Culture medium for enhancing tumor cell killing capability of CAR-T cells and application of culture medium

Abstract

The invention discloses a culture medium and a method for enhancing the tumor cell killing capability of CAR-T cells in a low-oxygen environment. Specifically, the culture medium comprises a CAR-T culture medium and a drug selected from the following group: palbociclib, rapamycin, ensidipine or a combination thereof. The CAR-T cells cultured by using the culture medium or the method disclosed by the invention have survival persistence and functional effects in a solid tumor microenvironment, so that the effect of a CAR-T therapy in solid tumor treatment is improved.

Description

Technical Field

[0001] This invention relates to the fields of cell immunotherapy and cell culture technology, specifically to a culture medium that enhances the ability of CAR-T cells to kill tumor cells and its application. Background Technology

[0002] Chimeric antigen receptor T-cell (CAR-T cell) therapy has become a breakthrough immunotherapy for treating various cancers. By genetically engineering T cells to express CAR-T cells that recognize specific tumor antigens, it precisely and effectively targets and eliminates cancer cells. This personalized treatment has achieved significant success in hematologic malignancies such as acute lymphoblastic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL). However, currently available CAR-T cell products for treating solid tumors are generally less effective than those for hematologic malignancies. A major reason for the difficulty in overcoming the challenges in treating solid tumors lies in their highly hypoxic tumor environment. Hypoxia is a prominent feature of the solid tumor microenvironment. Due to insufficient angiogenesis caused by rapid tumor proliferation, hypoxic areas often appear within the tumor. Hypoxic environments inhibit the effector functions of CAR-T cells through multiple mechanisms: First, hypoxia induces tumor cells to express more immunosuppressive molecules (such as PD-L1), enhancing their immune evasion ability; second, under hypoxic conditions, the proliferation, differentiation, and effector functions (such as cytotoxicity and cytokine secretion) of T cells are all inhibited; finally, hypoxic environments also promote the survival and expansion of tumor stem cell-like cells, which are generally more tolerant to CAR-T cell therapy.

[0003] To address this issue, several solutions have been proposed: 1. Increasing blood supply to the tumor region and improving the hypoxic environment by using angiogenesis promoters (such as VEGF). However, angiogenesis not only improves oxygen supply to the tumor region but may also promote tumor growth and metastasis, making the tumor more malignant. 2. Delivering oxygen to the tumor region using oxygen carriers such as artificial hemoglobin or hydrogen peroxide to alleviate hypoxia. However, oxygen carriers may induce oxidative stress, leading to damage to normal cells, and there are also concerns about uneven distribution and safety in vivo. 3. Adapting CAR-T cells to a hypoxic environment through hypoxia pretreatment in vitro to enhance their survival and function in vivo. However, hypoxia pretreatment may increase the metabolic burden on CAR-T cells, and the long-term efficacy and safety require further verification. 4. Using gene editing technology to enable CAR-T cells to express hypoxia-resistant genes (such as HIF-1α) to enhance their function in a hypoxic environment. However, this approach has significant drawbacks. Gene editing involves complex technical operations, carries potential off-target effects and immunogenicity risks, and requires rigorous safety assessment. 5. Adding antioxidants (such as N-acetylcysteine) to the culture medium can alleviate oxidative stress under hypoxic conditions, thereby improving the survival and function of CAR-T cells. However, the dosage and timing of antioxidant addition need to be precisely controlled; excessive amounts may inhibit CAR-T cell activity, thus reducing the therapeutic effect.

[0004] Therefore, there is an urgent need in this field for a culture medium that can enable CAR-T cells to maintain excellent tumor cell killing ability in solid tumors. Summary of the Invention

[0005] The purpose of this invention is to provide a culture medium and method that enhance the tumor cell killing ability of CAR-T cells under low oxygen concentration.

[0006] A first aspect of the present invention provides a culture medium for culturing CAR-T cells, comprising:

[0007] (a) Culture medium for CAR-T cells; and

[0008] (b) An active ingredient selected from two or three of the following groups: palbociclin, rapamycin, and enxidipine.

[0009] In another preferred embodiment, the concentration of palbociclin in the culture medium is 10–500 nM; the concentration of rapamycin in the culture medium is 0.1–100 nM; and / or the concentration of enxidipine in the culture medium is 0.1–10 μM.

[0010] In another preferred embodiment, the concentration of palbociclin in the culture medium is 50–300 nM, more preferably 50–200 nM, more preferably 100–200 nM, for example, about 100 nM.

[0011] In another preferred embodiment, the concentration of rapamycin in the culture medium is 1–80 nM, more preferably 1–50 nM, more preferably 10–50 nM, for example, about 50 nM.

[0012] In another preferred embodiment, the concentration of encilidipine in the culture medium is 0.5–5 μM, more preferably 1–5 μM, more preferably 3–5 μM, for example, about 5 μM.

[0013] In another preferred embodiment, the culture medium for the CAR-T cells comprises Gibco fetal bovine serum and X-VIVO medium containing IL-2.

[0014] In another preferred embodiment, the Gibco fetal bovine serum content in the X-VIVO medium is 1-10% (v / v), more preferably 3-8%, more preferably 4-6%, for example, about 5%.

[0015] In another preferred embodiment, the amount of IL-2 in the X-VIVO medium is 50 to 500 IU, more preferably 70 to 300 IU, more preferably 90 to 150 IU, for example, about 100 IU.

[0016] In another preferred embodiment, the active ingredients include palbociclin and rapamycin.

