T cell culture medium, cell culture, culture method therefor, and application

Abstract

Provided are a T cell culture medium, a cell culture, a culture method therefor, and an application. The culture medium comprises IL7 and TGFβ. A method for obtaining T cells comprises contacting immune cells with a culture medium, and resulting CD4 / CD8 positive mixed T cells and universal T cell cultures have high cell proliferation capacity, viability and in vivo efficacy after cryopreservation and thawing.

Description

A T cell culture medium, cell culture, culture method and application Technical Field

[0001] This application relates to the technical field of immune cell preparation, specifically to a T cell culture medium, cell culture, culture method, and application. Background Technology

[0002] Chimeric antigen receptor T (CAR-T) cell therapy involves genetically modifying patient or donor T lymphocytes in vitro to express tumor-specific antigen receptors. These receptors bind to specific antigens on the surface of tumor cells, exerting potent anti-tumor activity through the secretion of cytotoxic cytokines and the induction of immune responses. Conventional CAR-T cell preparation primarily involves activating T lymphocytes with CD3 / CD28 antibodies or magnetic beads containing CD3 / CD28 antibodies, transducing them with lentiviral vectors for genetic modification, and then expanding and culturing them in a medium containing one or more cytokines such as IL2, IL7, IL21, and IL15. Existing CAR-T culture protocols using the IL7+IL21 combination have achieved good anti-tumor efficacy; however, cell viability is impaired after cryopreservation and resuscitation, and cell proliferation is also severely compromised. As a cell therapy product, the cells are cryopreserved during the product stage, and resuscitation and the presence of live cells are necessary for them to exert their effects upon reinfusion into the patient. Therefore, these factors all impact clinical efficacy, necessitating the development of new T cell culture protocols.

[0003] TGFβ1 is a member of the TGFβ superfamily that regulates cell growth and differentiation. Current research indicates that TGFβ1 inhibits the proliferation of immune-active cells, such as cytotoxic T cells, and suppresses the production of IFN-γ and TNF-α in PBMCs. Some studies have shown that TGFβ1 and IL4 can promote the proliferation of CD4+ T cell subsets; however, these studies do not consider the viability and persistence of cells after cryopreservation and thawing. Furthermore, these culture protocols are only designed for CD4 subsets and do not consider the more common mixed-type T cells containing multiple subtypes, such as CD4 and CD8 single-positive mixed-type T cell populations like PBMCs, or CD4 and CD8 double-negative T cell populations, and other non-pure CD4 cell populations.

[0004] In summary, it is necessary to provide a method for preparing a population of mixed-type T cells that significantly improves cell viability and proliferation capacity after resuscitation. Summary of the Invention

[0005] This application provides a T-cell culture medium, cell culture, culture method, and application thereof. By culturing T-cells through a special combination of factors, a mixed T-cell population containing at least two subtypes can be obtained. After cryopreservation and thawing, the cell viability and factor-free proliferation capacity of the mixed T-cells are significantly improved. In addition, when the mixed T-cells are thawed and used in vivo, they have better expansion capacity and drug efficacy, thereby exerting a better therapeutic effect.

[0006] Furthermore, current conventional CAR-T products are all autologous products, meaning they are prepared using the patient's own immune cells. These autologous CAR-T cells must be generated on a customized basis. However, due to the high production costs and lengthy production process, this autologous T cell production platform remains a significant limiting factor for large-scale clinical application, and there are also risks such as production failure. Therefore, the development of universal allogeneic T cell products, such as universal CAR-T cells, is necessary. These cells can serve as "off-the-shelf" therapeutic agents. Many technical solutions are currently under development; however, most gene-edited universal T cell products exhibit reduced proliferation capacity and decreased in vivo persistence due to gene editing. Therefore, the culture protocols for universal T cells and adoptive cell therapies derived from them require further optimization.

[0007] The method described in this application can also be used to prepare universal CAR-T cells. The resulting hybrid T cells can promote the proliferation and persistence of allogeneic T cells and genetically modified allogeneic T cells in vivo, thereby promoting better in vivo effectiveness of allogeneic T cells and genetically modified allogeneic T cells.

[0008] This application involves the following:

[0009] 1. A T cell culture medium comprising any one, two or three of the cytokines IL7, IL21, and IL4, and a combination of TGFβ;

[0010] Preferably, the TGF-β includes any one or more of the following cytokines: TGF-β1, TGF-β2, and TGF-β3;

[0011] More preferably, the TGF-β is TGF-β1.

[0012] 2. The culture medium according to item 1, wherein the content of IL7 in the culture medium is 0.5 - 250 ng / ml, 0.5 - 100 ng / ml, 0.5 - 50 ng / ml, 0.5 - 30 ng / ml, 0.5 - 15 ng / ml, 0.5 - 10 ng / ml, 0.5 - 5 ng / ml, 1 - 250 ng / ml, 1 - 100 ng / ml, 1 - 50 ng / ml, 1 - 30 ng / ml, 1 - 15 ng / ml, 1 - 10 ng / ml, 1 - 5 ng / ml, 2 - 250 ng / ml, 2 - 100 ng / ml, 2 - 50 ng / ml, 2 - 30 ng / ml, 2 - 15 ng / ml, 2 - 10 ng / ml, 2 - 9 ng / ml, 2 - 8 ng / ml, 2 - 6 ng / ml, 2 - 5 ng / ml, 3 - 250 ng / ml, 3 - 100 ng / ml, 3 - 50 ng / ml, 3 - 30 ng / ml, 3 - 15 ng / ml, 3 - 10 ng / ml, 3 - 8 ng / ml, 3 - 9 ng / ml, 3 - 6 ng / ml, 3 - 5 ng / ml, 4 - 250 ng / ml, 4 - 100 ng / ml, 4 - 50 ng / ml, 4 - 30 ng / ml, 4 - 15 ng / ml, 4 - 10 ng / ml, 4 - 9 ng / ml, 4 - 8 ng / ml, 4 - 7 ng / ml, 4 - 6 ng / ml, 4 - 5 ng / ml, 5 - 250 ng / ml, 5 - 100 ng / ml, 5 - 50 ng / ml, 5 - 30 ng / ml, 5 - 15 ng / ml, 5 - 10 ng / ml, 5 - 8 ng / ml, 5 - 6 ng / ml, 6 - 250 ng / ml, 6 - 100 ng / ml, 6 - 50 ng / ml, 6 - 30 ng / ml, 6 - 15 ng / ml, 6 - 10 ng / ml, 6 - 8 ng / ml, 7 - 250 ng / ml, 7 - 100 ng / ml, 7 - 50 ng / ml, 7 - 30 ng / ml, 7 - 15 ng / ml, 7 - 10 ng / ml, 7 - 8 ng / ml, 8 - 250 ng / ml, 8 - 100 ng / ml, 8 - 50 ng / ml, 8 - 30 ng / ml, 8 - 15 ng / ml, 8 - 10 ng / ml, 9 - 250 ng / ml, 9 - 100 ng / ml, 9 - 50 ng / ml, 9 - 30 ng / ml, 9 - 15 ng / ml, 9 - 10 ng / ml or 50 - 250 ng / ml;

[0013] And / or, the TGFβ content in the culture medium is 0.5-100 ng / ml, 0.5-80 ng / ml, 0.5-60 ng / ml, 0.5-40 ng / ml, 0.5-20 ng / ml, 0.5-15 ng / ml, 0.5-10 ng / ml, 0.5-9 ng / ml, 0.5-8 ng / ml, 0.5-7 ng / ml, 0.5-6 ng / ml, 0.5-5 ng / ml, 0.5-4 ng / ml, 0.5-3 ng / ml, 0.5-2 ng / ml, 0.5 - 1 ng / ml, 1 - 100 ng / ml, 1 - 80 ng / ml, 1 - 60 ng / ml, 1 - 40 ng / ml, 1 - 20 ng / ml, 1 - 15 ng / ml, 1 - 10 ng / ml, 1 - 9 ng / ml, 1 - 8 ng / ml, 1 - 7 ng / ml, 1 - 6 ng / ml, 1 - 5 ng / ml, 1 - 4 ng / ml, 1 - 3 ng / ml, 1 - 2 ng / ml, 2 - 100 ng / ml, 2 - 80 ng / ml, 2 - 60 ng / ml, 2 - 40 ng / ml, 2 - 20 ng / ml, 2 - 15 ng / ml, 2 - 10 ng / ml, 2 - 9 ng / ml, 2 - 8 ng / ml, 2 - 7 ng / ml, 2 - 6 ng / ml, 2 - 5 ng / ml, 2 - 4 ng / ml, 2 - 3 ng / ml, 3 - 100 ng / ml, 3 - 80 ng / ml, 3 - 60 ng / ml, 3 - 40 ng / ml, 3 - 20 ng / ml, 3 - 15 ng / ml, 3 - 10 ng / ml, 3 - 9 ng / ml, 3 - 8 ng / ml, 3 - 7 ng / ml, 3 - 6 ng / ml, 3 - 5 ng / ml, 3 - 4 ng / ml, 4 - 100 ng / ml, 4 - 80 ng / ml, 4 - 60 ng / ml, 4 - 40 ng / ml, 4 - 20 ng / ml, 4 - 15 ng / ml, 4 - 10 ng / ml, 4 - 9 ng / ml, 4 - 8 ng / ml, 4 - 7 ng / ml, 4 - 6 ng / ml, 4 - 5 ng / ml, 5 - 100 ng / ml, 5 - 80 ng / ml, 5 - 60 ng / ml, 5 - 40 ng / ml, 5 - 20 ng / ml, 5 - 15 ng / ml, 5 - 10 ng / ml, 5 - 9 ng / ml, 5 - 8 ng / ml, 5 - 7 ng / ml, 5 - 6 ng / ml, 6 - 100 ng / ml, 6 - 80 ng / ml, 6 - 60 ng / ml, 6 - 40 ng / ml, 6 - 20 ng / ml, 6 - 15 ng / ml, 6 - 10 ng / ml, 6 - 9 ng / ml, 6 - 8 ng / ml, 6 - 7 ng / ml, 7 - 100 ng / ml, 7 - 80 ng / ml, 7 - 60 ng / ml, 7 - 40 ng / ml, 7 - 20 ng / ml, 7 - 15 ng / ml, 7 - 10 ng / ml, 7 - 9 ng / ml, 7 - 8 ng / ml, 8 - 100 ng / ml, 8 - 80 ng / ml, 8 - 60 ng / ml, 8 - 40 ng / ml, 8 - 20 ng / ml, 8 - 15 ng / ml, 8 - 10 ng / ml, 8 - 9 ng / ml, 9 - 100 ng / ml, 9 - 80 ng / ml, 9 - 60 ng / ml, 9 - 40 ng / ml, 9 - 20 ng / ml, 9 - 15 ng / ml, 9 - 10 ng / ml or 10 - 100 ng / ml;.

[0014] Preferably, the IL7 content in the culture medium is 1-5 ng / ml, 1-10 ng / ml, 3-8 ng / ml, 4-7 ng / ml, 5-6 ng / ml, or 5-50 ng / ml;

[0015] Preferably, the content of TGFβ in the culture medium is 0.5-1 ng / ml, 0.5-2 ng / ml, 0.5-3 ng / ml, 0.5-5 ng / ml, 0.5-10 ng / ml, 1-2 ng / ml, 1-5 ng / ml or 1-10 ng / ml.

[0016] 3. The culture medium according to item 1, wherein the content of IL4 in the culture medium is 1 - 2 ng / ml, 1 - 3 ng / ml, 1 - 4 ng / ml, 1 - 5 ng / ml, 1 - 6 ng / ml, 1 - 7 ng / ml, 1 - 8 ng / ml, 1 - 9 ng / ml, 1 - 10 ng / ml, 1 - 11 ng / ml, 1 - 12 ng / ml, 1 - 13 ng / ml, 1 - 14 ng / ml, 1 - 15 ng / ml, 1 - 20 ng / ml, 1 - 50 ng / ml, 1 - 70 ng / ml, 1 - 80 ng / ml, 1 - 90 ng / ml, 1 - 100 ng / ml, 1 - 120 ng / ml, 1 - 150 ng / ml, 1 - 200 ng / ml, 1 - 250 ng / ml, 2 - 3 ng / ml, 2 - 4 ng / ml, 2 - 5 ng / ml, 2 - 6 ng / ml, 2 - 7 ng / ml, 2 - 8 ng / ml, 2 - 9 ng / ml, 2 - 10 ng / ml, 2 - 11 ng / ml, 2 - 12 ng / ml, 2 - 13 ng / ml, 2 - 14 ng / ml, 2 - 15 ng / ml, 2 - 20 ng / ml, 2 - 50 ng / ml, 2 - 70 ng / ml, 2 - 80 ng / ml, 2 - 90 ng / ml, 2 - 100 ng / ml, 2 - 120 ng / ml, 2 - 150 ng / ml, 2 - 200 ng / ml, 2 - 250 ng / ml, 5 - 6 ng / ml, 5 - 7 ng / ml, 5 - 8 ng / ml, 5 - 9 ng / ml, 5 - 10 ng / ml, 5 - 11 ng / ml, 5 - 12 ng / ml, 5 - 13 ng / ml, 5 - 14 ng / ml, 5 - 15 ng / ml, 5 - 20 ng / ml, 5 - 40 ng / ml, 5 - 50 ng / ml, 5 - 70 ng / ml, 5 - 80 ng / ml, 5 - 90 ng / ml, 5 - 100 ng / ml, 5 - 150 ng / ml, 5 - 200 ng / ml, 5 - 250 ng / ml, 10 - 11 ng / ml, 10 - 12 ng / ml, 10 - 13 ng / ml, 10 - 14 ng / ml, 10 - 15 ng / ml, 10 - 20 ng / ml, 10 - 40 ng / ml, 10 - 50 ng / ml, 10 - 70 ng / ml, 10 - 80 ng / ml, 10 - 90 ng / ml, 10 - 100 ng / ml, 10 - 150 ng / ml, 10 - 200 ng / ml, 10 - 250 ng / ml, 20 - 40 ng / ml, 20 - 50 ng / ml, 20 - 70 ng / ml, 20 - 80 ng / ml, 20 - 90 ng / ml, 20 - 100 ng / ml, 20 - 150 ng / ml, 20 - 200 ng / ml, 20 - 250 ng / ml, 30 - 40 ng / ml, 30 - 50 ng / ml, 30 - 70 ng / ml,30-80ng / ml, 30-100ng / ml, 30-150ng / ml, 30-200ng / ml, 30-250ng / ml, 40-50ng / ml, 40-70ng / ml, 40-80ng / ml , 40-100ng / ml, 40-150ng / ml, 40-200ng / ml, 40-250ng / ml, 60-70ng / ml, 60-80ng / ml, 60-100ng / ml, 60-150ng / ml, 60-200ng / ml, 60-250ng / ml, 70-80ng / ml, 70-100ng / ml, 70-150ng / ml, 70-200ng / ml, 70-250ng / ml, 80-10 0ng / ml, 80-150ng / ml, 80-250ng / ml, 90-100ng / ml, 90-120ng / ml, 90-150ng / ml, 90-200ng / ml or 90-250ng / ml;,

[0017] Preferably, the IL4 content in the culture medium is 1-10 ng / ml, 1-20 ng / ml, 5-20 ng / ml, 5-40 ng / ml, 20-90 ng / ml, 30-80 ng / ml, 70-150 ng / ml, or 90-120 ng / ml.

[0018] Preferably, the content of TGFβ in the culture medium is 0.5-1 ng / ml, 0.5-10 ng / ml, 0.5-2 ng / ml, 0.5-3 ng / ml, 0.5-5 ng / ml, 1-2 ng / ml, 1-5 ng / ml or 1-10 ng / ml.

[0019] 4. The culture medium according to claim 1, wherein the content of IL21 in the culture medium is 0-1250 ng / ml, 1-1250 ng / ml, 1-1000 ng / ml, 1-750 ng / ml, 1-500 ng / ml, 1-250 ng / ml, 1-200 ng / ml, 1-150 ng / ml, 1-100 ng / ml, 1-50 ng / ml, 1-40 ng / ml, 1-30 ng / ml, 1-29 ng / ml, 1-28 ng / ml, 1-27 ng / ml, 1-26 ng / ml, 1-25 ng / ml, 1-20 ng / ml, 1-10 ng / ml, 1-8 ng / ml, 1-7 ng / ml, 1-6 ng / ml, 1-5 ng / ml, 1-4 ng / ml, 1-3 ng / ml. l, 2.5-1250ng / ml, 2.5-1000ng / ml, 2.5-750ng / ml, 2.5-500ng / ml, 2.5-250ng / ml, 2. 5-200ng / ml, 2.5-150ng / ml, 2.5-100ng / ml, 2.5-50ng / ml, 2.5-40ng / ml, 2.5-30ng / m l, 2.5-29ng / ml, 2.5-28ng / ml, 2.5-27ng / ml, 2.5-26ng / ml, 2.5-25ng / ml, 2.5-20ng / ml, 2.5-10ng / ml, 2.5-8ng / ml, 2.5-7ng / ml, 2.5-6ng / ml, 2.5-5ng / ml, 2.5-4ng / ml, 2.5 - 3 ng / ml, 5 - 1250 ng / ml, 5 - 1000 ng / ml, 5 - 750 ng / ml, 5 - 500 ng / ml, 5 - 250 ng / ml, 5 - 200 ng / ml, 5 - 150 ng / ml, 5 - 100 ng / ml, 5 - 50 ng / ml, 5 - 40 ng / ml, 5 - 30 ng / ml, 5 - 29 ng / ml, 5 - 28 ng / ml, 5 - 27 ng / ml, 5 - 26 ng / ml, 5 - 25 ng / ml, 5 - 20 ng / ml, 5 - 10 ng / ml, 5 - 8 ng / ml, 5 - 7 ng / ml, 5 - 6 ng / ml, 10 - 1250 ng / ml, 10 - 1000 ng / ml, 10 - 800 ng / ml, 10 - 750 ng / ml, 10 - 500 ng / ml, 10 - 400 ng / ml, 10 - 250 ng / ml, 10 - 200 ng / ml, 10 - 150 ng / ml, 10 - 100 ng / ml, 10 - 50 ng / ml, 10 - 40 ng / ml, 10 - 30 ng / ml, 10 - 29 ng / ml, 10 - 28 ng / ml, 10 - 27 ng / ml, 10 - 26 ng / ml, 10 - 25 ng / ml, 10 - 20 ng / ml, 20 - 1250 ng / ml, 20 - 1000 ng / ml, 20 - 800 ng / ml, 20 - 750 ng / ml, 20 - 500 ng / ml, 20 - 400 ng / ml, 20 - 250 ng / ml, 20 - 200 ng / ml, 20 - 150 ng / ml, 20 - 100 ng / ml, 20 - 50 ng / ml, 20 - 40 ng / ml, 20 - 30 ng / ml, 20 - 29 ng / ml, 20 - 28 ng / ml, 20 - 27 ng / ml, 20 - 26 ng / ml, 20 - 25 ng / ml, 30 - 1250 ng / ml, 30 - 1000 ng / ml, 30 - 800 ng / ml, 30 - 750 ng / ml, 30 - 500 ng / ml, 30 - 400 ng / ml, 30 - 250 ng / ml, 30 - 200 ng / ml, 30 - 150 ng / ml, 30 - 100 ng / ml, 30 - 50 ng / ml, 30 - 40 ng / ml, 100 - 1250 ng / ml, 100 - 1000 ng / ml, 100 - 800 ng / ml, 100 - 900 ng / ml, 100 - 750 ng / ml, 100 - 500 ng / ml, 100 - 400 ng / ml, 100 - 250 ng / ml, 100 - 200 ng / ml or 100 - 150 ng / ml;.

[0020] Preferably, the IL21 content is 10-30 ng / ml, 20-30 ng / ml, or 20-40 ng / ml.

[0021] Preferably, the content of TGFβ in the culture medium is 0.5-1 ng / ml, 0.5-10 ng / ml, 0.5-2 ng / ml, 0.5-3 ng / ml, 0.5-5 ng / ml, 1-2 ng / ml, 1-5 ng / ml or 1-10 ng / ml.

[0022] 5. The culture medium according to any one of items 1-4, further comprising any one or more of the following cytokines:

[0023] IL1, IL2, IL3, IL5, IL6, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL22, IL23, IL24, IL26, IL27, IL28, IL29, IL32, IL33, IL35, IL37, anti-IFNγ (interferon-gamma antibody), TNF-α;

[0024] Preferably, the culture medium comprises any one of the following groups:

[0025] (I) IL7, TGFβ, and any one or more of the group consisting of IL2, IL4, IL15, IL21 and anti-IFNγ;

[0026] (II) IL7, TGFβ, and any one or more of the group consisting of IL4, IL15 and IL21;

[0027] (III) IL7, TGFβ, and any one or more of the group consisting of IL4 and IL21;

[0028] More preferably, the culture medium comprises any one of the following groups:

[0029] 1) Combinations of IL7, TGFβ, and IL4;

[0030] 2) Combinations of IL7, TGFβ, and IL21;

[0031] 3) Combinations of IL7, TGFβ, IL2, and IL4;

[0032] 4) Combinations of IL7, TGFβ, IL2 and IL21;

[0033] 5) Combinations of IL7, TGFβ, IL4 and IL21;

[0034] 6) Combinations of IL7, TGFβ, IL2, IL4 and IL21;

[0035] 7) Combinations of IL7, TGFβ, and antiIFN-γ;

[0036] 8) Combinations of IL7, TGFβ, IL4 and antiIFN-γ;

[0037] 9) Combinations of IL7, TGFβ, IL21 and antiIFN-γ;

[0038] 10) A combination of IL7, TGFβ, IL4, IL21 and antiIFN-γ;

[0039] 11) Combinations of IL7, TGFβ, and IL15;

[0040] 12) Combinations of IL7, TGFβ, IL4 and IL15;

[0041] 13) Combinations of IL7, TGFβ, IL21 and IL15;

[0042] 14) Combinations of IL7, TGFβ, IL4, IL21 and IL15;

[0043] 15) Combinations of IL2, IL4 and TGFβ;

[0044] 16) Combinations of IL4, TGFβ, IL2 and antiIFN-γ;

[0045] 17) Combinations of IL4, TGFβ, and antiIFN-γ; and

[0046] 18) Combination of IL4, TGFβ, IL21 and antiIFN-γ.

