, thereby enhancing anti-tumor efficacy.

[0025] In the present invention, the preparation of CAR-T cells includes three steps: (1) activation of T cells; (2) CAR transfection of T cells and culturing of CAR-T cells; (3) late culture period, i.e., 1-2 days before the end of culture, the culture medium and culture method of the present invention are conventionally designed through this step, and then 2-deoxyglucose or Simvastatin is added to achieve the technical effect.

[0026] In the present invention, the T cells involved are all existing types of T cells; the CARs involved are all existing types of CARs (for example, all CARs targeting CD19, BCMA or CD123 and other targets in the applicant's other patents 201710613317.6, 201710618293.3 and 201810207761.2); the methods for transfecting T cells with CAR involved are all currently known technologies, such as lentiviral transfection, liposome transfection, etc.

[0027] In the present invention, the "late stage of CAR-T cell culture" refers to the period of time during which additives are added to the culture medium 1-2 days before the end of CAR-T cell culture and culture is continued.

[0028] In the present invention, for technical solutions involving numerical values, the numerical changes caused by operating errors and equipment errors are not within the numerical range recorded in the present invention. That is to say, the numerical changes caused by operating errors and equipment errors also belong to the technical solutions of the present invention.

[0029] The beneficial effects of the present invention are

[0030] The CAR-T cells / CAR-T cell products obtained by culturing T cell activation / CAR-T cell culture / CAR-T cell late culture medium containing 2-Deoxyglucose or Simvastatin provided by the present invention have a higher proportion of naive CAR-T cells and a significantly enhanced anti-tumor effect than those obtained by culturing the corresponding culture medium without 2-Deoxyglucose or Simvastatin. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Cell phenotype analysis of CAR-T cells activated by 2-Deoxyglucose.

[0032] Figure 2 Phenotypic analysis of T cells in a mixture of CAR-transfected T cells after administration of 2-Deoxyglucose for activation.

[0033] Figure 3 Cell phenotypic analysis of CAR-T cells activated by simvastatin.

[0034] Figure 4 Phenotypic analysis of T cells in a mixture of CAR-transfected T cells after activation with simvastatin.

[0035] Figure 5 Phenotypic analysis of T cells in a mixture of CAR-transfected T cells after continuous administration of 2-Deoxyglucose.

[0036] Figure 6 Phenotypic analysis of T cells in a mixture of CAR-transfected T cells after continuous administration of simvastatin.

[0037] Figure 7 Phenotypic analysis of T cells in a mixture of CAR-transfected T cells after administration of 2-Deoxyglucose in the late culture stage.

[0038] Figure 8 Effects of activated CAR-T cells administered with 2-Deoxyglucose or simvastatin on tumors in mice. DETAILED DESCRIPTION

[0039] The examples are provided to better illustrate the present invention, but are not intended to limit the present invention to the examples. Therefore, non-essential improvements and adjustments to the embodiments made by those skilled in the art based on the above-mentioned invention still fall within the scope of protection of the present invention.

[0040] In the examples of the present invention, both the T cell culture medium and the CAR-T cell culture medium are 1640 culture medium from Gibco.

[0041] In the embodiment of the present invention, the CAR used is: a CAR in the applicant's patent 201810207761.2, whose structure is CD19ScFv-CD8 hinge region-CD28 transmembrane region-CD137-CD3 intracellular signal.

[0042] Example 1 Activated Administration of 2-Deoxyglucose

[0043] During T cell activation, 1 mM 2-Deoxyglucose was added to the T cell culture medium. CAR-carrying lentivirus was added for infection 18-24 hours after activation. After 18-24 hours of infection, the medium was changed and the cells were cultured with CAR-T cell culture medium. The cell culture density was maintained at 1*10 6 cell / ml; culture to the 7th day, take 1*10 6 The cells were subjected to flow cytometry to detect the expression of CD3, PL, CD45RA, CD45RO, and CD197;

[0044] Among them, fluorescent antibodies were used to detect the cell subtypes in all CAR-T cells, and the proportion of cell subtypes was calculated based on the expression of cell surface antigens. The results are shown in Table 1 and Figure 1 shown.