[0017] In a second aspect, the present invention provides the use of a combination of active ingredients for preparing a formulation or composition for increasing the number of memory cell populations and / or enhancing the tumor cell killing ability of CAR-T cells in a low-oxygen environment.

[0018] The active ingredient combination is selected from two or three of the following groups: palbociclin, rapamycin, and enxidipine.

[0019] In another preferred embodiment, the formulation or composition is also used to improve the survival rate of CAR-T cells in a low-oxygen environment.

[0020] In another preferred embodiment, the low oxygen concentration refers to an oxygen content of ≤10%, more preferably ≤5%, more preferably ≤3%, for example, about 1%.

[0021] In a third aspect, the present invention provides a method for culturing CAR-T cells, comprising the steps of:

[0022] (s1) provides CAR-T cells; and

[0023] (s2) CAR-T cells are cultured under suitable culture conditions using the culture medium as described in the first aspect of the present invention.

[0024] In another preferred embodiment, the method can increase the number of memory cells and / or enhance the tumor cell killing ability of CAR-T cells in a low-oxygen environment.

[0025] In another preferred embodiment, in step (s1), the CAR-T cells are precultured in CAR-T cell culture medium for 1 to 10 days, more preferably 3 to 8 days, more preferably 4 to 7 days, for example, about 6 days or 4 days.

[0026] In another preferred embodiment, step (s2) includes steps (s2a) and (s2b):

[0027] (s2a) The CAR-T cells described herein are cultured in CAR-T cell culture medium containing palbocicillin and / or ensidipine for 1 to 10 days;

[0028] (s2b) Add rapamycin to the CAR-T culture medium in step (s2a) and culture the CAR-T cells for 1 to 5 days.

[0029] In another preferred embodiment, the concentration of palbociclin is 10–500 nM; the concentration of rapamycin is 0.1–100 nM; and / or the concentration of enxidipine is 0.1–10 μM.

[0030] In another preferred embodiment, in step (s2a), CAR-T cells are cultured for 1 to 6 days, preferably 1 to 5 days, for example, about 2 or 3 days.

[0031] In another preferred embodiment, in step (s2b), CAR-T cells are cultured for 1 to 4 days, preferably 1 to 3 days, for example, about 2 days.

[0032] In another preferred embodiment, the culture medium for the CAR-T cells comprises Gibco fetal bovine serum and X-VIVO medium containing IL-2.

[0033] In another preferred embodiment, the Gibco fetal bovine serum content in the X-VIVO medium is 1-10% (w / w), more preferably 3-8%, more preferably 4-6%, for example, about 5%.

[0034] In another preferred embodiment, the amount of IL-2 in the X-VIVO medium is 50 to 500 IU, more preferably 70 to 300 IU, more preferably 90 to 150 IU, for example, about 100 IU.

[0035] In a fourth aspect, the present invention provides a kit for culturing CAR-T cells, comprising:

[0036] (i) The first container and the culture medium for CAR-T cells located in the first container;

[0037] (ii) the second container, and the palbocicillin located in the second container; and

[0038] (iii) The third container, and rapamycin located in the third container.

[0039] In another preferred embodiment, the kit further includes a fourth container and ensidiapine located in the fourth container.

[0040] In another preferred embodiment, the culture medium for the CAR-T cells comprises Gibco fetal bovine serum and X-VIVO medium containing IL-2.

[0041] In another preferred embodiment, the Gibco fetal bovine serum content in the X-VIVO medium is 1-10% (v / v), more preferably 3-8%, more preferably 4-6%, for example, about 5%.

[0042] In another preferred embodiment, the amount of IL-2 in the X-VIVO medium is 50 to 500 IU, more preferably 70 to 300 IU, more preferably 90 to 150 IU, for example, about 100 IU.

[0043] In a fifth aspect, the present invention provides CAR-T cells or CAR-T cell products cultured using a culture medium as described in the first aspect of the present invention, a method as described in the third aspect of the present invention, or a kit as described in the fourth aspect of the present invention.

[0044] In another preferred embodiment, the survival rate of the CAR-T cells or CAR-T cell products in a hypoxic environment is ≥80%.

[0045] In another preferred embodiment, the relative cytotoxicity (Z1) of the CAR-T cells or CAR-T cell products is ≥1.2, preferably ≥1.5, and more preferably ≥1.7, compared to CAR-T cells (Z0) not cultured in the culture medium described in the first aspect of the invention.

[0046] In another preferred embodiment, the CAR-T cells or CAR-T cell product have a large number of memory cell populations. The large number of memory cell populations means that the ratio (N1) of the number of memory cell populations (N1) of the CAR-T cells or CAR-T cell product to that of CAR-T cells (N0) not cultured in the culture medium described in the first aspect of the present invention is ≥1.1, preferably ≥1.2, and more preferably ≥1.25.

[0047] In a sixth aspect, the present invention provides a method for improving the therapeutic effect of CAR-T cell therapy in vitro, comprising the steps of:

[0048] By contacting the CAR-T cells or CAR-T cell products described in the fifth aspect of this invention with cancer cells, the therapeutic effect of CAR-T cell therapy can be improved.

[0049] In another preferred embodiment, the CAR-T cell to cancer cell effector-target ratio is 0.1 to 20:1, more preferably 0.5 to 15:1, more preferably 1 to 13:1, for example, about 10:1 or about 2:1.

[0050] In another preferred embodiment, the CAR-T cells are contacted with cancer cells under low oxygen conditions.

[0051] In another preferred embodiment, the low oxygen concentration refers to an oxygen content of ≤10%, more preferably ≤5%, more preferably ≤3%, for example, about ≤1%.