[0047] 6. The culture medium according to claim 5, wherein the IL2 content in the culture medium is 0-1000 IU / ml, 10-1000 IU / ml, 50-1000 IU / ml, 100-1000 IU / ml, 200-1000 IU / ml, 300-1000 IU / ml, 400-1000 IU / ml, 410-1000 IU / ml, 420-1000 IU / ml, 430-1000 IU / ml, 440-1000 IU / ml, 450-1000 IU / ml, 460-1000 IU / ml, 470-1000 IU / ml, 480-1 000IU / ml, 490-1000IU / ml, 500-1000IU / ml, 800-1000IU / ml, 10-800IU / ml, 50-800IU / ml, 100-800IU / ml, 200-800IU / ml, 300-800IU / ml , 400-800IU / ml, 410-800IU / ml, 420-800IU / ml, 430-800IU / ml, 440-800IU / ml, 450-800IU / ml, 460-800IU / ml, 470-800IU / ml, 480-800IU / ml, 490-800IU / ml, 500-800IU / ml, 10-700IU / ml, 50-700IU / ml, 100-700IU / ml, 200-700IU / ml, 300-700IU / ml, 400-700IU / ml, 410-700 IU / ml, 420-700IU / ml, 430-700IU / ml, 440-700IU / ml, 450-700IU / ml, 460-700IU / ml, 470-700IU / ml, 480-700IU / ml, 490-700IU / ml, 500- 700IU / ml, 10-600IU / ml, 50-600IU / ml, 100-600IU / ml, 100-600IU / ml, 200-600IU / ml, 300-600IU / ml, 400-600IU / ml, 410-600IU / ml, 420 -600IU / ml, 430-600IU / ml, 440-600IU / ml, 450-600IU / ml, 460-600IU / ml, 470-600IU / ml, 480-600IU / ml, 490-600IU / ml or 500-600IU / ml;

[0048] And / or, the concentration of IL15 in the culture medium is 0-100 ng / ml, 10-100 ng / ml, 20-100 ng / ml, 30-100 ng / ml, 40-100 ng / ml, 50-100 ng / ml, 60-100 ng / ml, 70-100 ng / ml, 90-100 ng / ml, 10-90 ng / ml, 20-90 ng / ml, 30-90 ng / ml, 40-90 ng / ml, 50 -90ng / ml, 10-80ng / ml, 20-80ng / ml, 30-80ng / ml, 40-80ng / ml, 50-80ng / ml, 10-70ng / ml, 20-70ng / ml, 3 0-70ng / ml, 40-70ng / ml, 50-70ng / ml, 10-60ng / ml, 20-60ng / ml, 30-60ng / ml, 40-60ng / ml or 50-60ng / ml;

[0049] And / or, the antiIFN-γ content in the culture medium is 0-200 ng / ml, 50-200 ng / ml, 70-200 ng / ml, 80-200 ng / ml, 90-200 ng / ml, 100-200 ng / ml, 150-200 ng / ml, 50-150 ng / ml, 70-150 ng / ml, 80-150 ng / ml, 90-150 ng / ml, 100-150 ng / ml, 50-140 ng / ml, 70-140 ng / ml , 80-140ng / ml, 90-140ng / ml, 100-140ng / ml, 50-130ng / ml, 70-130ng / ml, 80-130ng / ml, 90-130ng / ml, 100-130ng / m l, 50-120ng / ml, 70-120ng / ml, 80-120ng / ml, 90-120ng / ml, 50-110ng / ml, 70-110ng / ml, 80-110ng / ml or 90-110ng / ml.

[0050] 7. The culture medium according to any one of items 1-6, comprising a basal culture medium for cell culture.

[0051] 8. The culture medium according to item 7, wherein the basal culture medium is a universal basal culture medium for cell culture; the universal basal culture medium is a serum-free culture medium or a serum-containing culture medium;

[0052] Preferably, the serum-free culture medium is selected from OpTmizer™ CTS™, Immunocult™ XF, CellGro™, TexMacs™, Stemline™, Xvivo15™, PrimeXV, and ImmunoCult. TM X-VF medium, SCGM medium, X-VIVO series serum-free immune cell medium, X-VIV010 serum-free immune cell medium, X-VIV015 serum-free immune cell medium, X-VIVO 20 serum-free cell medium, Serum-free culture medium for T cells, serum-free culture medium for lymphocytes HIPP-T009, and serum-free culture medium for lymphocytes KBM581. L500 serum-free lymphocyte culture medium and StemXVivo, or any one or more of them;

[0053] Preferably, the serum-containing culture medium is selected from any one or more of α-MEM medium, RPMI 1640 medium, AIM-V medium, DMEM medium, F-12 medium, X-vivo 15 medium, X-Vivo 20 medium, OpTmizer medium, and IMDM medium.

[0054] 9. A method for obtaining mixed T cells, comprising culturing peripheral blood mononuclear cells (PBMCs), CD4-positive T cells, CD8-positive T cells, CD4 / CD8 double-negative T cells, or any combination thereof, using the culture medium described in any one of items 1-8; wherein the mixed T cells comprise CD4 / CD8-positive mixed cells and / or CD4 / CD8 double-negative mixed cells;

[0055] Preferably, the culture time is 2-25 days;

[0056] More preferably, the culture time is 7-21 days.

[0057] 10. The method according to claim 9, wherein the T cell is a natural T cell and / or a genetically modified T cell;

[0058] Preferably, the genetically modified T cells contain the introduced exogenous gene.

[0059] 11. The method according to claim 10, wherein the genetically modified T cell comprises any one or more of the following: a gene segment encoding a chimeric antigen receptor CAR, a gene segment encoding a T cell receptor (TCR) that specifically recognizes tumor antigens, and a gene segment encoding a fusion protein of the T cell receptor;

[0060] Preferably, the genetically modified T cells contain a gene segment encoding a chimeric antigen receptor CAR;

[0061] Preferably, the gene-modified T cells are CAR-T cells.

[0062] 12. According to the method of item 11, the chimeric antigen receptor CAR includes at least one extracellular antigen-binding domain, at least one transmembrane domain and at least one signal transduction domain;

[0063] Preferably, the extracellular antigen-binding domain recognizes one or more target molecules expressed on the surface of tumor cells; preferably, the target molecules include CD19, CD20, CD22, CD33, CLL-1 (CLEC12A), CD7, CD5, CD70, CD123, CEA, CEACAM5, CEACAM6, CEACAM7, Mesothelin, MUC1, CLDN18.2, CDH17, Trop2, BCMA, NKG2D, PDL1, EGFR, EGFRVIII, PSCA, PSMA, MUC16, CD133, GD2, IL13R2, B7H3, Her2, CD30, SLAMF7, CD38, GPC3, WT1, AFP, FOLR1, c-Met, LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma-associated antigen 1), and TAG-72 are any one or more of these.

[0064] More preferably, the target molecule is selected from any one or more of CD19, CD70, CEA, and BCMA.

[0065] 13. According to the method of item 12, when the target molecule is CEA, the amino acid sequence of the chimeric antigen receptor CAR comprises the sequence shown in SEQ ID NO.1, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO.1;

[0066] And / or, when the target molecule is CD19, the amino acid sequence of the chimeric antigen receptor CAR includes the sequence shown in SEQ ID NO.2, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO.2;

[0067] And / or, when the target molecule is CD70, the amino acid sequence of the chimeric antigen receptor CAR includes the sequence shown in SEQ ID NO.3, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO.3.

[0068] 14. The method according to any one of items 9-13 further includes the step of activating T cells before culture;

[0069] Preferably, the step of activating T cells includes contacting the T cells with an activating agent; preferably, the activating agent comprises CD3 antibody and / or CD28 antibody; preferably, the activating agent comprising CD3 antibody and / or CD28 antibody is a magnetic bead or reagent conjugated with CD3 antibody and / or CD28 antibody, a virus loaded with CD3 antibody and / or CD28 antibody, nanoparticles or vesicles, or any combination thereof;

[0070] Preferably, the activation time is 5 min-48 h, 5-60 min, 1-6 h, 1-12 h, 1-24 h, 1-36 h, 6-12 h, 6-24 h, 6-36 h, 6-48 h, 12-24 h, 12-36 h, 12-48 h, or 24-48 h.

[0071] 15. A hybrid T cell obtained by the method described in any one of items 9-14.

[0072] 16. A method for culturing universal T cells, comprising culturing universal T cells using the culture medium described in any one of items 1-8;

[0073] Preferably, the culture time is 2-25 or 7-21 days;

[0074] Preferably, the universal T cells are CD4 and CD8 double-negative CAR-T cells;

[0075] More preferably, the universal T cell is a universal CAR-T cell (U CAR-T cell).

[0076] 17. A universal T cell culture, obtained by culturing using the method described in item 16.

[0077] 18. Use of the hybrid T cells described in item 15 or the universal T cell culture described in item 17 for the preparation of medicaments for the prevention and / or treatment of diseases.

[0078] 19. A pharmaceutical composition comprising the hybrid T cells of claim 15 and / or the universal T cell culture of claim 17.

[0079] 20. A treatment method for a disease, comprising administering to a subject in need an effective amount of the hybrid T cells of claim 15 and / or the universal T cell culture of claim 17, and / or its cryopreservation and resuscitation product.

[0080] Invention Effects

[0081] 1. The research in this application found that any one, two, or three of IL7, IL4, and TGFβ1 and IL21 can significantly improve the viability and factor-free proliferation capacity of T lymphocytes or cell populations containing T lymphocytes, such as PBMCs, after cryopreservation and resuscitation; it can also significantly improve the viability and factor-free proliferation capacity of genetically modified T cells (such as CAR-T cells) prepared from isolated mononuclear cells or T cells obtained by sorting mononuclear cells after cryopreservation and resuscitation, enabling them to have better expansion and persistence in the human body, and thus exert better functions.

[0082] 2. The study in this application found that the combination of TGFβ1+IL7+IL21+IL4 can promote the proliferation and persistence of allogeneic T cells and genetically modified allogeneic T cells in vivo, thereby promoting better efficacy of allogeneic T cells and genetically modified allogeneic T cells; and the above-mentioned allogeneic T cells or genetically modified allogeneic T cells are not limited by CD4 phenotype, and can be a mixed cell population of CD4 and CD8 positive cells, or a population of universal T cells or modified universal T cells in which CD4 and CD8 molecules are knocked down or deleted by gene editing technology.

[0083] 3. The study in this application found that using a combination of TGFβ1+antiIFNγ+IL21+IL4 or a combination of TGFβ1+antiIFNγ+IL4, with non-CD4 single-positive mononuclear cells or T cell populations generated by sorting mononuclear cells (such as those containing CD4) + CD8 + After CAR-T cells are prepared from T cells (either mononuclear T cells or T cells generated by sorting mononuclear T cells), the resulting CAR-T cells exhibit significantly enhanced cell viability and factor-free proliferation capacity after cryopreservation and thawing, resulting in better expansion and persistence in the human body, and thus better function. Attached Figure Description

[0084] Figure 1. Viability, fold increase, and proliferation curves of CAR-T cells prepared by different combinations of cryopreserved factors after thawing and factor-free culture in each group; Figure 1A shows the viability detection results of each group after 24 hours of factor-free culture; Figure 1B shows the total fold increase of CAR-T cells in each group on day 7 after 7 days of factor-free culture; Figure 1C shows the proliferation curves of each group during the 7-day factor-free culture process; Figure 1D shows the factor-free proliferation of the IL21+IL4+TGFβ1 and IL21+TGFβ1 combinations.

[0085] Figure 2. Proportion of CD4 and CD8 positive cells obtained after culturing with different combinations of cytokines.

[0086] Figure 3. The ability of CAR-T cells prepared with different combinations of cytokines to continuously kill tumor cells; Figure 3A shows the continuous tumor killing ability of the IL7+TGFβ1 and IL7+IL21+IL4+TGFβ groups; Figure 3B shows the continuous tumor killing ability of the IL21+IL4+TGFβ1 and IL21+TGFβ1 groups.

[0087] Figure 4. CEA expression in NCI-N87-Luc-GFP cells.

[0088] Figure 5. Viability and fold increase of CD19 CAR-T cells after cryopreservation and subsequent factor-free culture; Figure 5A shows the viability results; Figure 5B shows the total fold increase of cells on day 7 of factor-free culture.

[0089] Figure 6. CD19 expression in Nalm6-luc-GFP cells.

[0090] Figure 7. Sustained killing ability of cryopreserved CD19 CAR-T cells in vitro after thawing.

[0091] Figure 8. Antitumor activity of CEA CAR-T cells prepared with different factor combinations; Figure 8A is a fluorescence imaging image of mice; Figure 8B shows the copy number of CAR-T cells in the blood of mice after different days of CAR-T cell infusion, with the horizontal axis representing the number of days of CEA CAR-T cell infusion and the vertical axis representing the copy number of CEA CAR-T cells in the blood of mice.

[0092] Figure 9. Antitumor activity of CAR-T cells prepared with different combinations of anti-IFNγ factors; where Figure 9A is a mouse fluorescence imaging image; Figure 9B is the copy number of CEA CAR-T cells in mouse blood. The horizontal axis of Figure 9B represents different culture protocols, and the vertical axis represents the copy number of CEA CAR-T cells in mouse blood.

[0093] Figure 10. Antitumor activity of CD70 CAR-T cells prepared with different factor combinations in mice.

[0094] Figure 11. CD70 expression of SKOV3-Luc-GFP in target cells.

[0095] Figure 12. Effects of IL4 and TGFβ1 cytokine concentrations on the viability of CAR-T cells after cryopreservation and thawing.

[0096] Figure 13. Effects of different cytokine concentrations on the viability of CAR-T cells after cryopreservation and thawing.

[0097] Figure 14. Proliferation curves of CD19 UCAR-T cells cultured with different combinations of cytokines.

[0098] Figure 15. Cell viability detection of CD19 UCAR-T cells cultured with different cytokine combinations after cryopreservation and thawing, followed by 7 days of cytokine-free culture.

[0099] Figure 16. Proliferation curves of CD19 UCAR-T cells cultured with different cytokine combinations after cryopreservation and thawing, followed by 7 days of cytokine-free culture.

[0100] Figure 17. Proportion of Tcm-center memory cells in CD19 UCAR-T cells prepared with different combinations of cytokines.

[0101] Figure 18. Tumor fluorescence of CD19 UCAR-T prepared with different factor combinations in mice.

[0102] Figure 19. Proliferation curves of UCAR-T cells prepared with different combinations of factors.

[0103] Figure 20. Cell viability statistics on day 7 after factor-free culture.

[0104] Figure 21. Proliferation curves of UCAR-T cells cultured at different cytokine concentrations.

[0105] Figure 22. Viability of UCAR-T cells after cryopreservation and thawing at different cytokine concentrations.

[0106] Figure 23. Cell proliferation curves of UCAR-T cells cultured at different cytokine concentrations after cryopreservation and thawing, followed by cell culture without cytokine.

[0107] Figure 24. Cell viability of CAR-T cells or T cells derived from umbilical cord blood cultured in a culture medium containing the cytokine combination of this application after 24 hours of cytokine-free culture; wherein, Figure 24A shows the cell viability detection of CAR-T cells derived from umbilical cord blood cultured in the cytokine combination of this application after 24 hours of cytokine-free culture; Figure 24B shows the cell viability detection of T cells derived from umbilical cord blood cultured in the cytokine combination of this application after 24 hours of cytokine-free culture.

[0108] Figure 25. Cell status of cryopreserved CEA CAR-T cells after 24 hours of culture in cytokine-free basal medium; Figure 25A shows the CAR-T cell viability; Figure 25B shows the total fold increase of CAR-T cells.

[0109] Figure 26. Viability of the cytokine combinations described in this application and the culture medium without cytokine for 24 hours under different stimulation regimens.

[0110] Figure 27. In vivo efficacy verification of the cytokine combinations and culture media described in this application under different stimulation regimens.

[0111] Specific implementation methods

[0112] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in their functions.

[0113] As used throughout the specification and claims, the terms "comprising" or "including" are open-ended and should be interpreted as "comprising but not limited to". The subsequent descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0114] It should be understood that the embodiments of this application described herein include embodiments that are "composed of" and / or "substantially composed of". References to values ​​or parameters of "about" herein include (and describe) variations of that value or parameter itself. For example, a reference to "about X" includes a description of "X".

[0115] As used herein, references to “not” values ​​or parameters generally refer to and describe “except” values ​​or parameters. For example, “The method is not used to treat type X cancer” means that the method is used to treat cancers other than type X.

[0116] As used in this article, the term “approximately XY” has the same meaning as “approximately X to approximately Y”.

[0117] As used herein and in the appended claims, the singular forms “a / an” and “the” include the plural objects unless the context clearly indicates otherwise. It should also be noted that claims may be drafted to exclude any optional elements. Therefore, this statement is intended as a preliminary basis for the use of exclusive terms such as “only” or “merely” in conjunction with the description of the elements of the claim, or for the use of the limitation of “no”.

[0118] As used herein, the term "and / or" in words such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, as used herein, the term "and / or" in words such as "A, B and / or C" is intended to include each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0119] In this application, "cytokine" refers to a class of small molecule proteins with broad biological activity synthesized and secreted by immune cells (such as monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.) upon stimulation. Cytokines include, but are not limited to, any one or more of IL1, IL2, IL3, IL4, IL5, IL6, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL21, IL22, IL23, IL24, IL26, IL27, IL28, IL29, IL32, IL33, IL35, IL37, anti-IFNγ, and TNFα. In this application, "cytokine" can be the cytokine itself, and / or a subtype of a cytokine, and / or a functional variant of a cytokine.

[0120] As used herein, the term "functional variant" means a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide but retains the necessary characteristics. A typical functional variant of a polynucleotide differs from the nucleic acid sequence of another reference polynucleotide. Changes in the nucleic acid sequence of a functional variant may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes can result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence, as described below. A typical functional variant of a polypeptide differs from another reference polypeptide in its amino acid sequence. Typically, the differences are limited, making the sequences of the reference polypeptide and the functional variant very similar overall and identical in many regions. The amino acid sequences of the functional variant and the reference polypeptide can differ by any combination of one or more substitutions, additions, or deletions. The substituted or inserted amino acid residues may or may not be amino acid residues encoded by the genetic code. Functional variants of polynucleotides or polypeptides may be naturally occurring (such as allelic functional variants) or may be unknown naturally occurring functional variants. Non-naturally occurring functional variants of polynucleotides and polypeptides can be prepared by mutagenesis, by direct synthesis, and by other recombinant methods known to those skilled in the art.

[0121] As used herein, the terms "IL7", "IL-7", "interleukin 7", "interleukin-7", and "interleukin-7" are interchangeable in this application, referring to a cytokine belonging to the chemokine family. As used herein, the terms "IL2", "IL-2", "interleukin 2", "interleukin-2", and "interleukin-2" are interchangeable in this application, referring to a cytokine belonging to the chemokine family. As used herein, the terms "IL21", "IL-21", "interleukin 21", "interleukin-21", and "interleukin-21" are interchangeable in this application. It is mainly secreted by activated CD4+ T cells, has a molecular structure similar to IL-15, participates in regulating B cell proliferation, and synergistically promotes bone marrow progenitor cell proliferation and NK cell proliferation, differentiation, and cytotoxic activity in conjunction with IL-15. As used herein, the terms "IL4", "IL-4", "interleukin 4", "interleukin-4", and "interleukin-4" are interchangeable in this application, referring to a cytokine belonging to the chemokine family. As used herein, the terms “IL15,” “IL-15,” “interleukin 15,” “interleukin-15,” and “interleukin-15” are interchangeable and refer to a cytokine belonging to the chemokine family. As used herein, the terms “TGF-β1,” “TGFβ1,” and “transforming growth factor β1” are interchangeable and refer to a polypeptide member of the transforming growth factor β superfamily, possessing various cellular functions, including regulating cell growth, proliferation, differentiation, and apoptosis. As used herein, the terms “antiIFN-γ” and “interferon-γ antibody” are interchangeable.

[0122] PBMC (Peripheral blood mononuclear cell, referred to as mononuclear cell in this example) are cells with a single nucleus in peripheral blood, including lymphocytes and monocytes. Leukocytes (white blood cells, abbreviated as WBC) are colorless, spherical, nucleated, heterogeneous blood cells, including granulocytes, monocytes, and lymphocytes. TIL (Tumor Infiltrating Lymphocytes) refer to infiltrating lymphocytes isolated from tumor tissue; these lymphocytes include CD4-positive and CD8-positive cells. As understood by those skilled in the art, single positive (SP), i.e., CD4+CD8- or CD4-CD8+, is a developmental stage of T lymphocytes in the thymus based on the expression status of CD4 and CD8.

[0123] The term "cell" as used herein should be understood not only to a specific single cell, but also to its offspring or potential offspring. Because certain modifications may occur in offspring due to mutations or environmental influences, such offspring may indeed differ from the parent cell, but are still included within the scope of this terminology.

[0124] The terms “subject,” “individual,” or “patient” are used interchangeably herein. For therapeutic purposes, “individual” refers to any animal classified as a mammal, including humans, livestock, and farm animals, as well as zoo, farm, or pet animals such as dogs, horses, cats, and cattle. In some embodiments, “individual” refers to a human individual.

[0125] The term "cell cryopreservation" refers to a technique that places cells in an ultra-low temperature environment to put them into a "dormant" state, and then revives the cells when needed.

[0126] The term "cell resuscitation" refers to the process of thawing frozen cells.

[0127] In this application, the term "cytokine-free proliferation" refers to cell culture performed under conditions without any cytokines, followed by cell counting using cell counting equipment, counting, or protocols, and the determination of the proliferation fold of cultured cells relative to uncultured cells after direct resuscitation based on the cell counting results.

[0128] The term "cell population" or "cell group" refers to a composition comprising one or more cells. In some embodiments, each cell in the "cell population" has one or more identical characteristics, such as having the same nuclear typing, similar function, size within a similar range, having one or more identical cell markers (i.e., the one or more identical cell markers are positive), or not having one or more specific cell markers (i.e., the one or more specific cell markers are negative). The "not having" or "negative" may not necessarily mean the absolute absence of the marker. Those skilled in the art can readily compare cells to positive and / or negative controls, and / or set predetermined thresholds, and classify cells as "not having" or negative for the marker when they have expression levels below a predetermined threshold or below a predetermined threshold using conventional detection methods (e.g., using flow cytometry). For example, in some embodiments, the cell population is a T cell population. In some embodiments, the cell population is PBMCs. In some embodiments, the population is a CD4 single-positive cell population isolated from PBMCs; in some embodiments, the population is a CD8 single-positive cell population isolated from PBMCs; in some embodiments, the population is a mixed population of CD4 single-positive cells and CD8 single-positive cells isolated from PBMCs.

[0129] As used herein, the term "wildtype" has the meaning commonly understood by those skilled in the art as referring to the typical form of an organism, strain, gene, or trait that distinguishes it from mutants or variants when it exists in nature. It can be isolated from resources in nature and is not deliberately modified.

[0130] As used herein, the terms “non-naturally occurring,” “engineered,” and “engineered modified” are used interchangeably to refer to artificial intervention. When these terms are used to describe nucleic acid molecules or peptides, they mean that the nucleic acid molecule or peptide is at least substantially free of at least one other component that is naturally associated with or naturally present in it.

[0131] The term "adoptive cell therapy" (ACT) refers to a treatment method that uses cells from the body's own immune system, which are cultured and modified externally before being reinfused into the body to eliminate disease.

[0132] The terms “chimeric antigen receptor” and “CAR” are used interchangeably in this application and refer to a group of engineered peptides or proteins that, when in immune effector cells, bind to specific antigens contained on target cells and generate intracellular signals after recognizing the specific antigens, thereby activating downstream pathways in the cell where the receptor is located to initiate the killing effect of the immune effector cells on the target cells.