[0045] Table 1 Cell subtype proportions in CAR-T cells activated with 2-Deoxyglucose

[0046] TN TSCM TCM TEM TEFF others 2-Deoxyglucose 6.1 61.94 24.42 3.03 0.24 10.61 control 7.1 57.24 17.54 5.69 1.13 19.53 Increased ratio 8.21％ 39.22％

[0047] In Table 1, control is the blank control group of the cell subtype ratio in CAR-T cells. Figure 1 It can be seen that after adding 2-Deoxyglucose during activation, the proportion of memory phenotype CAR-T cells (CAR-T cell subtypes) increased, among which TSCM and TCM were improved.

[0048] Fluorescent antibodies were used to detect all T cell subtypes (transfected + untransfected) in the T cell mixture after transfection with CAR. The proportion of all T cell subtypes was calculated based on the expression of cell surface antigens. The results are shown in Table 2 and Figure 2 shown.

[0049] Table 2 The proportion of all T cell subtypes in the T cell mixture after activation administration of 2-Deoxyglucose transfected CAR

[0050] TN TSCM TCM TEM TEFF others 2-Deoxyglucose 11.43 53.67 24.14 3.8 1 5.96 control 13.78 50.98 13.53 4.8 3.48 13.43 Control-T 38.8 31.14 11.69 5.21 6.69 6.47 Improved ratio (compared with control) 5.27％ 78.42％

[0051] In Table 2, control is the blank control group for the proportion of all T cell subtypes in the T cell mixture after transfection with CAR; control-T is the blank control group for the proportion of all T cell subtypes before transfection with CAR. Figure 2 It can be seen that by comparing the control and control-T, it can be seen that CAR transfection has a certain impact on the T cell phenotype; by comparing the control and the addition of 2-Deoxyglucose, it can be seen that the proportion of memory phenotype CAR-T cells (CAR-T cell subtype) is significantly increased.

[0052] Example 2 Activated Administration of Simvastatin

[0053] When T cells are activated, 2μM simvastatin is added to the T cell culture medium. Lentivirus is added for infection 18-24 hours after activation. After 18-24 hours of infection, the medium is changed. Then, CAR-T cell culture medium is used for culture, and the cell culture density is maintained at 1*10 6 cell / ml, cultured to the 7th day, took 1*10 6 The cells were sent for flow cytometry to detect the expression of CD3, PL, CD45RA, CD45RO, and CD197. Fluorescent antibodies were used to detect the subtypes of CAR-T cells. The proportion of cell subtypes was calculated based on the expression of cell surface antigens. The results are shown in Table 3 and Figure 3 shown.

[0054] Table 3 Cell subtype proportions in activated CAR-T cells administered with Simvastatin

[0055]

[0056]

[0057] In Table 3, control is the blank control group of the cell subtype ratio in CAR-T cells.

[0058] Fluorescent antibodies were used to detect all T cell subtypes (transfected + untransfected) in the T cell mixture after CAR transfection, and the Figure 4 , according to the cell surface antigen expression, the proportion of T cell subtypes was calculated, and the results are shown in Table 4 and Figure 4 shown.

[0059] Table 4 The proportion of all T cell subtypes in the T cell mixture after activation administration of Simvastatin transfected CAR

[0060] TN TSCM TCM TEM TEFF others simvastatin 12.15 44.39 23.54 8.19 1.11 10.62 control 13.78 50.98 13.53 4.8 3.48 13.43 Control-T 38.8 31.14 11.69 5.21 6.69 6.47 Improved ratio (compared with control) 73.98％

[0061] In Table 4, control is the blank control group for the percentage of T cell subtypes of all T cells in the T cell mixture after CAR transfection; control-T is the blank control group for the percentage of T cell subtypes of all T cells before CAR transfection. Figure 3 and Figure 4 It was found that the proportion of memory phenotype CAR-T cells (CAR-T cell subtype) increased after the addition of simvastatin during activation.

