CN-121991897-A - Culture medium for enhancing ability of CAR-T cells to kill tumor cells and application thereof

Abstract

The invention discloses a culture medium and a method for enhancing the killing capacity of CAR-T cells to tumor cells in a hypoxia environment. In particular, the medium comprises a CAR-T medium and a drug selected from the group consisting of palbocicline, rapamycin, azepine, or a combination thereof. The persistence and functional effector properties of CAR-T cells cultured using the culture medium or method of the invention in a solid tumor microenvironment, thereby enhancing the efficacy of CAR-T therapy in the treatment of solid tumors.

Inventors

• GUO ZHIGANG
• Zhou Muya
• CHEN WENWEN
• XU LUXIA

Assignees

• 南京康和细胞基因工程研究院有限公司

Dates

Publication Date

20260508

Application Date

20241107

Claims (10)

1. 1. A culture medium for culturing CAR-T cells, comprising: (a) Culture medium for CAR-T cells, and (B) An active ingredient selected from two or three of the group consisting of palbocicline, rapamycin, and encilnidipine.
2. 2. The culture medium according to claim 1, wherein the concentration of the palbocicline in the culture medium is 10-500 nM, the concentration of the rapamycin in the culture medium is 0.1-100 nM, and/or the concentration of the azepine in the culture medium is 0.1-10 μΜ.
3. 3. Use of a combination of active ingredients for the preparation of a formulation or composition for increasing the number of memory cell populations and/or increasing the tumor cell killing capacity of CAR-T cells in a low concentration oxygen environment; wherein the active ingredient combination is selected from two or three of palbocicline, rapamycin and encilnidipine.
4. 4. A method of culturing CAR-T cells comprising the steps of: (s 1) providing a CAR-T cell, and (S 2) culturing the CAR-T cells using the medium of claim 1 under conditions suitable for culturing.
5. 5. The method of claim 4, wherein in step (s 1), the CAR-T cells are pre-cultured for 1-10 days using CAR-T cell medium.
6. 6. The method of claim 5, wherein step (s 2) comprises steps (s 2 a) and (s 2 b): (s 2 a) culturing the CAR-T cells in a CAR-T cell culture medium containing palbocicline and/or azepine for 1-10 days; (s 2 b) adding rapamycin to the CAR-T medium in the step (s 2 a), and culturing the CAR-T cells for 1-5 days.
7. 7. A kit for culturing CAR-T cells, comprising (I) A first container, and a culture medium for CAR-T cells located in the first container; (ii) A second container, and palbocicline in the second container, and (Iii) A third container, and rapamycin located in the third container.
8. 8. The kit of claim 7, further comprising a fourth container and exendin in the fourth container.
9. 9. A CAR-T cell or CAR-T cell preparation cultured using the medium of claim 1, the method of claim 4, or the kit of claim 7.
10. 10. A method of enhancing the therapeutic effect of CAR-T cell therapy in vitro comprising the steps of: Contacting the CAR-T cell of claim 9 with a cancer cell, thereby increasing the therapeutic effect of the CAR-T cell therapy.

Description

Culture medium for enhancing ability of CAR-T cells to kill tumor cells and application thereof Technical Field The invention relates to the technical field of cell immunotherapy and cell culture, in particular to a culture medium for enhancing the capability of killing tumor cells by CAR-T cells and application thereof. Background Chimeric antigen receptor T cell (CAR-T cell) therapies have become a breakthrough immunotherapeutic approach to treat various cancers. Through genetic engineering of T cells, the T cells express the CAR-T cells which can recognize specific tumor antigens, so that cancer cells can be precisely and effectively targeted and eliminated. This personalized treatment has achieved significant success in hematological malignancies, such as Acute Lymphoblastic Leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL). However, CAR-T cell products currently being developed to treat solid tumors are generally less effective than hematological tumors. The great cause of the difficulty in treating solid tumors is the highly hypoxic solid tumor environment. Hypoxia is a significant feature of the solid tumor microenvironment. Hypoxic areas often occur inside tumors due to insufficient angiogenesis caused by their rapid proliferation. The hypoxic environment inhibits the effector functions of CAR-T cells through a variety of mechanisms, firstly, hypoxia induces tumor cells to express more immunosuppressive molecules (such as PD-L1) and enhances immune escape capacity, secondly, proliferation, differentiation and effector functions (such as cytotoxicity and cytokine secretion) of T cells are inhibited under hypoxic conditions, and finally, the hypoxic environment promotes survival and expansion of tumor stem cell-like cells, which are generally more resistant to CAR-T cell therapy. In order to solve this problem, there have been proposed some solutions as shown in 1. By increasing blood supply to the tumor area using angiogenesis promoters such as VEGF, the hypoxic environment is improved, but angiogenesis can not only improve oxygen supply to the tumor area, but also promote tumor growth and metastasis, making the tumor more malignant. 2. Oxygen is delivered to the tumor area by using oxygen carriers such as artificial hemoglobin or hydrogen peroxide, and the like, so that the anoxic state of the tumor area is relieved. However, oxygen carriers may cause oxidative stress, leading to normal cell damage, with in vivo maldistribution and safety issues. 3. CAR-T cells are adapted to hypoxic environments in vitro by hypoxia pretreatment to enhance their survival and function in vivo. However, hypoxia pretreatment of the cells may result in increased metabolic burden of CAR-T cells, and long-term effects and safety are further verified. 4. The CAR-T cells express hypoxia-resistant related genes (such as HIF-1 alpha) through a gene editing technology, so that the functions of the CAR-T cells in a hypoxia environment are enhanced. However, the disadvantages are obvious, the gene editing involves complex technical operations, potential off-target effects and immunogenicity risks exist, and strict safety assessment is required. 5. The survival rate and the function of the CAR-T cells are improved by adding an antioxidant (such as N-acetylcysteine) into the culture medium to relieve the oxidative stress in the anoxic environment. However, the dosage and timing of the antioxidant needs to be precisely controlled, and excessive amounts may inhibit the activity of CAR-T cells, thereby reducing the efficacy. Thus, there is a need in the art for a medium that can maintain excellent tumor cell killing capacity of CAR-T cells in solid tumors. Disclosure of Invention The invention aims to provide a culture medium and a method for enhancing the killing capacity of CAR-T cells on tumor cells under low-concentration oxygen. In a first aspect of the invention, there is provided a medium for culturing CAR-T cells comprising: (a) Culture medium for CAR-T cells, and (B) An active ingredient selected from two or three of the group consisting of palbocicline, rapamycin, and encilnidipine. In another preferred example, the concentration of the palbociclib in the culture medium is 10-500 nM, the concentration of the rapamycin in the culture medium is 0.1-100 nM, and/or the concentration of the azepine in the culture medium is 0.1-10 mu M. In another preferred embodiment, the concentration of the palbociclin in the medium is 50 to 300nM, preferably 50 to 200nM, more preferably 100 to 200nM, for example, about 100nM. In another preferred embodiment, the rapamycin is present in the medium at a concentration of 1 to 80nM, preferably 1 to 50nM, more preferably 10 to 50nM, e.g., about 50nM. In another preferred embodiment, the concentration of the exendin in the culture medium is 0.5 to 5. Mu.M, preferably 1 to 5. Mu.M, more preferably 3 to 5. Mu.M, for example, about 5. Mu.M. In another preferred embodiment, the medium for the CA