[0052] In another preferred embodiment, the CAR-T cells are exposed to cancer cells for 10 to 100 hours, more preferably 15 to 85 hours, and even more preferably 20 to 75 hours, for example, about 24 hours, about 48 hours, or about 72 hours.

[0053] In another preferred embodiment, the cancer cells are solid tumor cells.

[0054] In another preferred embodiment, the cancer cells are selected from the group consisting of breast cancer cells, ovarian cancer cells, or combinations thereof.

[0055] In another preferred embodiment, the cancer cells are selected from the group consisting of 1806-luc cells, SKOV3-luc cells, or combinations thereof.

[0056] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0057] A seventh aspect of the present invention provides a method for treating cancer, comprising the steps of:

[0058] Cancer is treated by administering a therapeutically effective amount of CAR-T cells or CAR-T cell products as described in the fifth aspect of the invention to the desired subjects.

[0059] In another preferred embodiment, the cancer is a solid tumor.

[0060] In another preferred embodiment, the cancer is selected from the group consisting of breast cancer, ovarian cancer, or a combination thereof.

[0061] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0062] Figure 1The study demonstrates the detection of CAR expression on T cells and anti-MSLN CAR-T cells by flow cytometry.

[0063] Figure 2 This study demonstrated hypoxia-induced apoptosis and differentiation of CAR-T cells. Figure 2 In the middle A: On day 12 of normal culture, CAR-T cells were divided into two groups and cultured under normoxic (21% O2) or hypoxic (1% O2) conditions. Figure 2 B and C: Apoptosis rate (B) and memory phenotype (C) of conditional CAR-T cells were detected by flow cytometry.

[0064] Figure 3 The study demonstrated that using a culture medium with added drugs could reverse hypoxia-induced CAR-T damage. Figure 3 A: Flowchart for CAR-T cell culture, drug addition, and analysis. Figure 3 B and C: Apoptosis rate of conditioned CAR-T cells after 6 days of hypoxia culture (B) and percentage of CAR-T cells with central memory phenotype and terminal exhaustion phenotype (C).

[0065] Figure 4 The effect of culture media with different drug concentrations on cell proliferation is shown. Growth curves of CAR-T cells (left) and inhibition rates on days 2, 4, and 6 (right).

[0066] Figure 5 The optimal concentration of the drug added to the culture medium is shown. Figure 5 A: Flowchart of CAR-T cell culture, drug addition, and multiple rounds of tumor cell challenge. Figure 5 In the second round, under normoxic or hypoxic conditions, SKOV3-MSLN-luc cells were used to challenge CAR-T cells for three rounds at an effector cell:target cell (E:T) ratio of 10:1. Figure 5 C: CAR-T cells were treated as shown in (A) and challenged for 3 rounds with SKOV3-MSLN-luc cells at an effector cell:target cell (E:T) ratio of 10:1 under normoxic (upper) or hypoxic (lower) conditions.

[0067] Figure 6 The in vivo morphology of CAR-T cells treated with the drug combination is shown. Using a culture medium supplemented with the drug combination increased the memory phenotype of CAR-T cells (A) and reduced hypoxia-induced exhaustion (B).

[0068] Figure 7 The in vitro killing effect of drug combination treatment on CAR-T cells is shown. Under normoxic (left) or hypoxic (right) conditions, CAR-T cells were used to kill SKOV3-MSLN-luc cells in three rounds at an effector-to-target ratio of 10:1.

[0069] Figure 8 The study demonstrated the effects of combined drug treatments on cytokines secreted by CAR-T cells. Under hypoxic conditions, the levels of TNF-α (A) and IFN-γ (B) secreted by CAR-T cells after three rounds of stimulation were shown. Detailed Implementation

[0070] Through extensive and in-depth research, and after numerous experiments and screenings, the inventors have unexpectedly discovered for the first time a combination of active ingredients selected from two or three of the following groups: palbociclin, rapamycin, and enxidipine. This combination of active ingredients increases the number of memory cells and / or enhances the tumor cell-killing ability of CAR-T cells in a low-oxygen environment.

[0071] This invention also provides a culture medium comprising a combination of CAR-T culture medium and active ingredients. Experiments show that culturing CAR-T cells using the culture medium of this invention reduces the apoptosis rate of CAR-T cells under hypoxic conditions and enhances the tumor cell-killing ability of CAR-T cells under hypoxic conditions. The inventors have optimized culture conditions to enhance the survival persistence and functional efficacy of CAR-T cells in the solid tumor microenvironment, particularly their efficacy in hypoxic environments. By adjusting the components and proportions of the substances in the culture medium and the duration of their addition, CAR-T cells can be pre-optimized in vitro to achieve better tumor cell-killing ability and a higher number of memory cells in vivo, thereby improving the efficacy of CAR-T therapy in the treatment of solid tumors. This invention was completed based on these findings.

[0072] the term

[0073] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0074] As used herein, the term “comprising” or its variations such as “including” or “comprising” are understood to include the said element or component without excluding other elements or other components.

[0075] The term “about” can refer to a value or composition within an acceptable margin of error for a particular value or composition as determined by a person skilled in the art, depending in part on how the value or composition is measured or determined. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0076] As used herein, unless otherwise stated, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).

[0077] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0078] As used in this article, the term "T" SCM "T memory stem cells refer to a subset of T cells with a central memory phenotype, or CAR-T cells with a central memory phenotype." SCM These cells are a subset of memory T cells that are at the top of the memory T lymphocyte grading system and have the least differentiation. They possess stem cell-like self-renewal capabilities and strong immune reconstitution potential.