[0062] Example 3: Continuous administration of 2-Deoxyglucose in culture

[0063] In the CAR-T cell culture medium, 2-Deoxyglucose was added at a concentration of 1mM so that the drug was present in the entire CAR-T cell culture. On the 9th day of culture, 1*10 6 The cells were sent for flow cytometry to detect the expression of CD3, PL, CD45RA, CD45RO, and CD197. Fluorescent antibodies were used to detect the CAR-T cell subtypes in the T cell mixture after CAR transfection. The proportion of CAR-T cell subtypes was calculated based on the expression of cell surface antigens. The results are shown in Table 5 and Figure 5 shown.

[0064] Table 5 The proportion of CAR-T cell subtypes in the T cell mixture after continuous administration of 2-Deoxyglucose transfected CAR

[0065] TN TSCM TCM TEM TEFF other 2-Deoxyglucose 25.76 9.52 25.85 32.79 1.79 4.29 control 36.19 21.54 4.81 7.43 13.39 16.64 Increased ratio 437.42％

[0066] In Table 5, control is the blank control group for the proportion of CAR-T cell subtypes in the T cell mixture after CAR transfection. Figure 5 As shown in the figure, the proportion of memory phenotype CAR-T cells (CAR-T cell subtype) increased after adding 2-Deoxyglucose during the CAR-T cell culture process.

[0067] Example 4: Continuous administration of simvastatin in culture

[0068] In the CAR-T cell culture medium, simvastatin was added at a concentration of 2 μM so that the drug was present in the entire CAR-T cell culture. On the 9th day of culture, 1*10 6 The cells were sent for flow cytometry to detect the expression of CD3, PL, CD45RA, CD45RO, and CD197. Fluorescent antibodies were used to detect the CAR-T cell subtypes in the T cell mixture after CAR transfection. The proportion of cell subtypes was calculated based on the expression of cell surface antigens. The results are shown in Table 6 and Figure 6 shown.

[0069] Table 6 Proportion of CAR-T cell subtypes in the T cell mixture after continuous administration of simvastatin transfected CAR

[0070] TN TSCM TCM TEM TEFF other simvastatin 39.68 34.34 5.94 9.03 1.72 9.29 control 36.19 21.54 4.81 7.43 13.39 16.64 Increased ratio 9.64％ 59.42％ 23.19％

[0071] In Table 6, control is the blank control group for the proportion of CAR-T cell subtypes in the T cell mixture after CAR transfection. Figure 6 As shown in the figure, the proportion of memory phenotype CAR-T cells (CAR-T cell subtype) increased after adding simvastatin during CAR-T cell culture.

[0072] Example 5 Administration of 2-Deoxyglucose in the Late Culture Stage

[0073] At the end of CAR-T cell culture (after the culture), add 2-Deoxyglucose at a concentration of 1mM and remove the drug after 40 hours. 6 The cells were sent for flow cytometry to detect the expression of CD3, PL, CD45RA, CD45RO, and CD197. Fluorescent antibodies were used to detect the CAR-T cell subtypes in the T cell mixture after CAR transfection. The proportion of T cell subtypes was calculated based on the expression of cell surface antigens. The results are shown in Table 7 and Figure 7 shown.

[0074] Table 7 Proportions of CAR-T cell subtypes in the T cell mixture after administration of 2-Deoxyglucose transfected with CAR in the late culture period

[0075] TN TSCM TCM TEM TEFF other 2-Deoxyglucose 46.05 18.19 1.93 6.73 10.6 16.5 control 37.85 17.83 1.56 5.18 17.67 19.91 Increased ratio 21.66％ 2.01％ 23.72％

[0076] In Table 7, control is the blank control group for the proportion of CAR-T cell subtypes in the T cell mixture after CAR transfection. Figure 7 As shown in the figure, after adding 2-Deoxyglucose for 40 hours in the late culture period, it was found that the proportion of memory phenotype CAR-T cells (CAR-T cell subtype) increased.

[0077] Example 6 Animal in vivo experiment

[0078] The experiment was conducted using CAR-T cells obtained by activated administration.