[0133] For nucleic acid sequences, the "sequence identity percentage (%)" is defined as the percentage of nucleotides in a candidate sequence that are identical to nucleotides in a specific nucleic acid sequence after sequence alignment (if necessary) to achieve the maximum sequence identity percentage, allowing gaps (gaps). For peptide, polypeptide, or protein sequences, the "sequence identity percentage (%)" is the percentage of amino acid residues in a candidate sequence that are identically substituted to amino acid residues in a specific peptide or amino acid sequence after sequence alignment (if necessary) to achieve the maximum sequence homology percentage. For the purpose of determining the amino acid sequence identity percentage, alignment can be performed in various ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0134] This application provides a T cell culture medium comprising a basal culture medium for cell culture and a combination of any one, two or three of the cytokines IL7, IL21, and IL4 with TGFβ.

[0135] This application provides a T cell culture medium comprising IL7 and TGFβ.

[0136] This application provides a T cell culture medium containing the cytokines IL21 and TGFβ.

[0137] This application provides a T cell culture medium, including the cytokines IL4 and TGFβ.

[0138] In some embodiments, TGFβ includes any one or more of the following cytokines: TGFβ1, TGFβ2, and TGFβ3. In some embodiments, TGFβ is TGFβ1, TGFβ2, and TGFβ3; in some embodiments, TGFβ is a combination of TGFβ1 and TGFβ2, or a combination of TGFβ1 and TGFβ3.

[0139] In some embodiments, the IL7 concentration in the culture medium is 0.5-250 ng / ml; in other embodiments, the IL7 concentration in the culture medium is 0.5-5 ng / ml, 0.5-10 ng / ml, 0.5-15 ng / ml, 0.5-30 ng / ml, 0.5-50 ng / ml, 0.5-100 ng / ml, 0.5-250 ng / ml, 1-5 ng / ml, 1-10 ng / ml, 1-15 ng / ml, 1-20 ng / ml, 1-30 ng / ml, 1-50 ng / ml, 1-60 ng / ml, 1-70 ng / ml, 1-80 ng / ml, 1-90 ng / ml, or 1-100 ng / ml. / ml, 1-250ng / ml, 2-5ng / ml, 2-6ng / ml, 2-8ng / ml, 2-9ng / ml, 2-10ng / ml, 2-15ng / ml, 2-20ng / ml, 2-30ng / ml, 2-50ng / ml, 2-100ng / ml, 2-250ng / ml, 2.5-5ng / ml, 2.5-10ng / ml, 2.5-20ng / ml, 2.5-50ng / ml, 2.5-100ng / ml, 2.5-250ng / ml, 3-5ng / ml, 3-6ng / ml, 3-9ng / ml, 3-8ng / ml, 3-10ng / ml, 3-15ng / ml, 3-30ng / ml, 3-50ng / ml, 3-100ng / ml, 3-250ng / ml, 4-5ng / ml, 4-6ng / ml, 4-7ng / ml, 4-8ng / ml, 4-9ng / ml, 4-10ng / ml, 4-15ng / ml, 4-30ng / ml, 4-5 0ng / ml, 4-100ng / ml, 4-250ng / ml, 5-6ng / ml, 5-8ng / ml, 5-10ng / ml, 5-15ng / ml, 5-20ng / ml, 5-30ng / ml, 5-50ng / ml, 5-60ng / ml, 5-70ng / ml, 5-80ng / ml, 5-90ng / ml, 5-100ng / ml, 5-250ng / ml, 6-8ng / ml, 6-10ng / ml, 6-15ng / ml, 6-30ng / ml, 6-50ng / ml, 6-100ng / ml, 6-250ng / ml, 7-8ng / ml, 7-10ng / ml , 7-15ng / ml, 7-30ng / ml, 7-50ng / ml, 7-100ng / ml, 7-250ng / ml, 8-10ng / ml, 8-15ng / ml, 8-30ng / ml, 8-50ng / ml, 8-100ng / ml, 8-250ng / ml, 9-10ng / ml,9-15ng / ml, 9-30ng / ml, 9-50ng / ml, 9-100ng / ml, 9-250ng / ml, 10-15ng / ml, 10-20ng / ml, 10-30ng / ml, 10-50ng / ml, 10-100ng / ml, 10-250ng / ml, 1 5-25ng / ml, 15-30ng / ml, 15-50ng / ml, 15-100ng / ml, 15-250ng / ml, 20-30ng / ml, 20-50ng / ml, 20-70ng / ml, 20-90ng / ml, 20-100ng / ml, 20-250ng / ml, 25-30ng / ml, 25-40ng / ml, 25-50ng / ml, 25-70ng / ml, 25-100ng / ml, 25-250ng / ml, 30-50ng / ml, 30-70ng / ml, 30-100ng / ml, 30-250ng / ml, 40-6 0ng / ml, 40-100ng / ml, 40-250ng / ml, 50-70ng / ml, 50-80ng / ml, 50-90ng / ml, 50-100ng / ml, 50-250ng / ml, 60-80ng / ml, 60-100ng / ml, 60-250ng / m 1, 70-100 ng / ml, 70-250 ng / ml, 80-100 ng / ml, 80-250 ng / ml, 90-100 ng / ml, 90-250 ng / ml, 100-250 ng / ml or any content range within the range of 0.5-250 ng / ml; in a specific embodiment, the IL7 content in the culture medium is 0.1 ng / ml, 0.2 ng / ml, 0.3 ng / ml, 0.4 ng / ml, 0.5 ng / ml, 0.6 ng / ml, 0.7 ng / ml, 0.8 ng / ml, 1 ng / ml, 2.5 ng / ml, 3 ng / ml, 3.5 ng / ml, 4 ng / ml. Any concentration within the range of 0.5-250 ng / ml, 4.5 ng / ml, 4.8 ng / ml, 5 ng / ml, 5.5 ng / ml, 6 ng / ml, 8 ng / ml, 10 ng / ml, 25 ng / ml, 30 ng / ml, 45 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 120 ng / ml, 140 ng / ml, 160 ng / ml, 180 ng / ml, 200 ng / ml, 210 ng / ml, 220 ng / ml, 230 ng / ml, 240 ng / ml, 250 ng / ml, or any concentration within the range of 0.5-250 ng / ml.

[0140] In some implementations, the IL4 content in the culture medium is 1-250 ng / ml; in some embodiments, the IL4 content in the culture medium is 1-2 ng / ml, 1-3 ng / ml, 1-4 ng / ml, 1-5 ng / ml, 1-6 ng / ml, 1-7 ng / ml, 1-8 ng / ml, 1-9 ng / ml, 1-10 ng / ml, 1-11 ng / ml, 1-12 ng / ml, 1-13 ng / ml, 1-14 ng / ml, 1-15 ng / ml, 1-20 ng / ml, 1-30 ng / ml, 1-40 ng / ml, 1-50 ng / ml, 1-60 ng / ml, 1-70 ng / ml, 1-8 ng / ml, 1-8 ng / ml, 1-20 ng / ml, 1-30 ng / ml, 1-40 ng / ml, 1-50 ng / ml, 1-60 ng / ml, 1-70 ng / ml, 1-8 ng / ml, 1-250 ... 0ng / ml, 1-90ng / ml, 1-100ng / ml, 1-120ng / ml, 1-150ng / ml, 1-200ng / ml, 1-250ng / ml, 2-3ng / ml, 2-4ng / ml, 2-5ng / ml, 2-6ng / ml, 2-7ng / ml, 2-8ng / ml , 2-9ng / ml, 2-10ng / ml, 2-11ng / ml, 2-12ng / ml, 2-13ng / ml, 2-14ng / ml, 2-15ng / ml, 2-20ng / ml, 2-30ng / ml, 2-50ng / ml, 2-70ng / ml, 2-80ng / ml, 2-90n g / ml, 2-100ng / ml, 2-120ng / ml, 2-150ng / ml, 2-200ng / ml, 2-250ng / ml, 3-5ng / ml, 3-10ng / ml, 3-20ng / ml, 3-50ng / ml, 3-100ng / ml, 3-250ng / ml, 4-1 0ng / ml, 4-20ng / ml, 4-50ng / ml, 4-100ng / ml, 4-250ng / ml, 5-6ng / ml, 5-7ng / ml, 5-8ng / ml, 5-9ng / ml, 5-10ng / ml, 5-11ng / ml, 5-12ng / ml, 5-13ng / ml, 5-14ng / ml, 5-15ng / ml, 5-20ng / ml, 5-25ng / ml, 5-30ng / ml, 5-40ng / ml, 5-50ng / ml, 5-70ng / ml, 5-80ng / ml, 5-90ng / ml, 5-100ng / ml, 5-150ng / ml, 5-2 00ng / ml, 5-250ng / ml, 6-10ng / ml, 6-100ng / ml, 6-250ng / ml, 7-10ng / ml, 7-100ng / ml, 7-250ng / ml, 8-10ng / ml, 8-100ng / ml, 8-250ng / ml, 9-10ng / ml,9-100ng / ml, 9-250ng / ml, 10-11ng / ml, 10-12ng / ml, 10-13ng / ml, 10-14ng / ml, 10-15ng / ml, 10-20ng / ml, 10-40ng / ml, 10-50ng / ml, 10-70ng / ml, 10- 80ng / ml, 10-90ng / ml, 10-100ng / ml, 10-150ng / ml, 10-200ng / ml, 10-250ng / ml, 15-100ng / ml, 15-250ng / ml, 20-40ng / ml, 20-50ng / ml, 20-70ng / ml, 2 0-80ng / ml, 20-90ng / ml, 20-100ng / ml, 20-150ng / ml, 20-200ng / ml, 20-250ng / ml, 30-40ng / ml, 30-50ng / ml, 30-70ng / ml, 30-80ng / ml, 30-100ng / ml, 30-150ng / ml, 30-200ng / ml, 30-250ng / ml, 40-50ng / ml, 40-70ng / ml, 40-80ng / ml, 40-100ng / ml, 40-150ng / ml, 40-200ng / ml, 40-250ng / ml, 50-100ng / ml, 50-250ng / ml, 60-70ng / ml, 60-80ng / ml, 60-100ng / ml, 60-150ng / ml, 60-200ng / ml, 60-250ng / ml, 70-80ng / ml, 70-100ng / ml, 70-150ng / ml, 70- 200ng / ml, 70-250ng / ml, 80-100ng / ml, 80-150ng / ml, 80-250ng / ml, 90-100ng / ml, 90-120ng / ml, 90-150ng / ml, 90-200ng / ml, 90-250ng / ml or 1-250ng / Any concentration range within the ml range; in one specific embodiment, the concentration of IL4 in the culture medium is 1 ng / ml, 2 ng / ml, 2.5 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 9.5 ng / ml, 10 ng / ml, 10.5 ng / ml, 11 ng / ml, 12 ng / ml, 15 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 80 ng / ml, 100 ng / ml, 120 ng / ml, 130 ng / ml.Any concentration within the range of 140 ng / ml, 150 ng / ml, 250 ng / ml, or 1-250 ng / ml.

[0141] In some embodiments, the concentration of IL21 in the culture medium is 0-1250 ng / ml; in other embodiments, the concentration of IL21 in the culture medium is 0-1250 ng / ml, 1-1000 ng / ml, 1-750 ng / ml, 1-500 ng / ml, 1-250 ng / ml, 1-200 ng / ml, 1-150 ng / ml, 1-100 ng / ml, 1-50 ng / ml, 1-40 ng / ml, 1-30 ng / ml, 1-29 ng / ml, 1-28 ng / ml, 1-27 ng / ml, 1-26 ng / ml, 1-25 ng / ml, 1-20 ng / ml, 1-10 ng / ml, 1- 8ng / ml, 1-7ng / ml, 1-6ng / ml, 1-5ng / ml, 1-4ng / ml, 1-3ng / ml, 2.5-3ng / ml, 2.5-4ng / ml, 2.5-5ng / ml, 2.5-6ng / ml, 2.5-7ng / ml, 2.5-8ng / ml, 2.5-10n g / ml, 2.5-25ng / ml, 2.5-26ng / ml, 2.5-27ng / ml, 2.5-28ng / ml, 2.5-29ng / ml, 2.5-30ng / ml, 2.5-40ng / ml, 2.5-50ng / ml, 2.5-100ng / ml, 2.5-150ng / m l, 2.5-200ng / ml, 2.5-250ng / ml, 2.5-1250ng / ml, 5-6ng / ml, 5-7ng / ml, 5-8ng / ml, 5-10ng / ml, 5-15ng / ml, 5-20ng / ml, 5-25ng / ml, 5-26ng / ml, 5-27ng / ml, 5-28ng / ml, 5-29ng / ml, 5-30ng / ml, 5-40ng / ml, 5-50ng / ml, 5-80ng / ml, 5-100ng / ml, 5-150ng / ml, 5-200ng / ml, 5-250ng / ml, 5-500ng / ml, 5-750n g / ml, 5-1000ng / ml, 5-1250ng / ml, 10-20ng / ml, 10-25ng / ml, 10-30ng / ml, 10-40ng / ml, 10-50ng / ml, 10-80ng / ml, 10-100ng / ml, 10-150ng / ml, 10-200 ng / ml, 10-250ng / ml, 10-400ng / ml, 10-500ng / ml, 10-750ng / ml, 10-800ng / ml, 10-1000ng / ml, 10-1250ng / ml, 15-25ng / ml, 15-30ng / ml, 20-25ng / ml,20-26ng / ml, 20-27ng / ml, 20-28ng / ml, 20-29ng / ml, 20-30ng / ml, 20-40ng / ml, 20-50ng / ml, 20-100ng / ml, 20-150ng / ml, 20-200ng / ml, 20-250ng / m l, 20-400ng / ml, 20-500ng / ml, 20-750ng / ml, 20-800ng / ml, 20-1000ng / ml, 20-1250ng / ml, 25-30ng / ml, 25-40ng / ml, 25-50ng / ml, 25-80ng / ml, 25- 100ng / ml, 25-250ng / ml, 25-1250ng / ml, 30-40ng / ml, 30-50ng / ml, 30-100ng / ml, 30-150ng / ml, 30-200ng / ml, 30-250ng / ml, 30-400ng / ml, 30-500n g / ml,30-750ng / ml,30-800ng / ml,30-1000ng / ml,30-1250 / ml,40-80ng / ml,50-70ng / ml,50-100ng / ml,50-250ng / ml,50-1250ng / ml,70-90ng / ml,8 The concentration range is 0-100 ng / ml, 80-1250 ng / ml, 90-100 ng / ml, 90-1250 ng / ml, 100-900 ng / ml, 250-1250 ng / ml, or any concentration range within the range of 0-1250 ng / ml; in a specific embodiment, the concentration of IL21 in the culture medium is 0 ng / ml, 1 ng / ml, 2.5 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 21 ng / ml, 22 ng / ml, 23 ng / ml, or 24 ng / ml. / ml, 25ng / ml, 26ng / ml, 27ng / ml, 28ng / ml, 29ng / ml, 30ng / ml, 45ng / ml, 50ng / ml, 60ng / ml, 70ng / ml, 80ng / ml, 90ng / ml, 100ng / ml, 150ng / ml, 200ng / ml, 210ng / ml, 220ng / ml, 230ng / ml, 240ng / ml, 250ng / ml, 260ng / ml, 270ng / ml, 280ng / ml, 290ng / ml, 300ng / ml, 1000ng / ml, 1100ng / ml, 1200ng / ml,1250 ng / ml or any concentration within the range of 0-1250 ng / ml.

[0142] In some embodiments, the TGFβ content in the culture medium is 0.1-100 ng / ml; in other embodiments, the TGFβ content in the culture medium is 0.1-0.5 ng / ml, 0.1-1 ng / ml, 0.1-2 ng / ml, 0.1-5 ng / ml, 0.1-10 ng / ml, 0.1-100 ng / ml, 0.5-1 ng / ml, 0.5-2 ng / ml, 0.5-3 ng / ml, 0.5-4 ng / ml, 0.5-5 ng / ml, 0. 5-6ng / ml, 0.5-7ng / ml, 0.5-8ng / ml, 0.5-9ng / ml, 0.5-10ng / ml, 0.5-15ng / ml, 0.5-20ng / ml, 0.5-25ng / ml, 0.5 -30ng / ml, 0.5-40ng / ml, 0.5-50ng / ml, 0.5-60ng / ml, 0.5-70ng / ml, 0.5-80ng / ml, 0.5-90ng / ml, 0.5-100ng / ml, 1-2ng / ml, 1-3ng / ml, 1-4ng / ml, 1-5ng / ml, 1-6ng / ml, 1-7ng / ml, 1-8ng / ml, 1-9ng / ml, 1-10ng / ml, 1-15ng / ml, 1 -20ng / ml, 1-25ng / ml, 1-30ng / ml, 1-40ng / ml, 1-50ng / ml, 1-60ng / ml, 1-80ng / ml, 1-90ng / ml, 1-100ng / ml, 10-1 00ng / ml, 1.5-100ng / ml, 2-3ng / ml, 2-4ng / ml, 2-5ng / ml, 2-6ng / ml, 2-7ng / ml, 2-8ng / ml, 2-9ng / ml, 2-10ng / ml , 2-15ng / ml, 2-20ng / ml, 2-40ng / ml, 2-50ng / ml, 2-60ng / ml, 2-70ng / ml, 2-80ng / ml, 2-90ng / ml, 2-100ng / ml, 2.Any content range within the range of 5 - 100 ng / ml, 3 - 100 ng / ml, 3 - 80 ng / ml, 3 - 60 ng / ml, 3 - 40 ng / ml, 3 - 20 ng / ml, 3 - 15 ng / ml, 3 - 10 ng / ml, 3 - 9 ng / ml, 3 - 8 ng / ml, 3 - 7 ng / ml, 3 - 6 ng / ml, 3 - 5 ng / ml, 3 - 4 ng / ml, 4 - 100 ng / ml, 4 - 9 ng / ml, 4 - 8 ng / ml, 4 - 7 ng / ml, 4 - 6 ng / ml, 4 - 5 ng / ml, 5 - 100 ng / ml, 5 - 80 ng / ml, 5 - 60 ng / ml, 5 - 50 ng / ml, 5 - 40 ng / ml, 5 - 20 ng / ml, 5 - 15 ng / ml, 5 - 10 ng / ml, 5 - 9 ng / ml, 5 - 8 ng / ml, 5 - 7 ng / ml, 5 - 6 ng / ml, 6 - 100 ng / ml, 6 - 80 ng / ml, 6 - 60 ng / ml, 6 - 40 ng / ml, 6 - 20 ng / ml, 6 - 15 ng / ml, 6 - 10 ng / ml, 6 - 9 ng / ml, 6 - 8 ng / ml, 6 - 7 ng / ml, 7 - 100 ng / ml, 7 - 80 ng / ml, 7 - 60 ng / ml, 7 - 40 ng / ml, 7 - 20 ng / ml, 7 - 15 ng / ml, 7 - 10 ng / ml, 7 - 9 ng / ml, 7 - 8 ng / ml, 8 - 100 ng / ml, 8 - 80 ng / ml, 8 - 60 ng / ml, 8 - 40 ng / ml, 8 - 20 ng / ml, 8 - 15 ng / ml, 8 - 10 ng / ml, 8 - 9 ng / ml, 9 - 100 ng / ml, 9 - 80 ng / ml, 9 - 60 ng / ml, 9 - 40 ng / ml, 9 - 20 ng / ml, 9 - 15 ng / ml, 9 - 10 ng / ml, 10 - 100 ng / ml, 20 - 100 ng / ml, 25 - 100 ng / ml, 30 - 100 ng / ml, 40 - 100 ng / ml, 50 - 100 ng / ml, 60 - 100 ng / ml, 70 - 100 ng / ml, 90 - 100 ng / ml or 0.1 - 100 ng / ml; In a specific embodiment, the content of TGFβ in this culture medium is 0.1 ng / ml, 0.5 ng / ml, 1 ng / ml, 1.5 ng / ml, 2 ng / ml, 3 ng / ml, 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9 ng / ml, 10 ng / ml, 11 ng / ml, 12 ng / ml, 13 ng / ml, 15 ng / ml, 25 ng / ml, 30 ng / ml, 45 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml or 0.Any concentration within the range of 1-100 ng / ml. In one specific embodiment, the cytokines are 5 ng / ml IL7 and 1 ng / ml TGFβ1. In another specific embodiment, the cytokines are 25 ng / ml IL21 and 1 ng / ml TGFβ1.

[0143] In some embodiments, the culture medium further includes any one or more of the following cytokines:

[0144] IL1, IL2, IL3, IL4, IL5, IL6, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL21, IL22, IL23, IL24, IL26, IL27, IL28, IL29, IL32, IL33, IL35, IL37, anti-IFNγ (interferon-gamma antibody), TNFα.

[0145] In some embodiments, the culture medium comprises any one of the following groups:

[0146] (I) IL7, TGFβ, and any one or more of the group consisting of IL2, IL4, IL15, IL21 and anti-IFNγ; (II) IL7, TGFβ, and any one or more of the group consisting of IL4, IL15 and IL21; (III) IL7, TGFβ, and any one or more of the group consisting of IL4 and IL21.

[0147] In some embodiments, the culture medium further includes any one of the following groups: 1) a combination of IL7, TGFβ, and IL4; 2) a combination of IL7, TGFβ, and IL21; 3) a combination of IL7, TGFβ, IL2, and IL4; 4) a combination of IL7, TGFβ, IL2, and IL21; 5) a combination of IL7, TGFβ, IL4, and IL21; 6) a combination of IL7, TGFβ, IL2, IL4, and IL21; 7) a combination of IL7, TGFβ, and antiIFN-γ; 8) a combination of IL7, TGFβ, IL4, and antiIFN-γ; 9) a combination of IL7, TGFβ, IL21, and antiIFN-γ. Combinations; 10) Combination of IL7, TGFβ, IL4, IL21 and antiIFN-γ; 11) Combination of IL7, TGFβ and IL15; 12) Combination of IL7, TGFβ, IL4 and IL15; 13) Combination of IL7, TGFβ, IL21 and IL15; 14) Combination of IL7, TGFβ, IL4, IL21 and IL15; 15) Combination of IL2, IL4 and TGFβ; 16) Combination of IL4, TGFβ, IL2 and antiIFN-γ; 17) Combination of IL4, TGFβ and antiIFN-γ; and 18) Combination of IL4, TGFβ, IL21 and antiIFN-γ.

[0148] In some embodiments, the culture medium comprises any one of the following groups: a combination of IL7, TGFβ1 and IL21; a combination of IL7, TGFβ1 and IL4; a combination of IL7, TGFβ1, IL4 and IL21; or a combination of IL7, TGFβ1, IL21, IL4 and IL15.