[0079] As used in this article, the term "T" TE "Refers to terminal effector T cells." TE T cells are terminally differentiated cells formed after T cells undergo a series of differentiation processes in response to antigen stimulation. They possess high cytotoxicity and effector functions, and are key effector cells in adaptive immune responses.

[0080] Pabocillin

[0081] Palbociclib (Pal), also known as pebocilib and marketed as Ibrance, is an oral cyclin-dependent kinase (CDK) 4 and 6 inhibitor. Palbociclib is a highly specific CDK4 and CDK6 inhibitor that selectively inhibits both kinases, thereby restoring cell cycle control and blocking tumor cell proliferation. Cell cycle dysregulation is a hallmark of cancer, and CDK4 / 6 are overactive in many cancers. Palbociclib inhibits CDK4 / 6, thus suppressing the transition of cells from the G1 phase to the S phase and inhibiting tumor cell proliferation.

[0082] Rapamycin

[0083] Rapamycin (Rap), also known as sirolimus, is a macrolide immunosuppressant produced by Streptomyces hygroscopicus. Rapamycin primarily exerts its effects by inhibiting the mTOR (mammalian target of rapamycin) signaling pathway. mTOR is a serine / threonine protein kinase that plays a crucial role in cell growth, proliferation, differentiation, and metabolism. Rapamycin binds to FKBP12 (FK506-binding protein 12) to form the FKBP12-rapamycin complex. This complex then binds to mTOR, thereby inhibiting mTOR activity and subsequently suppressing downstream signaling pathways, thus inhibiting cell growth and proliferation.

[0084] Ensidipine

[0085] Enasidenib (Ena) is an oral targeted inhibitor for a specific type of acute myeloid leukemia (AML). Enasidenib is indicated for adult patients with relapsed or refractory AML who have been diagnosed with an isocitrate dehydrogenase-2 (IDH2) mutation by an FDA-approved test. Clinical studies have shown that enasidenib is significantly effective in patients with IDH2-mutant AML, improving remission rates and survival, while also exhibiting good safety and tolerability.

[0086] The culture medium of the present invention

[0087] As used herein, the terms "culture medium of the present invention" and "culture medium of the present invention with added drugs" are used interchangeably and both refer to culture media that can enhance the tumor cell killing ability of CAR-T cells under hypoxic conditions, including:

[0088] (a) Culture medium for CAR-T cells; and

[0089] (b) An active ingredient selected from two or three of the following groups: palbociclin, rapamycin, and enxidipine.

[0090] The culture medium of the present invention can increase the number of memory cells in CAR-T cells and / or improve the tumor cell killing ability of CAR-T cells in a low oxygen environment.

[0091] The culture medium of this invention can promote the secretion of cytokines, such as IFN-γ and TNF-α, by CAR-T cells. The culture medium of this invention can enhance the relative cytotoxicity of CAR-T cells, especially their cytotoxicity under hypoxic or hypoxic conditions.

[0092] In a preferred embodiment, the concentration of palbociclin in the culture medium is 10–500 nM; the concentration of rapamycin in the culture medium is 0.1–100 nM; and / or the concentration of enxidipine in the culture medium is 0.1–10 μM.

[0093] In a preferred embodiment, the concentration of palbociclin in the culture medium is 50–300 nM, more preferably 50–200 nM, more preferably 100–200 nM, for example, about 100 nM.

[0094] In a preferred embodiment, the concentration of rapamycin in the culture medium is 1–80 nM, more preferably 1–50 nM, more preferably 10–50 nM, for example, about 50 nM.

[0095] In a preferred embodiment, the concentration of encildipine in the culture medium is 0.5–5 μM, more preferably 1–5 μM, more preferably 3–5 μM, for example, about 5 μM.

[0096] In a preferred embodiment, the culture medium for the CAR-T cells comprises Gibco fetal bovine serum and IL-2 in X-VIVO medium.

[0097] In a preferred embodiment, the Gibco fetal bovine serum content in the X-VIVO medium is 1-10% (v / v), more preferably 3-8%, more preferably 4-6%, for example, about 5%.

[0098] In a preferred embodiment, the amount of IL-2 in the X-VIVO medium is 50 to 500 IU, more preferably 70 to 300 IU, more preferably 90 to 150 IU, for example, about 100 IU.

[0099] In a preferred embodiment, the active ingredients include palbociclin and rapamycin.

[0100] The method of the present invention

[0101] As used herein, the terms "method of the present invention," "method to enhance the tumor cell killing ability of CAR-T cells under low oxygen conditions," and "method of culturing CAR-T cells of the present invention" are used interchangeably, including the following steps:

[0102] (s1) provides CAR-T cells; and

[0103] (s2) CAR-T cells are cultured using the culture medium described in this invention under suitable culture conditions.

[0104] CAR-T cells cultured using the method of this invention can enhance their tumor cell killing ability in low oxygen environments and / or increase the number of memory cell populations.

[0105] In a preferred embodiment, in step (s1), the CAR-T cells are precultured in CAR-T cell culture medium for 1 to 10 days, more preferably 3 to 8 days, more preferably 4 to 7 days, for example, about 6 days or 4 days.

[0106] In a preferred embodiment, step (s2) includes steps (s2a) and (s2b):

[0107] (s2a) The CAR-T cells described herein are cultured in CAR-T cell culture medium containing palbocicillin and / or ensidipine for 1 to 10 days;

[0108] (s2b) Add rapamycin to the CAR-T culture medium in step (s2a) and culture the CAR-T cells for 1 to 5 days.