[0079] (1) Construction of mouse tumor model: NOG mice and NALM6-Luc-GFP cells were used. Cell viability was ≥70%, Luc-GFP positive rate was ≥90%, target antigen CD19 positive rate was ≥90%, and 1*10 6 Mice were injected with tumors via tail vein.

[0080] (2) Reinfusion of test article: On the third day after tumor bearing, the mice were reinfused with test article CAR-T cells. Each group of test articles consisted of 5 mice, and each mouse was reinfused with 1*10 6 Effective cell number.

[0081] (3) In vivo imaging: In vivo imaging was performed once before administration of the test article and every 7 days thereafter.

[0082] (4) Repeated injection of tumor cells: On the 76th day, the mice in the 2-Deoxyglucose and Rapamycin groups were injected with tumor cells NALM6-Luc-GFP for the second time; on the 104th day, the mice in the 2-Deoxyglucose and Rapamycin groups were injected with tumor cells NALM6-Luc-GFP for the third time.

[0083] The results are as follows Figure 8 As shown in the figure, Group-3 is simvastatin and Group-6 is 2-deoxyglucose. The results show that the CAR-T cells obtained by activating and administering simvastatin or 2-deoxyglucose were injected into mice. As can be seen from the figure, these CAR-T cells can achieve good therapeutic effects on tumors in mice.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A cell culture medium, characterized in that The cell culture medium is a CAR-T cell culture medium or a T cell culture medium, and the culture medium contains a 2-Deoxyglucose additive.

2. The cell culture medium according to claim 1, characterized in that The T cell culture medium is a T cell culture medium used in the T cell activation stage, which includes 2-Deoxyglucose; Or the CAR-T cell culture medium is one of the following CAR-T cell culture media at different culture stages: the CAR-T cell culture medium is the CAR-T cell culture medium a used in the culture stage, which includes 2-Deoxyglucose; or the CAR-T cell culture medium is the CAR-T cell culture medium b used in the late culture stage, which includes 2-Deoxyglucose.

3. The cell culture medium according to claim 2, characterized in that The T cell culture medium includes 2-Deoxyglucose at a concentration of 1-3 mM; or The CAR-T cell culture medium a includes 2-Deoxyglucose at a concentration of 1-3 mM; or Or the CAR-T cell culture medium b includes 2-Deoxyglucose at a concentration of 1-3 mM.

4. A method for improving the anti-tumor effect of CAR-T cells / CAR-T cell products, characterized in that: The method comprises: in the T cell activation stage, using the T cell culture medium in the cell culture medium according to claim 2 or 3 for activation; and / or in the CAR-T cell culture stage, using the CAR-T cell culture medium a in the cell culture medium according to claim 2 or 3 for culture; and / or in the late stage of CAR-T cell culture, using the CAR-T cell base b in the cell culture medium according to claim 2 or 3 for culture.

5. The method according to claim 4, characterized in that During the T cell activation stage, the cells are activated for 24-48 hours; and / or during the CAR-T cell culture stage, the cells are cultured for 1-14 days; and / or during the late CAR-T cell culture stage, the cells are cultured for 36-48 hours. 6.2-Application of Deoxyglucose in the preparation of CAR-T cells / CAR-T cell products.

7. The use according to claim 6, characterized in that Application of 2-Deoxyglucose as a culture medium additive in the preparation of CAR-T cells / CAR-T cell products.

8. The use according to claim 7, characterized in that When preparing CAR-T cells, different culture media are used according to different stages of CAR-T cell preparation, and the 2-Deoxyglucose is used as a culture medium additive in the culture media at different stages; The 2-Deoxyglucose is used as a culture medium additive in the culture medium used in the T cell activation stage and / or the CAR-T cell culture stage and / or the late stage of CAR-T cell culture.

9. The use according to claim 8, characterized in that The added amount of the 2-Deoxyglucose is 1-3 mM. 10.2-Deoxyglucose is used to prepare CAR-T cells / CAR-T cell products that can enhance anti-tumor effects.

11. CAR-T cells / CAR-T cell products prepared using the cell culture medium according to any one of claims 1 to 3.

Images