[0149] In some implementations, the concentration of IL2 in the culture medium is 0-1000 IU / ml;In some embodiments, the IL2 content in the composition is 0-1000 IU / ml, 10-1000 IU / ml, 50-1000 IU / ml, 100-1000 IU / ml, 200-1000 IU / ml, 300-1000 IU / ml, 400-1000 IU / ml, 410-1000 IU / ml, 420-1000 IU / ml, 430-1000 IU / ml, 440-1000 IU / ml, 450-1000 IU / ml, 460-1000 IU / ml, 470-1000 IU / ml, 480-1000 IU / ml, 490-1000 IU / ml, 5 00-1000IU / ml, 800-1000IU / ml, 700-900IU / ml, 10-800IU / ml, 50-800IU / ml, 100-800IU / ml, 200-800IU / ml, 300-800IU / ml, 400-800IU / ml, 410-80 0IU / ml, 420-800IU / ml, 430-800IU / ml, 440-800IU / ml, 450-800IU / ml, 460-800IU / ml, 470-800IU / ml, 480-800IU / ml, 490-800IU / ml, 500-800IU / m l, 10-700IU / ml, 50-700IU / ml, 100-700IU / ml, 200-700IU / ml, 300-700IU / ml, 400-700IU / ml, 410-700IU / ml, 420-700IU / ml, 430-700IU / ml, 440- 700IU / ml, 450-700IU / ml, 460-700IU / ml, 470-700IU / ml, 480-700IU / ml, 490-700IU / ml, 500-700IU / ml, 10-600IU / ml, 50-600IU / ml, 100-600IU / m l, any content range within the range of 100-600 IU / ml, 200-600 IU / ml, 300-600 IU / ml, 400-600 IU / ml, 410-600 IU / ml, 420-600 IU / ml, 430-600 IU / ml, 440-600 IU / ml, 450-600 IU / ml, 460-600 IU / ml, 470-600 IU / ml, 480-600 IU / ml, 490-600 IU / ml, 500-600 IU / ml, 300-500 IU / ml, 100-300 IU / ml, 10-100 IU / ml or 0-1000 IU / ml;In one specific embodiment, the IL2 concentration in the culture medium is any concentration within the range of 0 IU / ml, 50 IU / ml, 100 IU / ml, 200 IU / ml, 350 IU / ml, 450 IU / ml, 460 IU / ml, 470 IU / ml, 480 IU / ml, 490 IU / ml, 500 IU / ml, 510 IU / ml, 520 IU / ml, 530 IU / ml, 540 IU / ml, 550 IU / ml, 560 IU / ml, 570 IU / ml, 580 IU / ml, 590 IU / ml, 600 IU / ml, 650 IU / ml, 750 IU / ml, 800 IU / ml, 900 IU / ml, 1000 IU / ml, or 0-1000 IU / ml.

[0150] In some embodiments, the concentration of IL15 in the culture medium is 0-100 ng / ml; in other embodiments, the concentration of IL15 in the culture medium is 10-100 ng / ml, 20-100 ng / ml, 25-100 ng / ml, 30-100 ng / ml, 40-100 ng / ml, 50-100 ng / ml, 60-100 ng / ml, 70-100 ng / ml, 80-100 ng / ml, 90-100 ng / ml, or 10-90 ng / ml. g / ml, 20-90ng / ml, 30-90ng / ml, 40-90ng / ml, 50-90ng / ml, 10-80ng / ml, 20-80ng / ml, 30-80ng / ml, 40-80ng / m l, 50-80ng / ml, 60-80ng / ml, 10-70ng / ml, 20-70ng / ml, 30-70ng / ml, 40-70ng / ml, 50-70ng / ml, 10-60ng / ml, 20 The concentration range is any range within the range of -60 ng / ml, 30-60 ng / ml, 40-60 ng / ml, 25-40 ng / ml, 15-25 ng / ml, 5-15 ng / ml, or 0-100 ng / ml; in one specific embodiment, the concentration of IL15 in the culture medium is 0 ng / ml, 3 ng / ml, 5 ng / ml, 10 ng / ml, 25 ng / ml, 30 ng / ml, 41 ng / ml, 42 ng / ml, 43 ng / ml, 44 ng / ml, or any range within the range of -60 ng / ml, 30-60 ng / ml, 40-60 ng / ml, 25 ng / ml, 30 ng / ml, 41 ng / ml, 42 ng / ml, 43 ng / ml, 44 ng / ml, or any range within the range of 0 ng / ml, 3 ng / ml, 5 ng / ml, 10 ng / ml, 25 ng / ml, 30 ng / ml, 41 ng / ml, 42 ng / ml, 43 ng / ml, 44 ng / ml, or any range within the range of 0 ng / ml, 30-60 ng / ml, 40-60 ng / ml, 25-40 ng / ml, 15-25 ng / ml, 5-15 ng / ml, or 0-100 ng / ml. The concentration may be 45 ng / ml, 46 ng / ml, 47 ng / ml, 48 ng / ml, 49 ng / ml, 50 ng / ml, 51 ng / ml, 52 ng / ml, 53 ng / ml, 54 ng / ml, 55 ng / ml, 56 ng / ml, 57 ng / ml, 58 ng / ml, 59 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, or any concentration within the range of 0-100 ng / ml.

[0151] In some embodiments, the concentration of antiIFN-γ in the culture medium is 0-200 ng / ml; in other embodiments, the concentration of antiIFN-γ in the culture medium is 0-200 ng / ml, 50-200 ng / ml, 70-200 ng / ml, 80-200 ng / ml, 90-200 ng / ml, 100-200 ng / ml, 150-200 ng / ml, 5-150 ng / ml, 50-150 ng / ml, 70-150 ng / ml, 80-150 ng / ml, 90- 150ng / ml, 100-150ng / ml, 50-140ng / ml, 70-140ng / ml, 80-140ng / ml, 90-140ng / ml, 100-140ng / ml, 50-130ng / ml, 70-130 ng / ml, 80-130ng / ml, 90-130ng / ml, 100-130ng / ml, 50-120ng / ml, 70-120ng / ml, 80-120ng / ml, 90-120ng / ml, 50-110ng / ml 1, any content range within the range of 70-110 ng / ml, 80-110 ng / ml, 90-110 ng / ml, 5-15 ng / ml, 15-25 ng / ml, 25-40 ng / ml, 40-60 ng / ml, 60-80 ng / ml, 80-100 ng / ml, 100-125 ng / ml, 125-140 ng / ml, 140-160 ng / ml, 160-180 ng / ml, 180-200 ng / ml, or 0-200 ng / ml; in one specific embodiment, a The concentration of ntiIFN-γ in this culture medium is any concentration within the range of 0 ng / ml, 3 ng / ml, 5 ng / ml, 10 ng / ml, 25 ng / ml, 30 ng / ml, 45 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 110 ng / ml, 130 ng / ml, 145 ng / ml, 150 ng / ml, 170 ng / ml, 190 ng / ml, 200 ng / ml, or 0-200 ng / ml.

[0152] In one specific embodiment, the culture medium includes any one of the following combinations:

[0153] A combination of 5 ng / ml IL7, 1 ng / ml TGFβ1 and 25 ng / ml IL21;

[0154] A combination of 5 ng / ml IL7, 1 ng / ml TGFβ1 and 10 ng / ml IL4;

[0155] A combination of 5 ng / ml IL7, 1 ng / ml TGFβ1, 25 ng / ml IL21 and 10 ng / ml IL4;

[0156] A combination of 5 ng / ml IL7, 0.5 ng / ml TGFβ1, 25 ng / ml IL21, and 5 ng / ml IL4;

[0157] A combination of 5 ng / ml IL7, 5 ng / ml TGFβ1, 25 ng / ml IL21 and 50 ng / ml IL4;

[0158] A combination of 5 ng / ml IL7, 10 ng / ml TGFβ1, 25 ng / ml IL21 and 100 ng / ml IL4;

[0159] A combination of 5 ng / ml IL7, 10 ng / ml TGFβ1, 25 ng / ml IL21 and 5 ng / ml IL4;

[0160] A combination of 5 ng / ml IL7, 0.5 ng / ml TGFβ1, 25 ng / ml IL21, and 100 ng / ml IL4;

[0161] A combination of 1 ng / ml IL7, 0.5 ng / ml TGFβ1, 5 ng / ml IL21, and 2 ng / ml IL4;

[0162] A combination of 5 ng / ml IL7, 1 ng / ml TGFβ1, 5 ng / ml IL21 and 2 ng / ml IL4;

[0163] A combination of 5 ng / ml IL7, 1 ng / ml TGFβ1, 25 ng / ml IL21 and 10 ng / ml IL4;

[0164] A combination of 50 ng / ml IL7, 10 ng / ml TGFβ1, 25 ng / ml IL21 and 10 ng / ml IL4;

[0165] A combination of 50 ng / ml IL7, 10 ng / ml TGFβ1, 250 ng / ml IL21 and 100 ng / ml IL4;

[0166] A combination of 250 ng / ml IL7, 100 ng / ml TGFβ1, 1250 ng / ml IL21 and 250 ng / ml IL4;

[0167] A combination of 5 ng / ml IL7, 1 ng / ml TGFβ1, 25 ng / ml IL21, 10 ng / ml IL4 and 50 ng / ml IL15;

[0168] A combination of 1 ng / ml TGFβ1 and 10 ng / ml IL4;

[0169] A combination of 500 IU / ml IL2, 10 ng / ml IL4, 1 ng / ml TGFβ1 and 100 ng / ml anti IFN-γ;

[0170] A combination of 100 ng / ml IL21, 100 ng / ml IL4, 10 ng / ml TGFβ1 and 100 ng / ml anti IFN-γ;

[0171] A combination of 0 ng / ml IL4, 1 ng / ml TGFβ1, and 100 ng / ml anti IFN-γ;

[0172] A combination of 25 ng / ml IL21 and 1 ng / ml TGFβ1;

[0173] A combination of 25 ng / ml IL21, 1 ng / ml TGFβ1, and 10 ng / ml IL4;

[0174] Alternatively, a combination of 5 ng / ml IL7, 1 ng / ml TGFβ1, 25 ng / ml IL21, 10 ng / ml IL4, and 500 IU / ml IL2.

[0175] In some embodiments, the culture medium further comprises a basal culture medium for cell culture. The basal culture medium is a general basal culture medium for cell culture.

[0176] "Basal culture medium" refers to a self-prepared or commercially available universal basal culture medium for cell or immune cell culture that does not contain the cytokines or cytokine combinations described in this application. It is a universal basal culture medium for immune cell culture. Those skilled in the art should understand that this application does not impose any special restrictions on the selection of the universal basal culture medium, as long as it can achieve the purpose of immune cell culture. This universal basal culture medium can refer to any liquid, solid, or semi-solid culture medium containing essential nutrients (including inorganic and organic substances) that can provide cells with the necessary nutrients for in vitro survival.

[0177] In some embodiments, the universal basal culture medium and cytokines are commercially available. In this application, "cytokine" refers to the cytokine itself and / or its subtypes and / or its functional variants. A functional variant refers to a cytokine variant in which the wild-type cytokine sequence has been modified by gene mutation, truncation, or other manipulations to retain or obtain a function superior to that of the wild-type cytokine.

[0178] In some embodiments, the universal basal medium provides culture conditions that allow cells to survive under serum-free conditions. In some embodiments, the universal basal medium is a serum-free medium selected from one or more of OpTmizer™ CTS™ (LifeTech), Immunocult™ XF (Stemcell Technologies), CellGro™ (CellGenix), TexMacs™ (Miltenyi), Stemline™ (Sigma), Xvivo15™ (Lonza), PrimeXV (Irvine Scientific), StemXVivo, and (RandD Group). In some embodiments, the serum-free medium may be supplemented with a serum replacement agent, such as ICSR (immune cell serum replacement) from LifeTech. In some embodiments, the proportion of the serum replacement agent (e.g., ICSR) added may be about 0-5%; for example, about 1%, 2%, 3%, 4%, or 5%. In some embodiments, the serum-free medium may be supplemented with serum, which may be, for example, human serum, such as human AB serum. In some embodiments, the serum is human serum that is allowed to coagulate naturally after collection, for example, non-clotting (OTC) serum. In some embodiments, the serum is plasma-derived human serum. Plasma-derived serum can be produced by defibrinating a mixture of human plasma collected in the presence of an anticoagulant (e.g., sodium citrate). In some embodiments, the medium is a medium suitable for T cell culture; in some embodiments, the medium suitable for T cell culture is selected from any one or more of Minimal Essential Media, α-MEM, RPMI 1640, AIM-V, DMEM, F-12, X-vivo15 (Lonza), X-Vivo 20, OpTmizer, and IMDM. In some embodiments, the basal medium may also be ImmunoCult selected from STEMCELL. TM-XF, CellGenix SCGM medium (Germany), Lonza's X-VIVO series serum-free immune cell culture media (such as X-VIV010 serum-free immune cell culture medium, X-VIV015 serum-free immune cell culture medium, X-VIVO 20 serum-free cell culture medium), and Ekosei... Serum-free T-cell culture medium, Betaine's HIPP-T009 serum-free lymphocyte culture medium, Corning's KBM581 serum-free lymphocyte culture medium, and Dakwei Biotechnology Co., Ltd. Any one or more immune cell culture media including L500 lymphocyte serum-free culture medium.

[0179] In some implementations, the universal basal culture medium is prepared. Those skilled in the art will understand that the components of the universal basal culture medium may include, but are not limited to, the following: amino acids, proteins or peptides, vitamins, carbohydrates, inorganic salts, buffers; optionally, organic acids, antioxidants, trace elements, etc.

[0180] In some embodiments, the amino acid may be any one or more of glycine, arginine, asparagine, aspartic acid, cysteine ​​hydrochloride, glutamic acid, histidine, hydroxyproline, isoleucine, leucine, lysine hydrochloride, methionine, phenylalanine, proline, serine, threonine, tryptophan, valine, glutamine, and alanine dipeptide, as well as derivatives of any of the said amino acids. In some embodiments, the content of amino acids or their derivatives in the universal basal culture medium is any range within the range of 1000-1500 mg / L, 1100-1500 mg / L, 1200-1500 mg / L, 1300-1500 mg / L, 1400-1500 mg / L, 1100-1200 mg / L, 1100-1300 mg / L, 1100-1400 mg / L, 1100-1500 mg / L, 1200-1400 mg / L, 1300-1400 mg / L, 1000-1100 mg / L, 1050-1200 mg / L, 1150-1300 mg / L, 1250-1400 mg / L, 1350-1500 mg / L, or 1000-1500 mg / L.

[0181] In some embodiments, the vitamin may be any one or more of vitamin H, vitamin A, vitamin D, vitamin K, vitamin B7, vitamin B9, vitamin B12, vitamin B1, vitamin B2, vitamin B6, vitamin C, vitamin E, choline chloride, calcium pantothenate, folic acid, nicotinamide, para-aminobenzoic acid, pyridoxine hydrochloride, thiamine hydrochloride, and inositol, as well as derivatives of any of the said vitamins. In some embodiments, the vitamin or its derivative in the universal basal culture medium is in any range of 50-100 mg / L, 60-100 mg / L, 70-100 mg / L, 80-100 mg / L, 90-100 mg / L, 50-90 mg / L, 60-90 mg / L, 80-90 mg / L, 70-90 mg / L, 60-80 mg / L, 60-70 mg / L, or 50-100 mg / L.

[0182] In some embodiments, the carbohydrate is selected from one or more of sucrose, glucose, lactose, maltose, and fructose. In some embodiments, the protein is albumin or an albumin substitute.

[0183] In some embodiments, the protein is selected from albumin, transferrin, fibronectin, aprotinin, insulin, growth hormone, and fetoglobulin, as well as any one or more functional variants and / or substitutes of any protein.

[0184] In some embodiments, the albumin is a natural albumin; for example, human albumin, and more specifically, natural human serum albumin; or, for example, a non-human albumin. In some embodiments, the albumin is a recombinant albumin; for example, recombinant human albumin. In some embodiments, albumin substitutes may be, but are not limited to, bovine pituitary extracts, plant hydrolysates (e.g., rice hydrolysates), fetal bovine albumin (fetoglobulin), ovalbumin, human serum albumin (HSA), or albumin of non-human animal origin, chicken extracts, bovine embryo extracts, and any one or more functional variants or modified proteins of any of the proteins listed above. In some embodiments, the albumin content in the universal basal culture medium is any range within the range of 0-20 mg / L, 0.5-5 mg / L, 0.5-10 mg / L, 0.5-15 mg / L, 0.5-20 mg / L, 2-5 mg / L, 2-10 mg / L, 2-15 mg / L, 2-20 mg / L, 5-15 mg / L, 5-10 mg / L, 5-20 mg / L, 8-10 mg / L, 8-15 mg / L, 12-18 mg / L, 15-20 mg / L, or 0-20 mg / L.

[0185] In some embodiments, the transferrin is natural transferrin; for example, human transferrin; for example, non-human transferrin. In some embodiments, the transferrin is recombinant transferrin; for example, recombinant human transferrin. In some embodiments, exemplary examples of transferrin substitutes include, but are not limited to, any iron chelating compound. In some embodiments, the content of transferrin and / or its functional variants and / or its substitutes in the universal basal culture medium is 10-800 mg / L, 10-50 mg / L, 10-100 mg / L, 10-200 mg / L, 10-300 mg / L, 10-400 mg / L, 10-500 mg / L, 10-600 mg / L, 10-700 mg / L, 10-800 mg / L, 50-100 mg / L, 50-300 mg / L, 50-500 mg / L, 50-800 mg / L. / L, any content range within the range of 100-200mg / L, 100-300mg / L, 100-400mg / L, 100-500mg / L, 100-800mg / L, 200-400mg / L, 200-600mg / L, 200-800mg / L, 300-700mg / L, 300-500mg / L, 300-400mg / L, 400-700mg / L, 400-600mg / L, 500-700mg / L or 10-800mg / L.

[0186] In some embodiments, exemplary selections of the inorganic salt include, but are not limited to, the following: calcium nitrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, disodium hydrogen phosphate, sodium selenite, copper sulfate, ferrous sulfate, nickel chloride, stannous chloride, zinc sulfate, and hydrates of any of the above inorganic salts. In some embodiments, the components of the universal basal culture medium further include growth factors, with optional examples including, but not limited to, any one or more of the following: epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), and transforming growth factor (TGF).

[0187] This application provides a method for obtaining mixed T cells, including culturing peripheral blood mononuclear cells (PBMCs), T cells, CD4 single-positive T cells, CD8 single-positive T cells, CD4 / CD8 double-negative T cells, or any combination thereof using the culture medium; the mixed T cells include CD4 / CD8 positive mixed cells and / or CD4 / CD8 double-negative mixed cells.

[0188] The culture medium for culturing T cells is as described above and will not be repeated here.

[0189] In this application, the original cells used to prepare the hybrid T cells are immune cells.

[0190] In some embodiments, the immune cells are peripheral blood mononuclear cells (PBMCs) and / or T cells. The T cells are natural T cells and / or genetically modified T cells; preferably, the genetically modified T cells contain an introduced exogenous gene.

[0191] The terms "T lymphocyte" and "T cell" are used interchangeably in this application. T lymphocyte is a type of lymphocyte. The T cells used for cell culture in this application can be individual T cells, a mixture of cells containing T cells, or a cell population containing T lymphocytes. Further examples include T lymphocytes, leukocytes or PBMCs containing T cells, a population of T cells after CD3-positive sorting of leukocytes or PBMCs, a mixture of single-positive cells after CD4-positive and CD8-positive sorting, or a population of single-positive T cells (CD4-positive or CD8-positive) after CD3-positive sorting of leukocytes or PBMCs; or the immune cells can be CD4 / CD8 double-negative mixed cells. The aforementioned cell populations can be fresh or cryopreserved cells.

[0192] The term "genetically modified T cells" in this application means: T cells that express exogenous or endogenous genes and are suitable for adoptive cell therapy. In some embodiments, the T cells are natural T cells and / or modified T cells; in some embodiments, the modification is selected from any one or more of chemical modification, physical modification, and biological modification. In some embodiments, the genetically modified T cells comprise any one or more of the following: a gene fragment encoding a chimeric antigen receptor (CAR), a gene fragment encoding a T cell receptor (TCR) that specifically recognizes tumor antigens, and a gene fragment encoding a fusion protein of the T cell receptor.

[0193] For chimeric antigen receptors (CARs), a CAR typically includes at least one extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain. In some embodiments, the CAR also includes a co-stimulatory signaling domain.

[0194] The terms "extracellular antigen-binding domain" and "extracellular antigen-binding region" are used interchangeably in this application, referring to any peptide or polypeptide that can specifically bind to a target antigen, containing a naturally occurring antigen associated with a medical symptom (e.g., a disease), or an antigenic portion conjugated to a therapeutic agent targeting a disease-related antigen; an "extracellular antigen-binding domain" specifically recognizes an antigen and is generally located outside the cell. In some embodiments, the extracellular antigen-binding domain includes, but is not limited to, any one or more of the following: a single-chain variable fragment (scFv) derived from an antibody, a fragment antigen-binding region (Fab) selected from a library, a single-domain fragment or a natural ligand conjugated to its homologous receptor, or an artificially designed target-specific recognition domain that recognizes a specific target; in some embodiments, the artificially designed target-specific recognition domain that recognizes a specific target may be, for example, a combination of fibronectin type III (FN3) domains and / or a target-specific ankyrin repeat protein (DARPins) that recognizes a specific target.

[0195] In some implementations, the extracellular antigen-binding domain of the "chimeric antigen receptor" or "CAR" structure can recognize target molecules expressed on the surface of solid tumor or hematologic malignancy cells / tissues. These target molecules include, but are not limited to, any one or more of the following: CD19, CD20, CD22, CD33, CLL-1 (CLEC12A), CD7, CD5, CD70, CD123, CEACAM5, CEACAM6, CEACAM7, Mesothelin, MUC1, and CLDN18.2 CDH17, Trop2, BCMA, NKG2D, PDL1, EGFR, EGFRVIII, PSCA, PSMA, MUC16, CD133, GD2, IL13R2, B7H3, Her2, CD30, SLAMF7, CD38, GPC3, WT1, AFP, FOLR1, c-Met, LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma-associated antigen 1), and TAG-72, etc.

[0196] In some embodiments, the target molecule is selected from any one or more of the following: CD19, CD70, CEA, and BCMA.

[0197] In some embodiments, when the target molecule is CEA, the amino acid sequence of the chimeric antigen receptor CAR includes the sequence shown in SEQ ID NO.1, or has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO.1; in a specific embodiment, the amino acid sequence of the chimeric antigen receptor CAR is as shown in SEQ ID NO.1, or has 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO.1.

[0198] In some embodiments, when the target molecule is CD19, the amino acid sequence of the chimeric antigen receptor CAR includes the sequence shown in SEQ ID NO.2, or has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO.2; in some embodiments, the amino acid sequence of the chimeric antigen receptor CAR is as shown in SEQ ID NO.2, or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO.2.

[0199] In some embodiments, when the target molecule is CD70, the amino acid sequence of the chimeric antigen receptor CAR includes the sequence shown in SEQ ID NO.3, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO.3; in some embodiments, the amino acid sequence of the chimeric antigen receptor CAR is as shown in SEQ ID NO.3, or has 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the sequence shown in SEQ ID NO.3.

[0200] In this application, the terms "signal transduction domain" and "cytoplasmic signal transduction domain" are used interchangeably. It typically contains the tyrosine-based activation motif (ITAM) sequence of an immune receptor, with the basic composition being YXXL / V. Here, Y represents tyrosine, L / V refers to leucine or valine, and X can be any amino acid. When the receptor binds to its corresponding ligand, the tyrosine residue in the ITMA linked to it can be phosphorylated by a protein tyrosine kinase (PTK) connected to the cell membrane, thereby recruiting other free intracellular protein kinases or adaptor proteins to transduce activation signals into the cell. In some embodiments, the "signal transduction domain" is selected as the intracellular signal transduction domain of TCRζ (CD3ζ) or FcεRIγ.

[0201] In some embodiments, the costimulatory signal transduction domain is selected from any one or more of the group consisting of CD28, 4-1BB (CD137), OX40 (CD134), CD27, ICOS, and any of the above functional variants.