[0109] In a preferred embodiment, the concentration of palbociclin is 10–500 nM; the concentration of rapamycin is 0.1–100 nM; and / or the concentration of enxidipine is 0.1–10 μM.

[0110] In a preferred embodiment, in step (s2a), CAR-T cells are cultured for 1 to 6 days, preferably 1 to 5 days, for example, about 2 or 3 days.

[0111] In a preferred embodiment, in step (s2b), CAR-T cells are cultured for 1 to 4 days, preferably 1 to 3 days, for example, about 2 days.

[0112] In a preferred embodiment, under suitable culture conditions, CAR-T cells are cultured in CAR-T medium for 3–8 days, then cultured for 1–5 days with the addition of palbociclin and / or encilidipine, and then cultured for 1–5 days with the addition of rapamycin, thereby enhancing the tumor cell killing ability of CAR-T cells in low oxygen concentrations and / or increasing the number of memory cell populations.

[0113] Methods to improve the efficacy of CAR-T cell therapy

[0114] The method for achieving the therapeutic effect of CAR-T cell therapy of the present invention is in vitro and includes the following steps:

[0115] CAR-T cells cultured using the culture medium or method of the present invention are brought into contact with cancer cells, thereby improving the therapeutic effect of CAR-T cell therapy.

[0116] In a preferred embodiment, the CAR-T cell to cancer cell effector-target ratio is 0.1 to 20:1, more preferably 0.5 to 15:1, more preferably 1 to 13:1, for example, about 10:1 or about 2:1.

[0117] In a preferred embodiment, the CAR-T cells are contacted with cancer cells under low oxygen conditions.

[0118] In a preferred embodiment, the low oxygen concentration refers to an oxygen content of ≤10%, more preferably ≤5%, more preferably ≤3%, for example, about ≤1%.

[0119] In a preferred embodiment, the CAR-T cells are exposed to cancer cells for 10–100 hours, more preferably 15–85 hours, and even more preferably 20–75 hours, for example, about 24 hours, about 48 hours, or about 72 hours.

[0120] In a preferred embodiment, the cancer cells are solid tumor cells.

[0121] In a preferred embodiment, the cancer cells are selected from the group consisting of breast cancer cells, ovarian cancer cells, or combinations thereof.

[0122] In a preferred embodiment, the cancer cells are selected from the group consisting of 1806-luc cells, SKOV3-luc cells, or combinations thereof.

[0123] The main advantages of this invention include:

[0124] (1) CAR-T cells cultured using the culture medium or method of the present invention can enhance their survival rate, persistence and effector function in hypoxic environments.

[0125] (2) CAR-T cells cultured using the culture medium or method of the present invention improve the cytotoxicity and cytokine secretion capacity of CAR-T cells, and effectively enhance the killing effect on tumor cells.

[0126] (3) CAR-T cells cultured using the culture medium or method of the present invention have broad application prospects and make up for the current shortcomings of CAR-T cell therapy in solid tumors.

[0127] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0128] Materials and reagents

[0129] The materials and reagents used in this application are shown in Table 1.

[0130] Table 1

[0131] Materials and reagents supplier PEI transfection reagent Polysciences, 24765-100 X-VIVO medium Lonza, 04-418Q PBMC (Example 1) Heyousheng, EZ4096 Isolate antibodies Stemcell, 17951 Separating magnetic beads Stemcell, 17951 Activating magnetic beads Gibco, 11161D 1806 SKOV3 pMDLg / pRRE Addgene, 12251 pRSV-Rev Addgene, 12253 pMD2.G Addgene, 12259

[0132] Example 1: Preparation of CAR-T cells

[0133] 1.1 T cell sorting

[0134] Take out a vial containing 5×10 from the liquid nitrogen tank. 7 Thaw the PBMC cells in a 37°C water bath using a cryovial. Place a new, sterile cryovial in a magnetic rack and add 1 ml of X-VIVO medium (containing 5% Gibco fetal bovine serum and 100 IU IL-2; the X-VIVO medium is the same throughout). Then, take a 15 ml centrifuge tube, add 4 ml of X-VIVO medium, transfer the thawed PBMC cells to the 15 ml centrifuge tube, centrifuge (500×g, 5 min), discard the supernatant, add 50 μl of Stemcell easysep separation antibody and 800 μl of X-VIVO medium, incubate for 5 min, add 50 μl of magnetic separation beads, resuspend the cells, and then transfer them to the prepared cryovial. Mix well and incubate for 3 min.

[0135] After incubation, take a 15ml centrifuge tube, add 7ml of culture medium, transfer cells from the cryopreserved tube to the centrifuge tube, centrifuge at 500×g for 5min and count. After counting, centrifuge again (500×g, 5min), discard the supernatant, and count cells according to the count results. 7 Cells were added to 100 μl of activated magnetic beads and 50 μl of culture medium. After resuspending the cells several times by pipetting, they were incubated in an incubator for 7 min. This process was repeated three times. After incubation, the cells were transferred to a container at 2 × 10⁻⁶ cells / mL. 6 Cells were cultured in T25 flasks at a ratio of cells / ml of culture medium.

[0136] The T cells cultured overnight should be used promptly for lentivirus transfection the next day to construct CAR-T cells.

[0137] 1.2 Construction of CAR-T cells

[0138] 293T cells intended for virus packaging were cultured in a 37°C, 5% CO2 cell culture incubator. The culture medium used was DMEM containing 10% Gibco fetal bovine serum. The day before the actual virus packaging, the cultured 293T cells were sputtered at a rate of 1 × 10⁻⁶. 7 The cells were passaged three times in T75 cell flasks. When the 293T cells reached 70-80% confluence and were evenly distributed in the culture flasks, lentivirus packaging began.

[0139] Prepare plasmid and transfection reagent dilution solutions by vortexing the PEI 40K transfection reagent. Prepare two centrifuge tubes and prepare plasmid and transfection reagent dilution solutions in the order shown in Table 2.

[0140] Table 2

[0141] Centrifuge tube 1 (plasmid DNA) Centrifuge tube 2 (transfection reagent) Lentiviral vector 10μg PEI 40K transfection reagent 40μl VSVG carrier 7.5μg 460 μl of DMEM serum-free culture medium G-Puro carrier 2.5μg Total volume 500μl 480 μl of DMEM serum-free culture medium Total volume 500μl

[0142] Mix thoroughly. Add the transfection reagent dilution (centrifuge tube 2) dropwise to the plasmid DNA solution (centrifuge tube 1), mixing thoroughly after each addition. The order of addition is crucial. Incubate the transfection mixture at room temperature for 15–20 minutes.

[0143] Discard the old culture medium in the culture flask containing the 293T cells, wash once with PBS, and add 9 ml of fresh DMEM medium. Then add 1 ml of the incubated transfection mixture (carefully and slowly add dropwise from the opposite side of the cells to prevent them from floating). Incubate the cells at 37°C and 5% CO2 for 6 hours, then discard the culture medium in the flask and add 15 ml of fresh culture medium to continue culturing.

[0144] Forty-eight hours post-transfection, the cell culture supernatant was collected for the first time, and 15 ml of fresh culture medium was added for continued culturing. A second collection of cell culture supernatant was conducted 72 hours post-transfection, yielding a total of 30 ml. The supernatant was centrifuged at 1000×g for 8 min, then filtered through a 0.45 nm pore size filter to remove cell debris, and transferred to ultracentrifuge tubes. After centrifugation at 22,000 rpm for 2 h at 4°C, the virus location was immediately marked on the tube, and the supernatant was aspirated and discarded. The virus pellet was resuspended in 200 μl of X-VIVO medium, transferred to EP tubes, aliquoted, and stored overnight at 4°C. Storage at 4°C is recommended for one week; for longer storage, storage at 4°C overnight followed by storage at -80°C is recommended.

[0145] Remove 1×10 from the incubator 6T cells isolated the previous day (as described above) were added to 50 μl of viral resuspending solution and incubated at 37°C for 24 hours. After 24 hours, all cells were aspirated, centrifuged, and the medium was changed before continuing culture. The cultured cells are the desired CAR-T cells.

[0146] Flow cytometry was used to detect CAR expression on T cells and anti-MSLN CAR-T cells. Figure 1 As shown in Figure A, the results showed that the CAR positivity rate of anti-MSLN CAR-T cells reached 87%.

[0147] Example 2: CAR-T cell phenotype after hypoxia stress

[0148] 2.1 Methods

[0149] CAR-T cells were cultured normally in X-VIVO medium containing 5% (v / v) Gibco fetal bovine serum and 100 IU IL-2 until day 6 (day 1 during transfection), when the drugs listed in Table 3 were added:

[0150] Table 3

[0151] Group Add drugs Add concentration Add duration control group NA NA NA Single drug group 1 Palbociclib 1000nM 6d(D6-D12) Single drug group 2 Rapamycin 50nM 6d(D6-D12) Single drug group 3 Enasidebib 5μM 6d(D6-D12)

[0152] The CAR-T cells in each group in Table 3 were used at a rate of 2×10 5 Cells were incubated at a concentration of 100 cells / mL in a hypoxic incubator (1% O2, 5% CO2, 94% N2, the same below), with a total cell suspension volume of 6 mL for each group. After 6 days of hypoxia, the cells were removed from each group, centrifuged to remove the supernatant, washed with PBS, counted, and the cell concentration was adjusted. The cells were divided into multiple samples and labeled with specific antibodies, such as memory cell markers (CD45RO, CD62L) and apoptosis markers (Annexin V, 7-AAD). The labeled cells were incubated in the dark for a certain period of time, and then washed again with PBS to remove unbound antibodies. The fluorescence intensity of each marker was detected by flow cytometry, and the data were recorded and analyzed to assess the changes in memory and apoptosis phenotypes of CAR-T cells.

[0153] 2.2 Results

[0154] The diagrams illustrating the normoxic and hypoxic treatments of untreated CAR-T cells as described in Table 3 are shown below. Figure 2 As shown in Figure A. Cells in each group were cultured for 12 days and then placed in normoxic and hypoxic incubators respectively. After 6 days of static incubation, the cells were retrieved to detect memory and apoptosis phenotypes. Changes in cell memory and apoptosis phenotypes are shown below. Figure 2 As shown in B and C, the proportions of the memory groups are shown in Table 4.

[0155] Table 4

[0156] normoxic culture Hypoxia culture <![CDATA[T SCM ]]> 19.77％ 9.20％ <![CDATA[T CM ]]> 59.16％ 42.25％ <![CDATA[T EM ]]> 16.25％ 40.24％ <![CDATA[T TE ]]> 5.42％ 8.35％

[0157] The results showed that, compared with cells cultured under normoxic conditions, hypoxic stress led to an increase in CAR-T cell apoptosis levels and T cell proliferation. SCM and T CM The cell population is declining, and exhausted CAR-T cells (T cells) are increasing. TE The number of CAR-T cells increased. Based on this, it is speculated that 6 days of hypoxic stress led to the depletion of CAR-T cell phenotype and apoptosis.