[0202] As used herein, a "transmembrane domain," also known as a "transmembrane region," refers to a thermodynamically stable protein structural region anchored within the cell membrane. Transmembrane domains can be obtained from natural proteins, such as the transmembrane domain derived from the T-cell receptor (TCR); alternatively, transmembrane domains can be synthetic, non-naturally occurring protein segments or portions thereof, such as thermodynamically stable hydrophobic protein segments within the cell membrane. In some embodiments, the transmembrane domain is selected from the transmembrane domains of CD4, CD8α, CD28, and CD3ζ. The transmembrane domain belongs to any one or more of the following membrane proteomes: CD8α, CD8β, 4-1BB / CD137, CD27, CD28, CD34, CD4, FcεRIγ, CD16A, OX40 / CD134, CD3ζ, CD3ε, CD3γ, CD3δ, TCRα, TCRβ, TCRζ, CD32, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L / CD154, VEGFR2, FAS, FGFR2B, CD2, IL15, IL15R, IL21, DNAM-1, 2B4, NKG2D, NKp44, and NKp46.

[0203] A chimeric antigen receptor (CAR) can have various structures. In some embodiments, the structure of the chimeric antigen receptor is selected from, for example, secretible or membrane-expressed cytokines, antibody gene sequences, structures that can be regulated to activate or inactivate, inducible CAR structures, and any one or more of the following "logic gate" regulatory systems that bind to SynNotch receptors. In some embodiments, the structures that regulate activation or inactivation include, but are not limited to, any one or more of the following: a suicide switch, thymidine kinase (HSV-TK) and suicide epitopes in herpes simplex virus, truncated EGFR (EGFRt), and Fas-FasL apoptosis structures; in some embodiments, the suicide switch is inducible caspase-9 (iCasp9). In some embodiments, the inducible CAR structure includes, but is not limited to, any one or more of the following: Peptide neo-epitope (PNE), fluorescein (FITC), 10 amino acids (5B9 tag), FITC-HM-3 bifunctional molecule (FHBM) and scFv, Leucine ZipFv linked to antibody, Streptavidin 2 (mSA2) biotin-binding domain, VIPER CAR inducible structure, and biotin-biding immune receptor (BBIR) system.

[0204] In some implementations, the immune cells are private mononuclear cells (PBMCs).

[0205] In some implementations, the immune cells are natural T cells.

[0206] In some embodiments, the T cells are a population of T cells sorted for CD3 positivity from leukocytes or PBMCs, and more specifically, a population of single-positive T cells that are CD4-positive or CD8-positive. In some embodiments, the T cells are mixed cells resulting from a mixture of single-positive cells sorted for both CD4 and CD8 positivity. In some embodiments, the T cells are CD4 / CD8 double-negative mixed cells. In some embodiments, the immune cells are any combination of those listed above.

[0207] In some embodiments, the immune cells are modified T cells. In some embodiments, the gene-modified T cells are recombinant T cells and / or functionally treated T cells. Further, the recombinant T cells include T cells transduced with a target gene, such as CAR-T cells, TCR-T cells, STAR-T cells, etc. In some embodiments, the CAR-T cells are CEA CAR-T cells, CD19 CAR-T cells, CD70 CAR-T cells, BCMACAR-T cells, or any combination of the above-listed cells. In some embodiments, gene-modified T cells can be obtained by transducing activated T cells with a vector containing the CAR structural gene or target gene, such as plasmids, lentiviruses, or VLP vectors with modified viral envelopes (such as the vector described in patent 202310887156.5).

[0208] In some implementations, the method further includes the step of activating T cells before culture.

[0209] It should be noted that in this application, there are no further restrictions on the order of immune cell activation and immune cell culture. In some embodiments, immune cell activation and immune cell culture are performed simultaneously; in some embodiments, immune cell culture is started after immune cell activation. As explained above, the immune cells can be natural or modified. In some embodiments, a step of modifying the immune cells is also included. Those skilled in the art will understand that the order of immune cell activation and immune cell modification is not unique. For example, in some embodiments, immune cell modification is performed after immune cell activation; in some embodiments, immune cell activation and immune cell modification are performed simultaneously. When immune cell activation is performed independently, the activation time is 12-48 hours; in some embodiments, the activation time is any time range within 12-24 hours, 20-35 hours, 30-40 hours, 35-48 hours, and 12-48 hours; in one specific embodiment, the activation time is any time range within 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 48 ​​hours, and 12-48 hours. When immune cell activation and immune cell modification are performed simultaneously, the total activation and modification time is 5 min-48 h; in some embodiments, the total activation and modification time is any time range within 5-60 min, 1-6 h, 6-12 h, 12-24 h, 20-35 h, 30-40 h, 35-48 h, and 12-48 h; in a specific embodiment, the total activation and modification time is any time within 5 min, 30 min, 1 h, 5 h, 10 h, 12 h, 18 h, 24 h, 30 h, 36 h, 48 h, and 5 min-48 h.

[0210] T cell activation refers to the activation of downstream signals of the TCR molecule on the T cell surface, thereby generating an intracellular signaling cascade that breaks the T cell's quiescent state. Activation of downstream signals of the TCR molecule on the T cell surface can be achieved by any antibody, ligand, or small molecule compound that can bind to one or more subunits of CD3 or TCR, provided that its binding to the CD3 / TCR complex phosphorylates tyrosine residues in the ITAM (immunoreceptor tyrosine activation motif) of CD3, thereby activating downstream pathways. This can be achieved through the combination of one or more of the following: antibodies, small molecules, ligands (e.g., naturally occurring ligands, recombinant ligands, or chimeric ligands), or other possible reagents. In some embodiments, the activator is selected from conjugated magnetic beads and / or reagents (such as Dynabeads) containing CD3 and CD28 antibodies. TM Any one or more of the following groups: CD3 / CD28 magnetic beads, combinations of CD3 and CD28 antibodies, viruses loaded with CD3 and CD28 antibodies, and / or nanoparticles and / or vesicles.

[0211] In some implementations, the culture time is 5-25 days; in some implementations, the culture time is any time range of 5-10 days, 7-21 days, 15-20 days, 18-25 days, or 5-25 days; in one embodiment, the culture time is any time range of 5 days, 7 days, 12 days, 15 days, 18 days, 21 days, 24 days, or 5-25 days.

[0212] In some embodiments of this application, methods for obtaining hybrid T cells include:

[0213] Cell activation and expansion are carried out simultaneously; the cell population containing T lymphocytes (i.e., immune cells) is activated using an activator and cultured in a medium supplemented with the above-mentioned combination of cytokines for 48 hours to 21 days; for example, 48 hours, 72 hours, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, and 21 days; the culture medium is removed, the cells are collected, and the cells are resuspended in cryopreservation solution for cryopreservation; during the culture process, the culture medium can be changed or replenished according to the cell state and expansion.

[0214] In some embodiments of this application, methods for obtaining hybrid T cells include:

[0215] Cell activation: Activate the cell population (i.e. immune cells) containing T lymphocytes using an activator for 12-48 hours.

[0216] The activated immune cells are cultured in a culture medium containing the aforementioned cytokine combination for 48 hours to 21 days; the specific times may be 48 hours, 72 hours, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days. After 48 days, the culture medium is removed, the cells are collected, and the cells are resuspended in cryopreservation solution for cryopreservation. During the culture process, the culture medium may be changed or replenished according to the cell status and expansion.

[0217] In some embodiments of this application, methods for obtaining hybrid T cells include:

[0218] Cell activation and gene modification: Cell populations containing T lymphocytes (i.e. immune cells) are activated using an activator, and at the same time, gene transduction of immune cells is performed using vectors containing CAR structural genes or target genes, such as plasmids, lentiviruses, or VLP vectors with modified viral envelopes (such as the vectors described in patent 202310887156.5).

[0219] Cell culture: Continue culturing in a medium supplemented with the cytokine combination for 48 hours to 21 days; for example, 48 hours, 72 hours, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days; remove the medium, collect the cells, resuspend them in cryopreservation solution, and freeze for preservation.

[0220] During the culture process, the culture medium can be changed or replenished according to the state and expansion of the cells.

[0221] In some embodiments of this application, methods for obtaining hybrid T cells include:

[0222] Cell activation: Cell populations containing T lymphocytes are activated using an activator, then the culture medium is removed and the cells are collected.

[0223] Cellular gene modification: Vectors containing CAR structural genes or target genes, such as plasmids, lentiviruses, and VLP vectors with modified viral envelopes (such as the vector described in patent 202310887156.5), are used to transduce genes in activated immune cells.

[0224] Cell culture: Continue culturing in a medium supplemented with the cytokine combination for 48 hours to 21 days; for example, 48 hours, 72 hours, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days; remove the medium, collect the cells, resuspend them in cryopreservation solution, and freeze for preservation.

[0225] During the culture process, the culture medium can be changed or replenished according to the state and expansion of the cells.

[0226] This application provides a hybrid T cell obtained using the method described above.

[0227] This application provides a method for culturing universal T cells, including culturing universal T cells using the culture medium.

[0228] Universal T cells, also known as allogeneic T cells, refer to T cells extracted from the peripheral blood, umbilical cord blood, or tissue of a healthy donor. Gene editing technology is used to edit the genes of these T cells to address the issue of immune rejection between allogeneic individuals. In this application, universal T cells are defined as those resulting from gene knockout and / or knockdown and / or functional disruption of any one or more of the following T cell molecules: TCR molecules, MHC molecules, CD4, CD8, and CD3 molecules. Examples of TCR molecules include TRAC and TRBC; TRAC encodes the TCRα chain, and TRBC encodes the TCRβ chain. Examples of MHC molecules include MHC I and MHC II. In some embodiments, universal T cells are T cells generated by gene knockout and / or knockdown and / or functional disruption of T cell TCR-like molecules; in some embodiments, universal T cells are T cells generated by gene knockout and / or knockdown and / or functional disruption of T cell TCR-like molecules and MHC I; in some embodiments, universal T cells are T cells generated by gene knockout and / or knockdown and / or functional disruption of T cell TCR-like molecules and MHC II; in some embodiments, universal T cells are T cells generated by gene knockout and / or knockdown and / or functional disruption of T cell CD4; in some embodiments, universal T cells are T cells generated by gene knockout and / or knockdown and / or functional disruption of T cell CD8; in some embodiments, universal T cells are T cells generated by gene knockout and / or knockdown and / or functional disruption of both CD4 and CD8. In some embodiments, the universal T cells are CD4 and CD8 double-negative CAR-T cells.

[0229] In some implementations, the genome editing technology is selected from any one or more of ZFN (zinc finger nuclease), TALEN (transcription activator-like effector nuclease), and CRISPR; the gene editing technology is used to prepare universal T cells.

[0230] "Genetically modified allogeneic T cells" refers to allogeneic T cells that express exogenous or endogenous genes, including transducing target genes into allogeneic T lymphocytes, such as allogeneic CAR-T cells, allogeneic TCR-T cells, allogeneic STAR-T cells, and other adoptive cell therapies. In this application, universal CAR-T cells are a type of genetically modified allogeneic T cell. Universal CAR-T cells (U CAR-T cells) refer to universal T cells obtained by introducing a gene fragment encoding a chimeric antigen receptor (CAR), which can be used for adoptive cell therapy. Chimeric antigen receptor (CAR) has been described above and will not be repeated here. In some embodiments, universal CAR-T cells are CEA CAR-T cells, CD19 CAR-T cells, CD70 CAR-T cells, BCMACAR-T cells, or any combination of the above-listed cells, with CD4 and / or CD8 knockout, knockdown, and / or loss of function.

[0231] In some implementations, the culture time is any time range within the range of 2-25, 5-25, 10-25, 15-25, 20-25, 7-21, 7-14, 14-21, or 2-25 days; in one specific implementation, the culture time is any time range within the range of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 2-25 days.

[0232] In some implementation methods, methods for culturing universal T cells include:

[0233] Cell activation: A cell population containing T lymphocytes (i.e., immune cells) is activated using an activator and cultured in a medium supplemented with the above-mentioned combination of cytokines for any time range within the range of 12-72h, 12-24h, 12-36h, 12-48h, 12-54h, 24-48h, 24-56h, 24-72h, 36-48h, 36-56h, 36-72h, 48-56h, 48-72h, 54-72h, or 12-72h; for example, it can be any time range within 12, 15, 18, 21, 24, 27, 30, 32, 35, 27, 40, 43, 46, 49, 51, 54, 57, 60, 63, 66, 69, 72, or 12-72h.

[0234] Gene editing: The activated cells are modified using gene editing technology and then cultured in a culture medium containing the cytokines for 48 hours to 21 days, for example, any time within 48 hours, 72 hours, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 48 hours to 21 days.

[0235] Cryopreservation: After gene editing, cells are cultured for 3-21 days and then cryopreserved in cryopreservation solution. During the culture process, the culture medium can be changed or replenished according to the cell state and expansion.

[0236] In some implementation methods, methods for culturing universal T cells include:

[0237] Cell activation: A cell population containing T lymphocytes (i.e., immune cells) is activated using an activator and cultured in a medium supplemented with the above-mentioned combination of cytokines for any time range within the range of 12-72h, 12-24h, 12-36h, 12-48h, 12-54h, 24-48h, 24-56h, 24-72h, 36-48h, 36-56h, 36-72h, 48-56h, 48-72h, 54-72h, or 12-72h; for example, it can be any time range within 12, 15, 18, 21, 24, 27, 30, 32, 35, 27, 40, 43, 46, 49, 51, 54, 57, 60, 63, 66, 69, 72, or 12-72h.

[0238] Gene recombination: Vectors containing CAR structural genes or target genes, such as plasmids, lentiviruses, or VLP vectors with modified viral envelopes (such as the vector described in patent 202310887156.5), are used to transduce genes into activated immune cells, followed by cell culture for 12-24 hours.

[0239] Gene editing: Gene-recombinant cells are treated with gene editing technology and then cultured in a culture medium containing the cytokines for 48 hours to 21 days, for example, any time within 48 hours, 72 hours, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 48 hours to 21 days.

[0240] Cryopreservation: After gene editing, cells are cultured for 3-21 days and then cryopreserved in cryopreservation solution. During the culture process, the culture medium can be changed or replenished according to the cell state and expansion.

[0241] In some implementations, the cryopreservation method includes the step of subjecting the CD4 / CD8 positive mixed T cells to dormancy in an ultra-low temperature environment.

[0242] The ultra-low temperature environment described in this application refers to a temperature not higher than -20 degrees Celsius (hereinafter referred to as -20°C). In some embodiments, the ultra-low temperature environment refers to any temperature range below -25°C, -35°C, -60°C, -80°C, or below -20°C. In some embodiments, the ultra-low temperature environment refers to any temperature between -25°C, -30°C, -35°C, -40°C, or below -20°C. In some embodiments, the environment or device providing a temperature below -20°C may be a liquid nitrogen tank, a -80°C refrigerator, a -20°C refrigerator, etc.

[0243] In some implementations, the cryopreservation is carried out in a cryopreservation solution. It is understood that the cryopreservation solution is commercially available or prepared in-house.

[0244] In some embodiments, the cryopreservation solution is selected from any one or more of the following commercially available cryopreservation solutions: CS10, PRIME-XV FreezIS, Cellbanker2, Gibco Recovery and

[0245] In some embodiments, the cryopreservation solution comprises: a compound electrolyte solution, a glucose sodium chloride solution, an albumin mixture, a glucose solution, a dextran solution, and dimethyl sulfoxide.

[0246] In some embodiments, the electrolyte is a mineral salt containing sodium, potassium, chloride and bicarbonate, and the compound electrolyte is an injection containing one or more electrolytes such as sodium chloride, potassium chloride, magnesium chloride, sodium gluconate and sodium acetate.

[0247] In some embodiments, the volume percentages of the components in the cryopreservation solution are as follows:

[0248] Compound electrolyte 28-32 vol%; glucose sodium chloride solution 13-17 vol%; albumin mixture 18-22 vol%; glucose solution 7-10 vol%; dextran solution 15-18 vol%; and dimethyl sulfoxide 6-8 vol%.

[0249] In one specific embodiment, the cryopreservation solution comprises: 31.25 vol% compound electrolyte, 15.63 vol% glucose sodium chloride, 20 vol% human serum albumin, 8.96 vol% glucose injection, 16.67 vol% dextran 40, and 7.5 vol% dimethyl sulfoxide.

[0250] In some implementations, the resuscitation method includes the following steps: thawing the frozen immune cells at a temperature above -20°C.

[0251] In some implementations, cell resuscitation is carried out at a temperature of 15-40°C; for example, cell resuscitation at 37°C can be performed by placing the cells directly into a container filled with liquid at 37°C, such as a water bath, or by rapidly shaking the container to accelerate thawing; in some implementations, cell resuscitation is performed by directly thawing the cells to be resuscitated at room temperature.

[0252] This application provides a universal T cell culture, which is obtained by the above-described method.

[0253] This application provides the use of the above-described hybrid T cells or the above-described universal T cell culture for the preparation of drugs for the prevention and / or treatment of diseases.

[0254] This application provides a pharmaceutical composition comprising the above-described mixed T cells and / or the above-described cell cultures and / or the above-described universal T cell cultures.

[0255] This application provides a treatment method for a disease, including administering to a subject in need an effective amount of the above-mentioned mixed T cells, the above-mentioned cell culture, and / or the above-mentioned universal T cell culture, and / or its cryopreservation and thawing product. Example

[0256] Specific embodiments of the present application will now be described in more detail with reference to the accompanying drawings. While specific embodiments of the present application are shown in the drawings, it should be understood that the present application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0257] Example

[0258] The sources of the cytokines involved in Examples 1-9 below are as follows:

[0259] IL7 (Recombinant Human IL7, AF-200-07, Peprotech);

[0260] IL21 (Recombinant Human IL21, AF-200-21, Peprotech);

[0261] IL4 (Recombinant Human IL4, AF-200-04, Peprotech);

[0262] TGFβ1 (Recombinant Human TGFβ1, AF-200-21C, Peprotech);

[0263] anti-IFNγ: Biolegend 506532.

[0264] In all embodiments, the entire cell culture process, including activation, viral transduction, gene editing with or without, and cell preparation, uses a combination of culture media containing the aforementioned factors.

[0265] Example 1. Screening of Cytokine Combinations

[0266] Multiple T cell culture factor combinations were screened to form different cytokine combinations as described below:

[0267] 1) IL21+IL4 combination; 2) IL7+IL4 combination; 3) IL7+TGFβ1 combination; 4) IL7+IL21+TGFβ1 combination; 5) IL7+IL21+IL4 combination; 6) IL7+IL4+TGFβ1 combination; 7) IL7+IL21+IL4+TGFβ1 combination; 8) IL4+IL21+TGFβ1 combination; 9) IL21+TGFβ1 combination; 10) IL7+IL21 combination.

[0268] The concentrations of each cytokine in the above combinations are as follows:

[0269] IL7: 5ng / ml; IL21: 25ng / ml; IL4: 10ng / ml; TGFβ1: 1ng / ml.

[0270] Among the cytokines mentioned above, IL-7 has broad cell adaptability and is crucial for T cell development, especially the differentiation and survival of naive T cells, as well as the generation and maintenance of memory T cells. IL-21 can promote T cell proliferation. IL-4 is a cytokine secreted by CD4-positive cell subsets, B cells, mast cells, etc., and plays a role in regulating cell differentiation, proliferation, and apoptosis. IL-2 is produced by activated T cells and induces T cell activation, proliferation, and differentiation, stimulating long-term continuous T cell proliferation; it is also known as a T cell growth factor and also induces cytotoxicity. TGFβ1 inhibits the proliferation of immune-active cells such as cytotoxic T cells, suppressing the function of natural killer (NK) and cytotoxic T lymphocytes (CD8+ T cells). TGFβ1 also promotes Tregs and has a differentiation-regulating effect on various CD4+ T cell subtypes. CD4+ cells are helper T cells, whose main function is to regulate and coordinate the immune system response; their cytotoxic ability is not strong. CD8+ cells, on the other hand, are a type of cytotoxic T cell.

[0271] PBMCs (mononuclear cells) were isolated using Ficoll separation solution (Tianjin Haoyang, catalog number HY2015). The Ficoll separation solution was slowly added to normal human blood to maintain a clear separation interface between the Ficoll separation solution and the normal human blood. 50 mL centrifuge tubes containing the blood and separation solution were centrifuged at approximately 15°C for 20 min. After centrifugation, the liquid surface separated into four layers: an upper layer of plasma mixture, a lower layer of erythrocytes and granulocytes, and a middle layer of Ficoll fluid. At the boundary between the upper and middle layers, there was a narrow band of white, cloudy layer dominated by PBMCs, which was the PBMC cell layer. The upper plasma mixture was carefully aspirated using a sterile Pasteur pipette, and then PBMCs were aspirated using a new sterile Pasteur pipette to obtain the isolated PBMCs. Six PBMCs from different donors or T cells generated from PBMCs through sorting (or cells revived after cryopreservation of the above cells, where the PBMC cells and T cells were a mixed cell population containing CD4 and CD8 positive cells) were used.

[0272] Six samples from different donors were plated in 6-well plates with mononuclear cells (1–5 E+06 / ml) or T cells obtained from the isolated mononuclear cells or sorted mononuclear cells (the mononuclear cells and T cells were a mixed cell population containing CD4 and CD8 positive cells). Dynabeads (CD3CD28 antibody-conjugated magnetic beads) were used. TM CD3 / CD28 (40203D) activated T cells were activated for 18-27 hours. After activation, HIV-deficient lentiviral particles containing a chimeric antigen receptor (CAR) gene vector targeting CEA were co-incubated with the activated T cells for 18-27 hours. CEA CAR-T cells were obtained by transducing the CEA-targeting CAR gene and cultured for 7 days in a medium containing the above-described factor combination scheme. The basal medium was SCGM medium from CellGenix, Germany, 20802-0500. In some embodiments, CAR-T cells were harvested after culturing for 7-14 days in a medium containing the above-described factor combination scheme.

[0273] The structure of the CEA-CAR chimeric antigen receptor is: ScFv (anti-CEA)-8h-8TM-BBZ (where 8h is derived from the human CD8α hinge sequence, 8TM is derived from the human CD8α transmembrane sequence, BB is derived from the intracellular sequence of 4-1BB, also known as CD137, Z is derived from the intracellular sequence of CD3ζ, and BBZ is an abbreviation for 4-1BB-CD3ζ), and the amino acid sequence is shown in SEQ ID NO.1:

[0274] Following the above preparation protocol, CEA CAR-T cells were collected and cultured for 7 days in medium supplemented with different combinations of cytokines. The cells were then resuspended in cryopreservation solution and cryopreserved at ultra-low temperatures. As known to those skilled in the art, cryopreservation solutions such as CS10, PRIME-XV FreezIS, and Cellbanker2 can be used for cell cryopreservation. Gibco Recovery or Commercially available cell cryopreservation solutions can be used; alternatively, self-prepared cryopreservation solutions can be used, such as those used in this invention: the cell cryopreservation solution components are: 31.25 vol% compound electrolyte (Sichuan Kelun Pharmaceutical Co., Ltd.), 15.63 vol% glucose sodium chloride (Shijiazhuang No. 4 Pharmaceutical Co., Ltd.), 20 vol% human serum albumin (Guizhou Taibang Biological Products Co., Ltd.), 8.96 vol% glucose injection (Chenxin Pharmaceutical Co., Ltd.), 16.67 vol% dextran 40 (Sichuan Kelun Pharmaceutical Co., Ltd.), and 7.5 vol% dimethyl sulfoxide (OriGen Biomedical, Inc.).