[0158] The following is a schematic diagram of the phenotypic detection of CAR-T cells in each group after hypoxia treatment following the drug treatment described in Table 3. Figure 3 As shown in Figure A, the changes in cell memory and apoptosis phenotypes in each group are as follows: Figure 3 As shown in B and C. The apoptosis rate and the proportion of the memory population are shown in Tables 5 and 6, respectively.

[0159] Table 5. Cell apoptosis rate

[0160] Comparison Palbociclib Enasidenib Rapamycin 30.10％ 17.30％ 26.30％ 13.20％

[0161] Table 6. Proportion of the memory group

[0162] Comparison Palbociclib Enasidenib Rapamycin <![CDATA[T CM ]]> 33.91％ 70.73％ 45.21％ 59.11％ <![CDATA[T TE ]]> 6.25％ 0.73％ 4.14％ 2.00％

[0163] The results showed that, compared with the control group, each single-drug group could reduce hypoxia-induced apoptosis and cell exhaustion to a certain extent and enhance cellular memory. Therefore, the effects of each drug on enhancing CAR-T cell persistence were demonstrated at the phenotypic level.

[0164] Example 3: Optimization of drug concentration in culture medium

[0165] CAR-T cells were cultured normally in X-VIVO medium containing 5% (v / v) Gibco fetal bovine serum and 100 IU IL-2 until day 6, at which point the drugs listed in Table 7 were added. Cell counts were performed on each group in Table 7 every other day, and the drug inhibition rate was calculated. Drug inhibition rate = (number of cells in control group - number of cells in sample group) / number of cells in control group. Medium and drugs were replenished every other day.

[0166] Table 7

[0167] Group Add drugs Add concentration Add duration Single drug group 1 Palbociclib 0-2000nM 6d(D6-D12) Single drug group 2 Rapamycin 0-1000nM 6d(D6-D12) Single drug group 3 Enasidebib 0-20μM 6d(D6-D12)

[0168] The changes in CAR-T cell count and drug inhibition rate in each group after treatment are shown in Table 7. Figure 4 As shown in Table 8, the results indicate that different drug concentrations and treatment times have a significant impact on CAR-T inhibition rates. Therefore, drug concentrations and treatments with inhibition rates below 50% were selected for further screening.

[0169] Table 8

[0170]

[0171] Add SKOV3-MSLN-luc cells to each well of a 96-well plate according to the required sample volume, 2 × 10⁶ cells per well. 4 Cells, in a volume of 100 μl, were incubated in a normoxic incubator for 24 h. After discarding the original culture medium, the CAR-T cell suspension concentration for each group in Table 8 was adjusted according to different positive rates at a 10:1 (SKOV3-MSLN-luc) effector-to-target ratio. The total volume should always be 100 μl. The co-incubated cells were then placed in a normoxic or hypoxic incubator for 24 h. Stimulation was repeated three times. After each round, 100 μl of the E605A reagent from the ONE-Glo Luciferase Assay System was added to each well of cells, mixed thoroughly, and incubated at room temperature in the dark for 10 min. 100 μl of the solution from the cell culture plate was then transferred horizontally into a 96-well white plate. A multi-mode microplate reader and software were turned on, and the Luminescence mode was selected for plate layout. The 96-well white plate was placed in the microplate reader to read the data, which was then exported and saved for calculating the cell killing rate. Cell killing rate = (sample luminescence value - background luminescence value) / (control group luminescence value - background luminescence value) * 100% (same below).

[0172] The flowcharts for the killing of CAR-T cells in the control group and drug-treated groups under normoxic or hypoxic conditions, as described in Table 8, are as follows: Figure 5 As shown in Figure A, the result is as follows: Figure 5 As shown in B and C.

[0173] Compared with normoxic killing, the killing ability of control cells in Table 8 was significantly reduced after the third round of hypoxia killing following two rounds of hypoxia killing. This indicates that continuous hypoxia stress for 6 days significantly impairs the persistence of CAR-T cell killing. The killing ability of CAR-T cells in all groups treated with the drugs described in Table 8 was enhanced. Therefore, considering both safety and efficacy, the final drug concentration and addition time were determined (see Table 9).

[0174] Example 4: Treatment of CAR-T cells with the culture medium of the present invention

[0175] CAR-T cells were cultured normally in X-VIVO medium containing 5% (v / v) Gibco fetal bovine serum and 100 IU of IL-2 until day 6 (day 1 during viral transfection), and the groups were set up as shown in Table 9.

[0176] Table 9

[0177]

[0178] Culture medium and drugs were added every other day. On day 10, CAR-T cells in each group were centrifuged (500×g, 5min), resuspended in PBS, and centrifuged again (500×g, 5min) for subsequent confirmatory experiments.

[0179] Example 5: Phenotypic Changes in CAR-T Cells Treated with Drug Combinations

[0180] 5.1 Method

[0181] The CAR-T cells from each group in Table 9 of Example 4 were used at a rate of 2×10⁹ / L. 5 Cells were incubated at a concentration of 6 cells / mL in a hypoxic incubator, with a total cell suspension volume of 6 mL for each group. After 6 days of hypoxia, cells were removed from each group, centrifuged to remove the supernatant, washed with PBS, counted, and the cell concentration adjusted. Cells were divided into multiple samples and labeled with specific antibodies, such as memory cell markers (CD45RO, CD62L). The labeled cells were incubated in the dark for a certain period of time, and then washed again with PBS to remove unbound antibodies. The fluorescence intensity of each marker was detected using flow cytometry, and the data were recorded and analyzed to assess changes in the memory phenotype of CAR-T cells.

[0182] 5.2 Results

[0183] Table 10

[0184]

[0185] like Figure 6 As shown in Table 10, compared with the single-drug groups, the addition of the drug combination shown in Example 4 to the culture medium significantly increased the memory phenotype of CAR-T cells, and the results showed that the Pal+Rap and Pal+Ena+Rap drug combinations had significant effects in reducing hypoxia-induced exhaustion. Based on this analysis, the drug combination is more effective than the individual drugs in maintaining the memory population level of CAR-T cells and reducing exhaustion.

[0186] Example 6: Drug combination treatment for in vitro killing of CAR-T cells

[0187] 6.1 Method

[0188] In Example 4, CAR-T cells from each group were challenged three times with SKOV3-MSLN-luc cells under normoxic or hypoxic conditions at an effector cell:target cell (E:T) ratio of 10:1. Cytotoxicity was measured using luciferase reporter gene assay.

[0189] 6.2 Results

[0190] Table 11

[0191]

[0192]

[0193] like Figure 7 As shown in Table 11, the results of the third round of normoxic and hypoxic killing experiments revealed differences in killing ability between single-drug and multi-drug groups. CAR-T cells treated with the drug combinations in Example 4 were better able to overcome the limitations of hypoxic stress on the killing ability of CAR-T cells compared to cells in the single-drug group, with Pal+Rap exhibiting superior characteristics.

[0194] Example 7: Drug combination treatment for CAR-T cell cytokine release

[0195] 7.1 Method

[0196] The supernatant of CAR-T cells after each round of killing in Example 6 was stored at -80°C. The mixed capture microspheres and mixed detection antibody, along with concentration gradient standards from the CBA kit, were prepared. Cytokine standards and samples were mixed with the mixed capture microspheres separately according to the instructions and incubated under light-protected conditions. After incubation, fluorescently labeled mixed detection antibody was added, and incubation continued to form a capture microsphere-cytokine-detection antibody complex. After incubation, the mixture was washed with washing buffer to remove unbound detection antibody. After resuspending in washing buffer, flow cytometry was used for detection, and the cytokines IFN-γ and TNF-α were qualitatively and quantitatively analyzed using beads with different fluorescence intensities.

[0197] 7.2 Results

[0198] In Example 4, the IFN-γ and TNF-α secretion levels of CAR-T cells in each group after the third round of killing were as follows: Figure 8 As shown in Table 12.

[0199] Table 12

[0200] Group TNF-α (pg / mL) IFN-γ (pg / mL) Control 75.00 945.00 Palbociclib 89.30 1445.00 Enasidenib 67.20 1703.00 Rapamycin 86.70 1125.00 Pal+Ena 74.60 2293.00 Ena+Rap 113.00 923.00 Pal+Rap 131.00 1644.00 Pal+Ena+Rap 197.00 1562.00

[0201] The results showed that, compared with the single-drug group, the cells in the multi-drug group generally secreted more cytokines to kill cells, consistent with the killing results in Example 6.

[0202] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A culture medium for culturing CAR-T cells, characterized in that, include: (a) Culture medium for CAR-T cells; and (b) An active ingredient selected from two or three of the following groups: palbociclin, rapamycin, and enxidipine.

2. The culture medium as described in claim 1, characterized in that, The concentration of palbociclin in the culture medium is 10–500 nM; the concentration of rapamycin in the culture medium is 0.1–100 nM; and / or the concentration of enxidipine in the culture medium is 0.1–10 μM.

3. The use of a combination of active ingredients, characterized in that, This preparation or composition is used to increase the number of memory cell populations and / or enhance the tumor cell killing ability of CAR-T cells in a low-oxygen environment. The active ingredient combination is selected from two or three of the following groups: palbociclin, rapamycin, and enxidipine.

4. A method for culturing CAR-T cells, characterized in that, Including the following steps: (s1) provides CAR-T cells; and (s2) CAR-T cells are cultured using the culture medium as described in claim 1 under suitable culture conditions.

5. The method as described in claim 4, characterized in that, In step (s1), the CAR-T cells are pre-cultured in CAR-T cell culture medium for 1 to 10 days.

6. The method as described in claim 5, characterized in that, Step (s2) includes steps (s2a) and (s2b): (s2a) The CAR-T cells described herein are cultured in CAR-T cell culture medium containing palbocicillin and / or enxidipine for 1 to 10 days; (s2b) Add rapamycin to the CAR-T culture medium in step (s2a) and culture the CAR-T cells for 1 to 5 days.

7. A kit for culturing CAR-T cells, characterized in that, include (i) The first container and the culture medium for CAR-T cells located in the first container; (ii) the second container, and the palbocicillin located in the second container; and (iii) The third container, and rapamycin located in the third container.

8. The kit according to claim 7, characterized in that, The kit also includes a fourth container and ensidiapine located in the fourth container.

9. CAR-T cells or CAR-T cell products cultured using the culture medium as described in claim 1, the method as described in claim 4, or the kit as described in claim 7.

10. A method for enhancing the therapeutic effect of CAR-T cell therapy in vitro, characterized in that, Including the following steps: By contacting the CAR-T cells as described in claim 9 with cancer cells, the therapeutic effect of CAR-T cell therapy can be improved.

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