[0275] After cryopreservation, CEA CAR-T cells were rapidly thawed in a 37°C water bath and then cultured in basal medium without any added cytokines for 24 hours. Cell viability was assessed using AOPI. The thawed cells were then cultured in a cytokine-free environment (i.e., cultured in basal medium without any added cytokines) for 6 days, for a total of 7 days of cytokine-free culture. Cell count was then assessed using AOPI. The results are shown in Tables 1, 2, and 3, and Figure 1. Figure 1 shows the viability, fold increase, and proliferation curves of CEA CAR-T cells in each group after factor-free culture. Figure 1A shows the viability of CEA CAR-T cells prepared in each group after 24 hours of factor-free culture; Figure 1B shows the total fold increase of CEA CAR-T cells prepared in each group after 7 days of factor-free culture; Figure 1C shows the proliferation curves of CEA CAR-T cells prepared in each group after 7 days of factor-free culture; and Figure 1D shows the factor-free proliferation of IL21+IL4+TGFβ1 and IL21+TGFβ1 combinations.

[0276] Table 1 shows the cell viability data corresponding to Figure 1A. (Note: “—” in Table 1 indicates that no related experiments were conducted for this parallel sample in this group of experiments, therefore there are no data results.)

[0277] Table 2 shows the data results for the fold increases corresponding to Figures 1B and 1D.

[0278] Table 3 shows the data results for the fertilization rate corresponding to Figure 1C.

[0279] CAR-T cells function after being reinfused into the human body, but the human blood and tissue environment does not have the cytokine environment of in vitro culture. Therefore, culture medium without any added cytokines can better reflect the real environment in which CAR-T cells function in the human body.

[0280] Data from Tables 1, 2, and 3, as well as Figure 1, show that CEA CAR-T cells cultured with factor combinations containing TGFβ1 and IL7 (IL7+IL21+IL4+TGFβ1, IL7+IL4+TGFβ1, IL7+IL21+TGFβ1, IL7+TGFβ1), and combinations containing IL21+IL4+TGFβ1 or IL21+TGFβ1, exhibited higher cell viability after cryopreservation and resuscitation in factor-free cell culture medium, and also showed significantly greater proliferative capacity compared to other groups.

[0281] TGFβ1 is a member of the TGFβ superfamily that regulates cell growth and differentiation. Current research indicates that TGFβ1 inhibits the proliferation of immune-active cells such as cytotoxic T cells and suppresses the production of IFN-γ and TNF-α in PBMCs. Current studies also show that TGFβ1 is a cytokine required for the proliferation of CD4+ T cell subsets. Furthermore, studies have shown that co-addition of TGFβ1 and IL4 can promote the proliferation of CD4+ T cell subsets, but this requires separate isolation and culture of CD4+ cells for induction. Based on this, this application further analyzed the proportion of CD4+ and CD8+ positive cells after culture using the aforementioned cytokine combination regimen using flow cytometry. CEA CAR-T cells were prepared using the culture protocol described in Example 1. Cells were collected after 11 days of culture, and the proportion of CD4+ and CD8+ subsets was analyzed using flow cytometry. The results are shown in Figure 2.

[0282] As shown in Figure 2, the cytokine combinations containing TGFβ1 and IL7 selected in this application—specifically IL7+IL21+IL4+TGFβ1, IL7+IL4+TGFβ1, IL7+IL21+TGFβ1, and IL7+TGFβ1—are suitable for culturing mononuclear cells containing CD4 and CD8 positive T cells, or T cells generated from sorted mononuclear cells. Furthermore, the cultured CAR-T cells showed no CD4 predisposition, with a CD4:CD8 cell ratio of approximately 3:2, consistent with existing culture protocols. After cryopreservation and resuscitation in factor-free cell culture medium, the viability was higher, and all groups exhibited significantly greater proliferative capacity compared to other groups. We also verified that the CD4:CD8 cell ratio of IL21+IL4+TGFβ1 and IL21+TGFβ1 was also approximately 3:2, consistent with existing culture protocols.

[0283] Example 2. Verification of the ability of cells cultured with different combinations of cytokines to resist multiple rounds of tumor cell stimulation.

[0284] CEA CAR-T cells were prepared, collected, and cryopreserved according to the method in Example 1.

[0285] Among them, the CAR structure of CEA CAR-T cells is a CAR structure targeting CEA. After the above-mentioned cryopreserved CEA-targeting CAR-T cells were rapidly thawed in a 37°C water bath, the CEA-targeting CAR-T cells were co-cultured with NCI-N87-Luc-GFP (human gastric cancer cells, NCI-N87 purchased from ATCC) tumor cells at an effector-to-target ratio of 3:1 in 1640 basal medium. Cells were harvested every 3 days, the medium was removed by centrifugation, and repeated tumor killing tests were performed to compare the continuous tumor killing ability of different CAR-T cells.

[0286] The main implementation procedure is as follows: After resuspending the cell pellet in PBS, the proportion of GFP+ tumor cells in the cell population is detected by flow cytometry. When the proportion of GFP+ cells is less than 10%, it indicates that the tumor cells have been killed by CAR-T cells. At this point, the cells are collected and counted, and tumor cells are added again at the same effector-to-target ratio for repeated stimulation and killing. The endpoint of the killing test is defined as the proportion of GFP+ tumor cells in the cell population exceeding 90% as detected by flow cytometry. Each addition of tumor cells is counted as one continuous tumor killing round. The number of continuous tumor killing rounds for each test group is recorded. The continuous tumor killing rounds of CAR-T cells reflect the sustained anti-tumor function of each group of cells.

[0287] The results are shown in Figure 3. The horizontal axis represents the number of sustained tumor-killing rounds of CEA CAR-T cells in vitro, and the vertical axis represents CEA CAR-T cells prepared with different cytokine combinations. Each round of stimulation on the horizontal axis is represented by one bar, and alternating black and white bars are used to facilitate differentiation of each round. Figure 3A shows the sustained tumor-killing ability of the IL7+TGFβ1 and IL7+IL21+IL4+TGFβ groups; Figure 3B shows the sustained tumor-killing ability of the IL21+IL4+TGFβ1 and IL21+TGFβ1 groups. Figure 3 shows that, compared to the combinations of IL7+IL21, IL7+IL4, IL7+TGFβ1, IL21+IL4, IL7+IL21+IL4, IL7+IL21+IL4, IL7+IL21+IL4+TGFβ, IL21+IL4+TGFβ1, and IL21+TGFβ1, the combinations of IL7+TGFβ1, IL7+IL21+IL4+TGFβ, IL21+IL4+TGFβ1, and IL21+TGFβ1 exhibit stronger continuous tumor-killing ability. Combinations with TGFβ1 and IL7 or TGFβ1 and IL-21 are more effective. Combinations containing TGFβ1 and IL7 or TGFβ1 and IL21, specifically the combinations of IL7+IL21+IL4+TGFβ1, IL21+IL4+TGFβ1, IL21+TGFβ1, and IL7+TGFβ1, show superior continuous tumor-killing ability.

[0288] Flow cytometry was used to detect CEA expression in NCI-N87-Luc-GFP cells. The CEA detection was performed using an anti-CEA antibody (Thermo, 14-0669-82). The results are shown in Figure 4. The results show that 46.63% of NCI-N87-Luc-GFP cells expressed CEA, which is suitable for this experiment.

[0289] Example 3. Validation of Factor-Free Proliferation Capacity of Different CAR Structures and Factor Combinations

[0290] Existing technologies disclose the combination of TGF-β and IL4 to promote the differentiation of natural human CD4 T cells, but do not disclose the ability and effect of the combination of TGF-β and IL4 on the population culture of non-CD4 single-positive cells. Therefore, the applicant further validated the combination of TGF-β and IL4 against the IL7+IL21+IL4+TGFβ1 combination scheme screened in Example 2 and the traditional IL7+IL21 scheme. Factor concentrations: IL7: 5 ng / ml; IL21: 25 ng / ml; IL4: 10 ng / ml; TGFβ1: 1 ng / ml. The CAR structure used is a CD19-targeting CAR molecule, and the amino acid sequence is shown in SEQ ID NO.2.

[0291] Six samples from different donors were used, with cytokine combinations of IL7+IL21, IL7+IL21+IL4+TGFβ1, and IL4+TGFβ1. CD19 CAR-T cells were prepared according to the culture method in Example 1, with the difference being that in this example, after activation, lentiviral particles containing a chimeric antigen receptor (CAR) gene vector targeting CD19 were co-incubated with activated T cells for 18-27 hours, and the cells were collected for cryopreservation. The cryopreserved cells were thawed and seeded in 1640 medium without any added cytokines for 7 days. Cell viability and number were detected using AOPI. The results are shown in Tables 4 and 5, and Figures 5A and 5B. Figure 5A shows the viability of CAR-T cells cultured with different factor combinations after 7 days of factor-free culture, and Figure 5B shows the fold increase of CAR-T cells cultured with different factor combinations after 7 days of factor-free culture.

[0292] Data corresponding to Figure 5A in Table 4

[0293] Table 5 Data corresponding to Figure 5B

[0294] The results show that the combination of TGF-β1 and IL4 is also applicable to populations of non-CD4 single-positive cells, such as mononuclear cells containing CD4 and CD8 positive T cells in this embodiment, or T cells generated from sorted mononuclear cells; and the factor-free proliferation capacity after cryopreservation and thawing is superior to the existing IL7+IL21 CAR-T culture protocol. The IL7+IL21+IL4+TGFβ1 combination protocol exhibits more significant factor-free proliferation capacity after cryopreservation (Paired t test **p=0.0071, *p=0.0237) and long-term viability after proliferation (Paired t test **p=0.0010, *p=0.0168).

[0295] Example 4. Verification of the ability of different CAR structures and different factor combinations to resist multiple rounds of tumor cell stimulation

[0296] The CAR-T cells prepared in Example 3 were used in this example. CAR-T cells targeting CD19 were co-cultured with tumor cells expressing CD19 antigen, Nalm6-luc-GFP. Flow cytometry was used to detect CD19 expression in Nalm6-luc-GFP. An anti-CD19 antibody (Biolegend 302212) was used for CD19 expression. The results are shown in Figure 6. The CD19 expression of Nalm6-luc-GFP was 99.97%, suitable for this experiment.

[0297] CAR-T cells targeting CD19 were co-cultured with Nalm6-Luc-GFP tumor cells at an effector-to-target ratio of 1:1, with the remaining steps being the same as in Example 2. The results are shown in Figure 7, where the horizontal axis represents the number of sustained killing cycles of CAR-T cells in vitro (denoted by R), and the vertical axis represents the fold increase in cell proliferation after tumor killing by different preparation methods.

[0298] The results showed that the TGF-β1 + IL4 combination can be used to prepare CAR-T cells from mononuclear cells containing CD4 and CD8 positive T cells, or from T cells generated by sorting mononuclear cells, and can resist multiple rounds of tumor stimulation, which is superior to the existing IL7 + IL21 preparation method. CAR-T cells prepared by the IL7 + IL21 + IL4 + TGFβ1 cytokine combination method showed the strongest cell expansion capacity in continuous tumor killing experiments.

[0299] Example 5. Validation of the in vivo antitumor ability of CAR-T mice prepared with different factor combinations

[0300] For the factor combination (IL7+IL21, IL7+IL21+IL4+TGFβ, IL4+TGFβ) and the concentrations of each factor (IL7: 5 ng / ml; IL21: 25 ng / ml; IL4: 10 ng / ml; TGFβ1: 1 ng / ml) of Example 3, CEA CAR-T cells were prepared using a CAR structure targeting CEA, and then collected and cryopreserved.

[0301] Six- to eight-week-old female NCG mice were subcutaneously (sc) inoculated with N87-Luc-GFP (human gastric cancer cells, N87 purchased from ATCC) tumor cells expressing CEA at a dose of 1.0E+06 / mouse to establish a subcutaneous solid tumor model. Two weeks after tumor bearing, CAR-T cells cultured using the above three methods were revived and intraperitoneally infused at a dose of 2.0E+06CAR+ / mouse (0d). In vivo imaging was performed weekly to compare the in vivo anti-tumor effect of CEA CAR-T cells. Blood samples were collected from mice, and the survival of CEA CAR-T cells in the blood was detected by RT-PCR.

[0302] Figure 8 shows the anti-tumor ability of CEA CAR-T cells cultured with different factor combinations in mice; Figure 8A is a fluorescence imaging image of mice; Figure 8B shows the copy number of CEA CAR-T cells in mouse blood after different numbers of days of CEA CAR-T cell infusion, with the horizontal axis representing different numbers of days of CEA CAR-T cell infusion and the vertical axis representing the copy number of CEA CAR-T cells in mouse blood.

[0303] The results are shown in Figure 8. The results show that the cytokine culture regimen of IL7+IL21+IL4+TGFβ1 has better in vivo anti-tumor effect and CAR-T cell survival ability. Unpaired t test**p=0.0044.

[0304] Example 6. Validation of the anti-tumor ability of different factor combinations in mice

[0305] Interferon-γ (IFN-γ) is mainly produced by activated T cells and NK cells. IFN-γ promotes the activity of cytotoxic T cells (CD8-positive T cells). Some published literature uses IL21+IL4+TGFβ1+antiIFN-γ for the differentiation and culture of CD4-positive cell subsets, but it has not disclosed the further ability and effect of this combination on the population culture of non-CD4 single-positive cells. Based on this, this embodiment designs the following factor combination:

[0306] Option 1 (IL7+IL21): Cytokines 5 ng / ml IL7 and 25 ng / ml IL21;

[0307] Option 2 (IL7+IL21+IL4+TGFβ1): Cytokines 5 ng / ml IL7, 25 ng / ml IL21, 10 ng / ml IL4 and 1 ng / ml TGFβ1;

[0308] Option 3 (IL2+IL4+TGFβ1+antiIFN-γ): Cytokines 500 IU / ml IL2, 10 ng / ml IL4, 1 ng / ml TGFβ1 and 100 ng / ml anti IFN-γ;

[0309] Option 4 (IL21+IL4+TGFβ1+antiIFN-γ): Cytokines 100 ng / ml IL21, 100 ng / ml IL4, 10 ng / ml TGFβ1 and 100 ng / ml antiIFN-γ;

[0310] Option 5 (IL4+TGFβ1+antiIFN-γ): 10 ng / ml IL4, 1 ng / ml TGFβ1 and 100 ng / ml anti IFN-γ.

[0311] On the one hand, this study aims to verify the ability and effect of IL21+IL4+TGFβ1+antiIFN-γ on the population culture of non-CD4 single-positive cells, and on the other hand, to compare the factor combinations screened in this application.

[0312] Following the aforementioned factor combination scheme, CEA CAR-T cells were prepared using the scheme of Example 1, collected, and cryopreserved. Mouse tumor-bearing models were established using the scheme of Example 5, and CEA CAR-T cells prepared using different schemes were resuscitated and reinfused. The results are shown in Figure 9, which illustrates the antitumor activity of CEA CAR-T cells prepared with different combinations of anti-IFNγ factors in mice. Figure 9A shows a fluorescence imaging image of mice; Figure 9B shows the copy number of CAR-T cells in mouse blood under different culture schemes, with the horizontal axis representing different culture schemes and the vertical axis representing the copy number of CAR-T cells in mouse blood; the copy number data are shown in Table 6 below.

[0313] Table 6 shows the copy number data corresponding to Figure 9B. (Note: “—” in Table 6 indicates that no related experiments were conducted for this parallel sample in this group of experiments, therefore there are no data results.)

[0314] The results showed that the combinations of IL21+IL4+TGFβ1+antiIFN-γ and IL4+TGFβ1+antiIFN-γ can be used to prepare CEA CAR-T cells from mononuclear cells containing CD4 and CD8 positive T cells or from T cells generated by sorting mononuclear cells, and can inhibit tumor proliferation in vivo. The cytokine combination culture regimen of IL7+IL21+IL4+TGFβ1 screened in this application has better in vivo anti-tumor effect and CAR-T cell survival ability. Unpaired t test ****p<0.0001, ***p=0.0008.

[0315] Example 7. Validation of the in vivo antitumor activity of CD70CAR-T mice prepared with different combinations of factors

[0316] The combination of T cell culture factors involved in this embodiment is as follows:

[0317] Option 1 (IL7+IL21): Cytokines 5 ng / ml IL7 and 25 ng / ml IL21;

[0318] Option 2 (IL7+IL21+IL4+TGFβ1): Cytokines 5 ng / ml IL7, 25 ng / ml IL21, 10 ng / ml IL4 and 1 ng / ml TGFβ1;

[0319] CD70 CAR-T cells were prepared according to the method in Example 1. In this example, after activation, lentiviral particles containing a chimeric antigen receptor (CAR) gene vector targeting CD70 were co-incubated with activated T cells and cultured for 7 days in a culture medium containing the above-mentioned factor combination scheme before harvesting and cryopreservation.

[0320] Six- to eight-week-old female NCG mice were intraperitoneally (ip) inoculated with CD70-expressing SKOV3-Luc-GFP tumor cells (SKOV3 cell line purchased from ATCC) at a dose of 1.0E+06 / mouse to establish a peritoneal tumor model. Seven days after tumor bearing, CD70 CAR-T cells cultured under two different protocols were resuscitated and then intraperitoneally infused at a dose of 3.0E+05CAR+ / mouse (day 0). Uninfected Control-T cells served as a control. In vivo imaging was used weekly to compare the in vivo antitumor effects of CD70 CAR-T cells.

[0321] The amino acid sequence of the CD70 CAR chimeric antigen receptor is shown in SEQ ID NO.3;

[0322] Figure 10 shows the antitumor activity of CD70 CAR-T cells prepared with different factor combinations in mice. The IL7+IL21+IL4+TGFβ1 cytokine combination culture regimen described in this invention exhibits superior in vivo antitumor effects in the preparation of CD70 CAR-T cells. CD70 expression in target cells SKOV3-Luc-GFP was detected using flow cytometry, and the results are shown in Figure 11. The CD70 expression of SKOV3-Luc-GFP was 78.02%.

[0323] The above examples 3, 5, and 7 demonstrate that the IL7+IL21+IL4+TGFβ1 cytokine combination screened in this application can be applied to the culture of CAR-expressing T cells with any CAR structure, and can improve the effectiveness of CAR-T cells, as well as increase the factor-free proliferation and viability of CAR-T cells after cryopreservation.

[0324] Example 8. Validation of the factor-free proliferation ability of different factor combinations on T cells

[0325] In addition to the CAR-T cells mentioned above, this application also verified the factor-free proliferation capacity of mononuclear cells (PBMCs) after culturing them using the traditional IL7+IL21 protocol and the IL7+IL21+IL4+TGFβ1 protocol of this application. The results are shown in Table 7 below.

[0326] PBMCs (mononuclear cells) were separated using Ficoll separation solution (Tianjin Haoyang, catalog number HY2015). The Ficoll separation solution was slowly added to normal human blood to maintain a clear separation interface between the Ficoll separation solution and the normal human blood. A 50 mL centrifuge tube containing the blood and separation solution was centrifuged at approximately 15°C for 20 minutes. After centrifugation, the liquid surface separated into four layers: an upper layer of plasma mixture, a lower layer of erythrocytes and granulocytes, and a middle layer of Ficoll fluid. At the boundary between the upper and middle layers, there was a narrow band of white, cloudy layer dominated by PBMCs, which was the PBMC cell layer. The upper plasma mixture was carefully aspirated using a sterile Pasteur pipette, and then the PBMCs were aspirated using a new sterile Pasteur pipette to obtain the separated PBMCs. This PBMC cell population, containing a mixed population of CD4 and CD8 positive T cells, was used in this experiment (or it could be a cryopreserved PBMC sample prepared using this method, which required pre-thawing). In some embodiments, the above PBMCs could be further recruited to obtain CD4 positive T cells and CD8 positive T cells, which were then mixed.

[0327] The PBMC cells obtained above were plated at a concentration of 1–5 E+06 / ml and then coated with CD3CD28 antibody-conjugated magnetic beads (Dynabeads). TM CD3 / CD28 (40203D) cells were activated for 18-27 hours. Throughout the culture process, including plating, culture media containing either IL7+IL21 or IL7+IL21+IL4+TGFβ1 were used (IL7: 5 ng / ml; IL21: 25 ng / ml; IL4: 10 ng / ml; TGFβ1: 1 ng / ml). The basal medium was SCGM medium from CellGenix, Germany, 20802-0500. After activation, cells were cultured for 7 days using the same medium, then collected and cryopreserved. After cryopreservation, PBMC cells were rapidly thawed in a 37°C water bath and then seeded into basal medium without any added cytokines for 7 days. Cell count was assessed using AOPI, and the cell proliferation capacity without added cytokines is shown in Table 7.

[0328] Table 7. Fold-free proliferation of mononuclear cells after cryopreservation and thawing under different culture protocols

[0329] The results show that the method described in this application can also maintain the ability of PBMCs to proliferate without factors after cryopreservation and thawing.

[0330] Example 9. Validation of Factor Concentrations in Different Factor Combinations

[0331] Following the cell preparation process described in Example 1, T cells from donor PBMCs were activated using antibodies or magnetic beads containing CD3 / CD28 at a bead:cell ratio of 1:1 (CD3 / CD28 Dynabeads 40203D). 12–24 hours after activation, the cells were transduced into a CEA-targeting CAR lentiviral vector and expanded and cultured for 7 days in a culture system containing 5 ng / ml IL7, 25 ng / ml IL21, and different concentrations of IL4 (5 ng / ml–100 ng / ml) and TGFβ (0.5 ng / ml–10 ng / ml). CAR-T cells were then harvested and cryopreserved. The effects of different cytokine concentrations on maintaining cell viability after cryopreservation and thawing were evaluated.

[0332] The combination of cytokines involved in this embodiment is as follows:

[0333] Option 1: 5 ng / ml IL7 + 25 ng / ml IL21, no IL4 and TGFβ1 (0 ng);

[0334] Option 2: 5 ng / ml IL7 + 25 ng / ml IL21 + 5 ng / ml IL4 + 0.5 ng / ml TGFβ1;

[0335] Option 3: 5 ng / ml IL7 + 25 ng / ml IL21 + 10 ng / ml IL4 + 1 ng / ml TGFβ1;

[0336] Option 4: 5 ng / ml IL7 + 25 ng / ml IL21 + 50 ng / ml IL4 + 5 ng / ml TGFβ1;

[0337] Option 5: 5 ng / ml IL7 + 25 ng / ml IL21 + 100 ng / ml IL4 + 10 ng / ml TGFβ1;

[0338] Option 7: 5 ng / ml IL7 + 25 ng / ml IL21 + 5 ng / ml IL4 + 10 ng / ml TGFβ1;

[0339] Option 8: 5 ng / ml IL7 + 25 ng / ml IL21 + 100 ng / ml IL4 + 0.5 ng / ml TGFβ1.

[0340] The results are shown in Figure 12. Figure 12 shows the viability of CEA CAR-T cells after cryopreservation and thawing obtained from different concentrations of IL4 and TGFβ1. The horizontal axis represents the concentration of different factors, and the vertical axis represents the cell viability after cryopreservation and thawing. The results show that IL4 at concentrations of 5 ng / ml to 100 ng / ml and TGFβ1 at concentrations of 0.5 ng / ml to 10 ng / ml can maintain better cell viability of CEA CAR-T cells after cryopreservation.

[0341] Based on this, we further validated the different concentrations of the overall factor combination, as shown in Table 8.

[0342] Table 8. Combinations of different concentrations of IL7+IL4+IL21+TGFβ1 cytokines

[0343] The results are shown in Figure 13. The viability of CEA CAR-T cells prepared by different cytokine concentrations after cryopreservation and thawing and culture without factors for 24 hours was shown. The results showed that the concentration of IL7 in the range of 5 ng / ml to 250 ng / ml and the concentration of IL21 in the range of 25 ng / ml to 1250 ng / ml could maintain the superior viability of CEA CAR-T cells after cryopreservation without factors (viability), and were significantly better than the conventional IL7+IL21 regimen.

[0344] Referring to Figures 12 and 13, it can be seen that the factor combination containing IL7 and TGFβ1 described in this application has the following concentrations: IL7 concentration is 1 ng / ml to 250 ng / ml, preferably 5 ng / ml to 250 ng / ml; TGFβ1 concentration is 0.5 ng / ml to 100 ng / ml, preferably 1 ng / ml to 100 ng / ml. IL21 and IL4 can be further added to form a combination of IL7 + IL21 + IL4 + TGFβ1; wherein the concentration of IL7 is 1 ng / ml to 250 ng / ml, the concentration of TGFβ1 is 0.5 ng / ml to 100 ng / ml; the concentration of IL4 is 2 ng / ml to 250 ng / ml, preferably 5 ng / ml to 250 ng / ml; and the concentration of IL21 is 5 ng / ml to 1250 ng / ml, preferably 25 ng / ml to 1250 ng / ml.

[0345] Example 10. Cell proliferation of UCAR-T cells under different combinations of cytokines

[0346] The concentrations and sources of the cytokines used in this section are shown in Table 9 below:

[0347] Table 9 shows the concentrations and sources of cytokines involved in Examples 10-14.

[0348] Throughout the entire cell culture process in all embodiments, including activation, viral transduction, gene editing with or without, and cell preparation, a combination of culture media containing the aforementioned factors is used.

[0349] Mononuclear cells obtained according to the protocol in Example 1, or T cells generated by sorting mononuclear cells (or cells thawed after cryopreservation as described above), were used with CD3CD28 antibody-conjugated magnetic beads (Dynabeads). TM CD3 / CD28 (40203D) activated T cells were activated for 12-48 hours, specifically 24 hours in this embodiment. The activated T cells were then co-incubated with lentiviral particles containing a chimeric antigen receptor (CAR) gene vector targeting CD19 for 24 hours. This process was used to transduce the CD19-targeting lentiviral gene to prepare CD19 CAR-T cells. Using a Lonza electroporator, a suitable electroporation program was selected for T cell electroporation. The molar ratio of Cas9 protein (Kaika Biotechnology, Cat: CAS-EE109):sgRNA (the sgRNA sequence from Example 1 in patent 202111657717.X is incorporated into this application, and was custom-synthesized by GenScript) was 1:2 or another suitable ratio. CD4 and CD8 were knocked out using sgRNA targeting CD4 and CD8 to obtain allogeneic CD19 UCAR-T cells. The sgRNAs that knock out CD4 are sg4-1, sg4-2, sg4-3, sg4-4, sg4-5, and sg4-6; the sequences of each sgRNA are as follows:

[0350] sg4-1: TGAACCGGGGAGTCCCTTTT, SEQ ID NO: 4;

[0351] sg4-2:CCCTTTTAGGCACTTGCTTC, SEQ ID NO:5;

[0352] sg4-3: AGTGGTGCTGGGCAAAAAAG, SEQ ID NO: 6;

[0353] sg4-4:TCTGGTTGGAGTTTTCCAG, SEQ ID NO:7;

[0354] sg4-5:TCAGGGAAAGAAAGTGGTGC, SEQ ID NO:8;

[0355] sg4-6: CTCCTCCCAGCAGCCACTCA, SEQ ID NO:9.

[0356] The sgRNAs that knock out CD8 are sg8-1, sg8-2, sg8-3, sg8-48, and sg8-5; the sequences of each sgRNA are as follows:

[0357] sg8-1: GACCGCCTTGCTCCTGCCGC, SEQ ID NO: 10;

[0358] sg8-2: GGCAGGAGCAAGGCGGTCAC, SEQ ID NO: 11;

[0359] sg8-3: TCACGGAGCAGCAAGGCCAG, SEQ ID NO: 12;

[0360] sg8-4: GGCCACGGCAGGAGCAAGG, SEQ ID NO: 13;

[0361] sg8-5: CAAGGCCAGCGGCAGGAGCA, SEQ ID NO: 14.

[0362] The culture medium used was 1640 medium with 10% FBS supplemented with a combination of IL7+IL21+IL4+TGF-β1 or IL7+IL21 factors. The culture was carried out for 11 days and 21 days from the start of activation, and then harvested and frozen.

[0363] AOPI was used to detect cell viability and number in the culture. The results are shown in Figure 14, which shows the fold increase of CD19 UCAR-T cells cultured for different number of days and different combinations of cytokines. The horizontal axis of Figure 14 represents the number of days of culture, and the vertical axis represents the fold increase of CD19 UCAR-T cells.

[0364] Table 10 shows the fertilization data corresponding to Figure 14.

[0365] Table 10 shows that cell death and insufficient proliferation occurred in the IL7+IL21 culture group by day 17, therefore cell counting was not continued on days 19 and 21. Combining Figure 14 and Table 10, it can be seen that the cell proliferation rate of the IL7+IL21 combination culture protocol was significantly weaker than that of the IL7+IL21+TGFβ1+IL4 group. This indicates that the IL7+IL21+TGFβ1+IL4 factor combination described in this application can be applied to the culture of universal CAR-T cells and can significantly improve the proliferation capacity of universal CAR-T cells, solving the problem of insufficient industrial production capacity of universal CAR-T cells.

[0366] The universal CAR-T cells described in this embodiment refer to CAR-T cells obtained by using T cells extracted from the peripheral blood, umbilical cord blood, or tissue of a healthy donor as the starting sample, and employing gene editing technology to address the issue of immune rejection between allogeneic individuals. Gene editing technology can be used to knock out, knock down, or disrupt the function of TCR-related molecules such as TRAC and TRBC; it can also be used to remove the function of MHCI and class II molecules, such as B2M and CIITA; and it can also be used to knock out, knock down, or disrupt the function of CD4, CD8, or CD3 molecules. Genome editing technologies, including ZFN (zinc finger nucleases), TALEN (transcription activator-like effector nucleases), and CRISPR-related technologies, are used to prepare universal T cells. This embodiment uses a CD4-CD8 double-negative universal CAR-T cell obtained through CRISPR technology as an example.

[0367] Example 11. Factor-free proliferation detection of CD19 UCAR-T cells prepared with different cytokines

[0368] The cells cryopreserved in Example 10 above were rapidly thawed in a 37°C water bath and then cultured for 7 days in a medium without any added cytokines. Cell viability and cell count were assessed using AOPI. The results are shown in Figures 15 and 16. Figure 15 shows the cell viability of cryopreserved and thawed CD19 UCAR-T cells after 7 days of cytokine-free culture, and Figure 16 shows the fold increase of cryopreserved and thawed CD19 UCAR-T cells after 7 days of cytokine-free culture. The corresponding data are shown in Tables 11 and 12.

[0369] Table 11 Data corresponding to Figure 15

[0370] Data corresponding to Figure 16 in Table 12

[0371] As can be seen from the results in Tables 11 and 12, Figures 15 and 16, the allogeneic CD19 UCAR-T cells cultured with the factor combination of this application have higher viability and persistence in the factor-free state after cryopreservation and thawing.

[0372] For cells cultured under two different protocols, the proportion of memory cells was detected by flow cytometry. Allogeneic UCAR-T cells were labeled with anti-CD45RA antibody (BV421, Biolegend, 304130), anti-CD45RO antibody (PerCP-CY5.5, Biolegend, 304222), and anti-CD197 antibody (APC, Biolegend, 353214). Cells with CD45RA+CD197+ were defined as Tscm stem central memory cells, CD45RA-CD45RO+CD197+ were central memory cells, and CD197- cells were Tem+Tef effector memory cells and effector T cells. The results of statistical analysis of allogeneic CAR-T cells in central memory are shown in Figure 17, with the horizontal axis representing different factor protocols and the vertical axis representing the proportion of Tcm central memory cells.

[0373] Table 13 Data corresponding to Figure 17

[0374] The results showed that the UCAR-T cells prepared according to the protocol described in this application had a higher proportion of Tcm compared to cells cultured with conventional IL7+IL21, indicating a significantly increased proportion of memory cell subsets in the cell population. These results also demonstrate that the allogeneic CAR-T cells cultured using the factor combination described in this application have a higher proportion of Tcm-centered memory cells.

[0375] Example 12. Functional validation of CD19 UCAR-T cells prepared with different factor combinations

[0376] Six- to eight-week-old female NCG mice were inoculated with CD19-expressing NALM6 tumor cells via the tail vein (iv) at a rate of 1.0E+06 per mouse to establish an acute lymphoblastic leukemia tumor model. Three days after tumor bearing, allogeneic UCAR-T cells prepared in Example 10 were rapidly thawed in a 37°C water bath and then reinfused into tumor-bearing mice via the tail vein (iv) at a rate of 1.0E+06 per mouse (0d). In vivo imaging was performed weekly to compare the in vivo antitumor effect of CAR-T cells.

[0377] Figure 18 shows the tumor fluorescence spectrum; the horizontal axis represents the number of days the cells were cultured with different cytokine combinations, and the vertical axis represents the number of days the cells were cryopreserved, thawed, and reinfused into tumor-bearing mice. The results showed that the IL7+IL21+IL4+TGFβ1 regimen described in this application exhibited a significant proliferative advantage during culture and demonstrated superior long-term in vivo persistence (anti-tumor effect in mice on day 32) compared to the IL7+IL21 group.

[0378] Example 13. Effect of IL15 on allogeneic CD19UCAR-T proliferation

[0379] In addition to the above factor combinations, other T cell activity or functional cytokines, such as IL15, can be added to the IL7+IL21+TGFβ1+IL4 combination. Allogeneic UCAR-T cells targeting CD19 were prepared using the method in Example 10 and cryopreserved after 21 days of culture. The culture medium used throughout the activation and culture process was SCGM medium from CellGenix, Germany, 20802-0500. The cytokine combinations were: IL7+IL21, IL7+IL21+TGFβ1+IL4, and IL7+IL21+TGFβ1+IL4+IL15. The concentrations of each cytokine in each combination were: IL7: 5 ng / ml; IL21: 25 ng / ml; IL4: 10 ng / ml; TGFβ1: 1 ng / ml; and IL15: 50 ng / ml.

[0380] AOPI was used to detect cell viability and number in culture. The results are shown in Figure 19 (the horizontal axis of Figure 19 represents the number of culture days, and the vertical axis represents the fold increase of CD19 UCAR-T cells). The results show that the combination of IL7+IL21+TGFβ1+IL4 and IL7+IL21+TGFβ1+IL4+IL15 can significantly increase the fold increase of allogeneic CAR-T cells.

[0381] Furthermore, following the method described in Example 11, allogeneic CD19 CAR-T cells frozen after 11 days of culture were revived and cultured for 7 days in SCGM medium without any cytokines. Cell viability and proliferation were then assessed, and the results are shown in Figure 20. Figure 20 shows the statistical results of cell viability on day 7 of factor-free culture. The results indicate that the combinations of IL7+IL21+TGFβ1+IL4 and IL7+IL21+TGFβ1+IL4+IL15 can significantly improve the cell viability of allogeneic CD19 UCAR-T cells after factor-free culture.

[0382] This indicates that the combination of IL7+IL21+TGFβ1+IL4 can serve as a basis for further optimization by adding new cytokines.

[0383] Example 14. Validation of factor concentrations in different cytokine combinations

[0384] Following the cell preparation process described in Example 10, allogeneic CD19 UCAR-T cells targeting CD19 were prepared. Cells were cultured for 21 days, collected, and cryopreserved. The cytokine regimen in the culture medium is shown in Table 14 below.

[0385] Table 14 Concentrations of various cytokines in the culture medium during CD19 UCAR-T cell culture.

[0386] AOPI was used to detect cell viability and number in the culture. The results are shown in Figure 21 (the horizontal axis of Figure 21 represents the number of culture days, and the vertical axis represents the fold increase of CD19 UCAR-T cells). The results in Figure 21 show that IL7 concentrations of 1 ng / ml to 250 ng / ml, IL21 concentrations of 5 ng / ml to 1250 ng / ml, IL4 concentrations of 2 ng / ml to 250 ng / ml, and TGFβ1 concentrations of 0.5 ng / ml to 100 ng / ml all maintained the in vitro proliferation capacity of universal CAR-T cells compared to the traditional IL7+IL21 regimen.

[0387] Furthermore, CD19 UCAR-T cells cryopreserved for 21 days were revived and cultured in cytokine-free SCGM medium for 7 days. Cell viability was assessed on days 1-7, and cell counts were performed on day 7 to calculate the fold increase in cell proliferation without cytokines. The results are shown in Figures 22 and 23. Figure 22 shows the viability of universal CAR-T cells after cryopreservation and revival under different cytokine concentration combinations for 1-7 days of cytokine-free culture; Figure 23 shows the proliferation of universal CAR-T cells after cryopreservation and revival under different cytokine concentration combinations for 7 days of cytokine-free culture.

[0388] The results showed that IL7 concentrations of 1 ng / ml to 250 ng / ml, IL21 concentrations of 5 ng / ml to 1250 ng / ml, IL4 concentrations of 2 ng / ml to 250 ng / ml, and TGFβ1 concentrations of 0.5 ng / ml to 100 ng / ml all maintained high cell viability and factor-free survival after cryopreservation and thawing of universal CAR-T cells, which were superior to the traditional IL7+IL21 regimen.

[0389] Example 15. Umbilical cord-derived T cells adapted to the cytokine combination of this application

[0390] The cytokine sources and concentrations are as shown in Example 1, and the basal culture medium is: In this embodiment, GMP SCGM specifically uses a combination of IL7+IL21+IL14+TGFβ1 (cytokine concentrations are the same as in Example 1) to generate umbilical cord blood-derived T cells, which are different from the peripheral blood-derived T cells mentioned above, to verify the suitability of the cytokine combination described in this application.

[0391] After cryopreservation, CD19 CAR-T or T cells (CAR structure and sequence are described in Example 3, and cell preparation protocol is the same as in Example 1) were rapidly thawed in a 37°C water bath and then cultured in basal medium without any added cytokines for 24 hours. Cell viability was detected using AOPI (experimental principle and purpose are described in Example 1). The results are shown in Figure 24 below. Figure 24A: Viability detection of umbilical cord blood-derived CAR-T cells cultured with the cytokine combination described in this application after 24 hours of cytokine-free culture; Figure 24B: Viability detection of umbilical cord blood-derived T cells cultured with the cytokine combination described in this application after 24 hours of cytokine-free culture.

[0392] The results show that the cytokine combination described in this application, and the T cell culture medium containing the cytokine combination described in this application, can improve the cell viability of cells cultured without factors for 24 hours after cryopreservation and thawing, regardless of the source of the T cells or CAR-T cells. The cytokine combination described in this application, and the T cell culture medium containing the cytokine combination described in this application, are suitable for T cells of any source and can improve the cell viability of cells cultured without factors for 24 hours.

[0393] Example 16. Adaptation of the Cytokine Combinations of this Application to Different Basal Culture Media

[0394] The cytokines described in this application can be present in any basal culture medium, which includes basal culture media for cell culture. In this embodiment, we tested the compatibility of the cytokine combination described in this application with basal culture media for eight different media; we also verified possible culture media containing the cytokine combination described in this application. Specific culture media are shown in Table 15 below.

[0395] Table 15 Different basal culture media and the culture media described in this application formed based on the basal culture media and the cytokines described in this application.

[0396] The cytokine sources and concentrations were as shown in Example 1. CEA CAR-T cells were prepared according to the protocol of Example 1, including the cytokine combination described in this application and different basal culture media, to verify the advantages of the cytokine combination described in this application compared to the traditional IL7+IL21 protocol for CAR-T cells. Specific experimental steps and verification methods were the same as in Example 1, and the results are shown in Figure 25 and Table 16.

[0397] Table 16 Viability of CEA CAR-T cells prepared in each group after 24 hours of factor-free culture

[0398] After cryopreservation, CEA CAR-T cells were rapidly thawed in a 37°C water bath and then cultured in basal medium without any added cytokines for 24 hours. Cell viability was assessed using AOPI. Figure 25A shows the viability of CEA CAR-T cells prepared in each group after 24 hours of factor-free culture. Cell viability is shown in Table 16. The thawed cells were then cultured in factor-free medium (i.e., cultured in basal medium without any added cytokines) for 6 days, for a total of 7 days of factor-free culture. Cell number was assessed using AOPI. Figure 25B shows the total fold increase of CEA CAR-T cells prepared in each group after 7 days of factor-free culture on day 7.

[0399] For cells cultured under different protocols, the proportion of memory cells was detected by flow cytometry. CAR-T cells cultured in the above different media were labeled with anti-CD45RA antibody (BV421, Biolegend, 304130), anti-CD45RO antibody (PerCP-CY5.5, Biolegend, 304222), and anti-CD197 antibody (APC, Biolegend, 353214). Cells with CD45RA+CD197+ were defined as Tscm stem central memory cells, and cells with CD45RA-CD197+ were defined as Tcm central memory cells. The phenotypes of memory cells were statistically analyzed, and the results are shown in Table 17 below.

[0400] Table 17 shows that the cytokine combination described in this application can increase the population of memory T cells in different culture media.

[0401] The above results indicate that the cytokine combination described in this application can be added to any basal culture medium, preferably an immune cell basal culture medium, and most preferably a T cell culture medium, to form the T cell culture medium containing the cytokine combination described in this application. This can promote the viability of CAR-T cells cultured without factors for 24 hours after cryopreservation and thawing, promote the proliferation potential of CAR-T cells after cryopreservation and thawing, and promote the increase of CAR-T cell memory cell phenotype.

[0402] In summary, the cytokine combination described in this application is suitable for mixing with any basal culture medium to form the T cell culture medium containing the cytokine combination described in this application.

[0403] Example 17. Adapting different types of T cell activating agents to the cytokine combination of this application.

[0404] In the production of some cell therapy products, T cell activation is crucial. T cell activation can be achieved by antigen presentation to the TCR receptor, providing the first activation signal, commonly using CD3 monoclonal antibodies, or by non-specific activation of T cells through the binding of mitogens (such as phytohemagglutinin PHA and concanavalin A) to glycoproteins (such as CD3) on the surface of T cells. Co-stimulatory molecules such as CD28, 4-1BB, CD40, and OX40 can also provide co-stimulatory signals to assist T cell activation. Currently, the most common method for T cell activation is to use CD3 / CD28 soluble antibody complexes, or to load antibody complexes onto magnetic nanospheres or micron-sized magnetic beads to simultaneously activate the first and co-stimulatory signals of T cells.

[0405] This embodiment uses CD3CD28 nanomagnetic beads (T Cell TransAct). TM ,200-076-202) and CD3CD28 micron magnetic beads (Dynabeads) TM CD3 / CD28, 40203D) activated T cells were cultured with CEA-CAR-T cells. The basal culture medium was: GMP SCGM. A comparison was made between the IL7+IL21+IL14+TGFβ combination (cytokine concentration as in Example 1) described in this application and the traditional IL7+IL21 regimen in terms of cell viability and in vivo antitumor function 24 hours after cryopreservation and thawing. The experimental steps and methods were as described in Examples 1 and 5. The experimental results are shown in Figures 26 and 27 below; Figure 26: Cell viability of the cytokine combination described in this application and the culture medium without cytokine under different stimulation regimens for 24 hours; Figure 27: In vivo efficacy verification of the cytokine combination described in this application and the culture medium under different stimulation regimens.

[0406] The results show that the cytokine combination described in this application and the culture medium containing the cytokine combination described in this application have better post-freeze survival rate and efficacy than the traditional IL7+IL21 regimen under different activation schemes. The cytokine combination described in this application and the culture medium containing the cytokine combination described in this application are suitable for different T cell activation schemes.

[0407] Example 18. IL-9 can be used in combination with the factors described in this application.

[0408] The cytokine combination described in this application can serve as the basis for T cell culture, upon which other cytokines can be added. This embodiment verifies the feasibility of adding IL-9 cytokine to the combination of IL-7+IL21+IL14+TGFβ (cytokine concentrations as in Example 1), wherein the IL-9 cytokine concentration is 10 ng / ml, and the sources and concentrations of other cytokines are as shown in Example 1. The basal culture medium is: GMP SCGM.

[0409] CAR-T cells were prepared according to the protocol of Example 1. Cell viability and proliferation potential were verified after cryopreservation and thawing. The effect on the phenotype of CD45RA+CD45RO-CD197+ Tnaive memory cells was verified according to the method of Example 19. The results are shown in Table 18 below.

[0410] Table 18 Effects of IL9 addition on post-cryopreservation thawing, proliferation, and cell phenotype.

[0411] The results show that the addition of IL9 to the cytokine combination in this application can significantly increase the cell expansion rate after cryopreservation and thawing and culture for 7 days without cytokine, improve the cell proliferation potential after cryopreservation and thawing, and also improve the cell proliferation potential of cell therapy product formulations.

[0412] The description in this disclosure is provided for illustrative and descriptive purposes only and is not intended to be exhaustive or to limit the disclosure to its forms. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to better illustrate the principles and practical application of this disclosure and to enable those skilled in the art to understand this disclosure and to design various embodiments with various modifications suitable for a particular purpose.

Claims

1. A T cell culture medium comprising any one, two or three of the cytokines IL7, IL21, and IL4, and a combination of TGFβ; Preferably, the TGF-β includes any one or more of the following cytokines: TGF-β1, TGF-β2, and TGF-β3; More preferably, the TGF-β is TGF-β1.

2. The culture medium according to claim 1, wherein the content of IL7 in the culture medium is 0.5-250 ng / ml, 0.5-100 ng / ml, 0.5-50 ng / ml, 0.5-30 ng / ml, 0.5-15 ng / ml, 0.5-10 ng / ml, 0.5-5 ng / ml, 1-250 ng / ml, 1-100 ng / ml, 1-50 ng / ml, 1-30 ng / ml, 1-15 ng / ml, 1-10 ng / ml, 1-5 ng / ml, 2-250 ng / ml, 2-100 ng / ml, 2 -50ng / ml, 2-30ng / ml, 2-15ng / ml, 2-10ng / ml, 2-9ng / ml, 2-8ng / ml, 2-6ng / ml, 2-5ng / ml, 3-250ng / ml, 3-100ng / ml, 3-50ng / ml , 3-30ng / ml, 3-15ng / ml, 3-10ng / ml, 3-8ng / ml, 3-9ng / ml, 3-6ng / ml, 3-5ng / ml, 4-250ng / ml, 4-100ng / ml, 4-50ng / ml, 4-30ng / m l, 4-15ng / ml, 4-10ng / ml, 4-9ng / ml, 4-8ng / ml, 4-7ng / ml, 4-6ng / ml, 4-5ng / ml, 5-250ng / ml, 5-100ng / ml, 5-50ng / ml, 5-30ng / ml, 5-15ng / ml, 5-10ng / ml, 5-8ng / ml, 5-6ng / ml, 6-250ng / ml, 6-100ng / ml, 6-50ng / ml, 6-30ng / ml, 6-15ng / ml, 6-10ng / ml, 6-8n g / ml, 7-250ng / ml, 7-100ng / ml, 7-50ng / ml, 7-30ng / ml, 7-15ng / ml, 7-10ng / ml, 7-8ng / ml, 8-250ng / ml, 8-100ng / ml, 8-50ng / m l, 8-30ng / ml, 8-15ng / ml, 8-10ng / ml, 9-250ng / ml, 9-100ng / ml, 9-50ng / ml, 9-30ng / ml, 9-15ng / ml, 9-10ng / ml, or 50-250ng / ml; and / or the TGFp is present in the medium at a concentration of 0.5-100 ng / ml, 0.5-80 ng / ml, 0.5-60 ng / ml, 0.5-40 ng / ml, 0.5-20 ng / ml, 0.5-15 ng / ml, 0.5-10 ng / ml, 0.5-9 ng / ml, 0.5-8 ng / ml, 0.5-7 ng / ml, 0.5-6 ng / ml, 0.5-5 ng / ml, 0.5-4 ng / ml, 0.5-3 ng / ml, 0.5-2 ng / ml, 0.5 - 1 ng / ml, 1 - 100 ng / ml, 1 - 80 ng / ml, 1 - 60 ng / ml, 1 - 40 ng / ml, 1 - 20 ng / ml, 1 - 15 ng / ml, 1 - 10 ng / ml, 1 - 9 ng / ml, 1 - 8 ng / ml, 1 - 7 ng / ml, 1 - 6 ng / ml, 1 - 5 ng / ml, 1 - 4 ng / ml, 1 - 3 ng / ml, 1 - 2 ng / ml, 2 - 100 ng / ml, 2 - 80 ng / ml, 2 - 60 ng / ml, 2 - 40 ng / ml, 2 - 20 ng / ml, 2 - 15 ng / ml, 2 - 10 ng / ml, 2 - 9 ng / ml, 2 - 8 ng / ml, 2 - 7 ng / ml, 2 - 6 ng / ml, 2 - 5 ng / ml, 2 - 4 ng / ml, 2 - 3 ng / ml, 3 - 100 ng / ml, 3 - 80 ng / ml, 3 - 60 ng / ml, 3 - 40 ng / ml, 3 - 20 ng / ml, 3 - 15 ng / ml, 3 - 10 ng / ml, 3 - 9 ng / ml, 3 - 8 ng / ml, 3 - 7 ng / ml, 3 - 6 ng / ml, 3 - 5 ng / ml, 3 - 4 ng / ml, 4 - 100 ng / ml, 4 - 80 ng / ml, 4 - 60 ng / ml, 4 - 40 ng / ml, 4 - 20 ng / ml, 4 - 15 ng / ml, 4 - 10 ng / ml, 4 - 9 ng / ml, 4 - 8 ng / ml, 4 - 7 ng / ml, 4 - 6 ng / ml, 4 - 5 ng / ml, 5 - 100 ng / ml, 5 - 80 ng / ml, 5 - 60 ng / ml, 5 - 40 ng / ml, 5 - 20 ng / ml, 5 - 15 ng / ml, 5 - 10 ng / ml, 5 - 9 ng / ml, 5 - 8 ng / ml, 5 - 7 ng / ml, 5 - 6 ng / ml, 6 - 100 ng / ml, 6 - 80 ng / ml, 6 - 60 ng / ml, 6 - 40 ng / ml, 6 - 20 ng / ml, 6 - 15 ng / ml, 6 - 10 ng / ml, 6 - 9 ng / ml, 6 - 8 ng / ml, 6 - 7 ng / ml, 7 - 100 ng / ml, 7 - 80 ng / ml, 7 - 60 ng / ml, 7 - 40 ng / ml, 7 - 20 ng / ml, 7 - 15 ng / ml, 7 - 10 ng / ml, 7 - 9 ng / ml, 7 - 8 ng / ml, 8 - 100 ng / ml, 8 - 80 ng / ml, 8 - 60 ng / ml, 8 - 40 ng / ml, 8 - 20 ng / ml, 8 - 15 ng / ml, 8 - 10 ng / ml, 8 - 9 ng / ml, 9 - 100 ng / ml, 9 - 80 ng / ml, 9 - 60 ng / ml, 9 - 40 ng / ml, 9 - 20 ng / ml, 9 - 15 ng / ml, 9 - 10 ng / ml or 10 - 100 ng / ml;. Preferably, the IL7 content in the culture medium is 1-5 ng / ml, 1-10 ng / ml, 3-8 ng / ml, 4-7 ng / ml, 5-6 ng / ml, or 5-50 ng / ml; Preferably, the content of TGFβ in the culture medium is 0.5-1 ng / ml, 0.5-2 ng / ml, 0.5-3 ng / ml, 0.5-5 ng / ml, 0.5-10 ng / ml, 1-2 ng / ml, 1-5 ng / ml or 1-10 ng / ml.

3. The culture medium according to claim 1, wherein the content of IL4 in the culture medium is 1 - 2 ng / ml, 1 - 3 ng / ml, 1 - 4 ng / ml, 1 - 5 ng / ml, 1 - 6 ng / ml, 1 - 7 ng / ml, 1 - 8 ng / ml, 1 - 9 ng / ml, 1 - 10 ng / ml, 1 - 11 ng / ml, 1 - 12 ng / ml, 1 - 13 ng / ml, 1 - 14 ng / ml, 1 - 15 ng / ml, 1 - 20 ng / ml, 1 - 50 ng / ml, 1 - 70 ng / ml, 1 - 80 ng / ml, 1 - 90 ng / ml, 1 - 100 ng / ml, 1 - 120 ng / ml, 1 - 150 ng / ml, 1 - 200 ng / ml, 1 - 250 ng / ml, 2 - 3 ng / ml, 2 - 4 ng / ml, 2 - 5 ng / ml, 2 - 6 ng / ml, 2 - 7 ng / ml, 2 - 8 ng / ml, 2 - 9 ng / ml, 2 - 10 ng / ml, 2 - 11 ng / ml, 2 - 12 ng / ml, 2 - 13 ng / ml, 2 - 14 ng / ml, 2 - 15 ng / ml, 2 - 20 ng / ml, 2 - 50 ng / ml, 2 - 70 ng / ml, 2 - 80 ng / ml, 2 - 90 ng / ml, 2 - 100 ng / ml, 2 - 120 ng / ml, 2 - 150 ng / ml, 2 - 200 ng / ml, 2 - 250 ng / ml, 5 - 6 ng / ml, 5 - 7 ng / ml, 5 - 8 ng / ml, 5 - 9 ng / ml, 5 - 10 ng / ml, 5 - 11 ng / ml, 5 - 12 ng / ml, 5 - 13 ng / ml, 5 - 14 ng / ml, 5 - 15 ng / ml, 5 - 20 ng / ml, 5 - 40 ng / ml, 5 - 5 ng / ml, 5 - 70 ng / ml, 5 - 80 ng / ml, 5 - 90 ng / ml, 5 - 100 ng / ml, 5 - 150 ng / ml, 5 - 200 ng / ml, 5 - 250 ng / ml, 10 - 11 ng / ml, 10 - 12 ng / ml, 10 - 13 ng / ml, 10 - 14 ng / ml, 10 - 15 ng / ml, 10 - 20 ng / ml, 10 - 40 ng / ml, 10 - 50 ng / ml, 10 - 70 ng / ml, 10 - 80 ng / ml, 10 - 90 ng / ml, 10 - 100 ng / ml, 10 - 150 ng / ml, 10 - 200 ng / ml, 10 - 250 ng / ml, 20 - 40 ng / ml, 20 - 50 ng / ml, 20 - 70 ng / ml, 20 - 80 ng / ml, 20 - 90 ng / ml, 20 - 100 ng / ml, 20 - 150 ng / ml, 20 - 200 ng / ml, 20 - 250 ng / ml, 30 - 40 ng / ml, 30 - 50 ng / ml,30 - 70 ng / ml, 30 - 80 ng / ml, 30 - 100 ng / ml, 30 - 150 ng / ml, 30 - 200 ng / ml, 30 - 250 ng / ml, 40 - 50 ng / ml, 40 - 70 ng / ml, 40 - 80 ng / ml, 40 - 100 ng / ml, 40 - 150 ng / ml, 40 - 200 ng / ml, 40 - 250 ng / ml, 60 - 70 ng / ml, 60 - 80 ng / ml, 60 - 100 ng / ml, 60 - 150 ng / ml, 60 - 200 ng / ml, 60 - 250 ng / ml, 70 - 80 ng / ml, 70 - 100 ng / ml, 70 - 150 ng / ml, 70 - 200 ng / ml, 70 - 250 ng / ml, 80 - 100 ng / ml, 80 - 150 ng / ml, 80 - 250 ng / ml, 90 - 100 ng / ml, 90 - 120 ng / ml, 90 - 150 ng / ml, 90 - 200 ng / ml or 90 - 250 ng / ml; Preferably, the IL4 content in the culture medium is 1-10 ng / ml, 1-20 ng / ml, 5-20 ng / ml, 5-40 ng / ml, 20-90 ng / ml, 30-80 ng / ml, 70-150 ng / ml, or 90-120 ng / ml.

4. The culture medium according to claim 1, wherein the content of IL21 in the culture medium is 0-1250 ng / ml, 1-1250 ng / ml, 1-1000 ng / ml, 1-750 ng / ml, 1-500 ng / ml, 1-250 ng / ml, 1-200 ng / ml, 1-150 ng / ml, 1-100 ng / ml, 1-50 ng / ml, 1-40 ng / ml, 1-30 ng / ml, 1-29 ng / ml, 1-28 ng / ml, 1-27 ng / ml, 1-26 ng / ml, 1-25 ng / ml, 1-20 ng / ml, 1-10 ng / ml, 1-8 ng / ml, 1-7 ng / ml, 1-6 ng / ml, 1-5 ng / ml, 1-4 ng / ml, or 1-3 ng / ml. / ml, 2.5-1250ng / ml, 2.5-1000ng / ml, 2.5-750ng / ml, 2.5-500ng / ml, 2.5-250ng / ml, 2.5-200ng / ml, 2.5-150ng / ml, 2.5-100ng / ml, 2.5-50ng / ml, 2.5-40ng / ml, 2.5-30ng / ml, 2.5-29ng / ml, 2.5-28ng / ml, 2.5-27ng / ml, 2.5-26ng / ml, 2.5-25ng / ml, 2.5-20ng / ml, 2.5-10ng / ml, 2.5-8ng / ml, 2.5-7ng / ml, 2.5-6ng / ml, 2.5-5ng / ml, 2.5-4ng / ml, 2.5 - 3 ng / ml, 5 - 1250 ng / ml, 5 - 1000 ng / ml, 5 - 750 ng / ml, 5 - 500 ng / ml, 5 - 250 ng / ml, 5 - 200 ng / ml, 5 - 150 ng / ml, 5 - 100 ng / ml, 5 - 50 ng / ml, 5 - 40 ng / ml, 5 - 30 ng / ml, 5 - 29 ng / ml, 5 - 28 ng / ml, 5 - 27 ng / ml, 5 - 26 ng / ml, 5 - 25 ng / ml, 5 - 20 ng / ml, 5 - 10 ng / ml, 5 - 8 ng / ml, 5 - 7 ng / ml, 5 - 6 ng / ml, 10 - 1250 ng / ml, 10 - 1000 ng / ml, 10 - 800 ng / ml, 10 - 750 ng / ml, 10 - 500 ng / ml, 10 - 400 ng / ml, 10 - 250 ng / ml, 10 - 200 ng / ml, 10 - 150 ng / ml, 10 - 100 ng / ml, 10 - 50 ng / ml, 10 - 40 ng / ml, 10 - 30 ng / ml, 10 - 29 ng / ml, 10 - 28 ng / ml, 10 - 27 ng / ml, 10 - 26 ng / ml, 10 - 25 ng / ml, 10 - 20 ng / ml, 20 - 1250 ng / ml, 20 - 1000 ng / ml, 20 - 800 ng / ml, 20 - 750 ng / ml, 20 - 500 ng / ml, 20 - 400 ng / ml, 20 - 250 ng / ml, 20 - 200 ng / ml, 20 - 150 ng / ml, 20 - 100 ng / ml, 20 - 50 ng / ml, 20 - 40 ng / ml, 20 - 30 ng / ml, 20 - 29 ng / ml, 20 - 28 ng / ml, 20 - 27 ng / ml, 20 - 26 ng / ml, 20 - 25 ng / ml, 30 - 1250 ng / ml, 30 - 1000 ng / ml, 30 - 800 ng / ml, 30 - 750 ng / ml, 30 - 500 ng / ml, 30 - 400 ng / ml, 30 - 250 ng / ml, 30 - 200 ng / ml, 30 - 150 ng / ml, 30 - 100 ng / ml, 30 - 50 ng / ml, 30 - 40 ng / ml, 100 - 1250 ng / ml, 100 - 1000 ng / ml, 100 - 800 ng / ml, 100 - 900 ng / ml, 100 - 750 ng / ml, 100 - 500 ng / ml, 100 - 400 ng / ml, 100 - 250 ng / ml, 100 - 200 ng / ml or 100 - 150 ng / ml;. Preferably, the IL21 content is 10-30 ng / ml, 20-30 ng / ml, or 20-40 ng / ml.

5. The culture medium according to any one of claims 1-4, further comprising any one or more of the following cytokines: IL1, IL2, IL3, IL5, IL6, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL19, IL22, IL23, IL24, IL26, IL27, IL28, IL29, IL32, IL33, IL35, IL37, anti-IFNγ (interferon-gamma antibody), TNF-α; Preferably, the culture medium comprises any one of the following groups: (I) IL7, TGFβ, and any one or more of the group consisting of IL2, IL4, IL15, IL21 and anti-IFNγ; (II) IL7, TGFβ, and any one or more of the group consisting of IL4, IL15 and IL21; (III) IL7, TGFβ, and any one or more of the group consisting of IL4 and IL21; More preferably, the culture medium comprises any one of the following groups: 1) Combinations of IL7, TGFβ, and IL4; 2) Combinations of IL7, TGFβ, and IL21; 3) Combinations of IL7, TGFβ, IL2, and IL4; 4) Combinations of IL7, TGFβ, IL2 and IL21; 5) Combinations of IL7, TGFβ, IL4 and IL21; 6) Combinations of IL7, TGFβ, IL2, IL4 and IL21; 7) Combinations of IL7, TGFβ, and antiIFN-γ; 8) Combinations of IL7, TGFβ, IL4 and antiIFN-γ; 9) Combinations of IL7, TGFβ, IL21 and antiIFN-γ; 10) A combination of IL7, TGFβ, IL4, IL21 and antiIFN-γ; 11) Combinations of IL7, TGFβ, and IL15; 12) Combinations of IL7, TGFβ, IL4 and IL15; 13) Combinations of IL7, TGFβ, IL21 and IL15; 14) Combinations of IL7, TGFβ, IL4, IL21 and IL15; 15) Combinations of IL2, IL4 and TGFβ; 16) Combinations of IL4, TGFβ, IL2 and antiIFN-γ; 17) Combinations of IL4, TGFβ, and antiIFN-γ; and 18) Combination of IL4, TGFβ, IL21 and antiIFN-γ.

6. The culture medium according to claim 5, wherein the IL2 content in the culture medium is 0-1000 IU / ml, 10-1000 IU / ml, 50-1000 IU / ml, 100-1000 IU / ml, 200-1000 IU / ml, 300-1000 IU / ml, 400-1000 IU / ml, 410-1000 IU / ml, 420-1000 IU / ml, 430-1000 IU / ml, 440-1000 IU / ml, 450-1000 IU / ml, 460-1000 IU / ml, 470-1000 IU / ml, 480 -1000IU / ml, 490-1000IU / ml, 500-1000IU / ml, 800-1000IU / ml, 10-800IU / ml, 50-800IU / ml, 100-800IU / ml, 200-800IU / ml, 300-800IU / m l, 400-800IU / ml, 410-800IU / ml, 420-800IU / ml, 430-800IU / ml, 440-800IU / ml, 450-800IU / ml, 460-800IU / ml, 470-800IU / ml, 480-800I U / ml, 490-800IU / ml, 500-800IU / ml, 10-700IU / ml, 50-700IU / ml, 100-700IU / ml, 200-700IU / ml, 300-700IU / ml, 400-700IU / ml, 410-700 IU / ml, 420-700IU / ml, 430-700IU / ml, 440-700IU / ml, 450-700IU / ml, 460-700IU / ml, 470-700IU / ml, 480-700IU / ml, 490-700IU / ml, 500- 700IU / ml, 10-600IU / ml, 50-600IU / ml, 100-600IU / ml, 100-600IU / ml, 200-600IU / ml, 300-600IU / ml, 400-600IU / ml, 410-600IU / ml, 420 -600IU / ml, 430-600IU / ml, 440-600IU / ml, 450-600IU / ml, 460-600IU / ml, 470-600IU / ml, 480-600IU / ml, 490-600IU / ml or 500-600IU / ml; And / or, the concentration of IL15 in the culture medium is 0-100 ng / ml, 10-100 ng / ml, 20-100 ng / ml, 30-100 ng / ml, 40-100 ng / ml, 50-100 ng / ml, 60-100 ng / ml, 70-100 ng / ml, 90-100 ng / ml, 10-90 ng / ml, 20-90 ng / ml, 30-90 ng / ml, 40-90 ng / ml, 50 -90ng / ml, 10-80ng / ml, 20-80ng / ml, 30-80ng / ml, 40-80ng / ml, 50-80ng / ml, 10-70ng / ml, 20-70ng / ml, 3 0-70ng / ml, 40-70ng / ml, 50-70ng / ml, 10-60ng / ml, 20-60ng / ml, 30-60ng / ml, 40-60ng / ml or 50-60ng / ml; And / or, the concentration of antiIFN-γ in the culture medium is 0-200 ng / ml, 50-200 ng / ml, 70-200 ng / ml, 80-200 ng / ml, 90-200 ng / ml, 100-200 ng / ml, 150-200 ng / ml, 50-150 ng / ml, 70-150 ng / ml, 80-150 ng / ml, 90-150 ng / ml, 100-150 ng / ml, 50-140 ng / ml, 70-140 ng / ml , 80-140ng / ml, 90-140ng / ml, 100-140ng / ml, 50-130ng / ml, 70-130ng / ml, 80-130ng / ml, 90-130ng / ml, 100-130ng / m l, 50-120ng / ml, 70-120ng / ml, 80-120ng / ml, 90-120ng / ml, 50-110ng / ml, 70-110ng / ml, 80-110ng / ml or 90-110ng / ml.

7. The culture medium according to any one of claims 1-6, comprising a basal culture medium for cell culture.

8. The culture medium according to claim 7, wherein the basal culture medium is a universal basal culture medium for cell culture; the universal basal culture medium is a serum-free culture medium or a serum-containing culture medium; Preferably, the serum-free culture medium is selected from OpTmizer™ CTS™, Immunocult™ XF, CellGro™, TexMacs™, Stemline™, Xvivo15™, PrimeXV, and ImmunoCult. TM X-VF medium, SCGM medium, X-VIVO series serum-free immune cell medium, X-VIV010 serum-free immune cell medium, X-VIV015 serum-free immune cell medium, X-VIVO 20 serum-free cell medium, Serum-free culture medium for T cells, serum-free culture medium for lymphocytes HIPP-T009, and serum-free culture medium for lymphocytes KBM581. L500 serum-free lymphocyte culture medium and StemXVivo, or any one or more of them; Preferably, the serum-containing culture medium is selected from any one or more of α-MEM medium, RPMI 1640 medium, AIM-V medium, DMEM medium, F-12 medium, X-vivo 15 medium, X-Vivo 20 medium, OpTmizer medium, and IMDM medium.

9. A method for obtaining mixed T cells, comprising culturing peripheral blood mononuclear cells (PBMCs), CD4-positive T cells, CD8-positive T cells, CD4 / CD8 double-negative T cells, or any combination thereof, using the culture medium described in any one of claims 1-8; wherein the mixed T cells comprise CD4 / CD8-positive mixed cells and / or CD4 / CD8 double-negative mixed cells; Preferably, the culture time is 2-25 days; More preferably, the culture time is 7-21 days.

10. The method according to claim 9, wherein the T cell is a natural T cell and / or a genetically modified T cell; Preferably, the genetically modified T cells contain the introduced exogenous gene.

11. The method of claim 10, wherein the genetically modified T cell comprises any one or more of the following: a gene segment encoding a chimeric antigen receptor CAR, a gene segment encoding a T cell receptor (TCR) that specifically recognizes tumor antigens, and a gene segment encoding a fusion protein of the T cell receptor; Preferably, the genetically modified T cells contain a gene segment encoding a chimeric antigen receptor CAR; Preferably, the gene-modified T cells are CAR-T cells.

12. The method according to claim 11, wherein the chimeric antigen receptor CAR comprises at least one extracellular antigen-binding domain, at least one transmembrane domain and at least one signal transduction domain; Preferably, the extracellular antigen-binding domain recognizes one or more target molecules expressed on the surface of tumor cells; preferably, the target molecules include CD19, CD20, CD22, CD33, CLL-1 (CLEC12A), CD7, CD5, CD70, CD123, CEA, CEACAM5, CEACAM6, CEACAM7, Mesothelin, MUC1, CLDN18.2, CDH17, Trop2, BCMA, NKG2D, PDL1, EGFR, EGFRVIII, PSCA, PSMA, MUC16, CD133, GD2, IL13R2, B7H3, Her2, CD30, SLAMF7, CD38, GPC3, WT1, AFP, FOLR1, c-Met, LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma-associated antigen 1), and TAG-72 are any one or more of these. More preferably, the target molecule is selected from any one or more of CD19, CD70, CEA and BCMA.

13. The method according to claim 12, wherein when the target molecule is CEA, the amino acid sequence of the chimeric antigen receptor CAR comprises the sequence shown in SEQ ID NO.1, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO.1; And / or, when the target molecule is CD19, the amino acid sequence of the chimeric antigen receptor CAR includes the sequence shown in SEQ ID NO.2, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO.2; And / or, when the target molecule is CD70, the amino acid sequence of the chimeric antigen receptor CAR includes the sequence shown in SEQ ID NO.3, or a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence shown in SEQ ID NO.

3.

14. The method according to any one of claims 9-13, further comprising the step of activating T cells prior to culture; Preferably, the step of activating T cells includes contacting the T cells with an activator; preferably, the activator comprises CD3 antibody and / or CD28 antibody; preferably, the activator comprising CD3 antibody and / or CD28 antibody is a magnetic bead or reagent conjugated with CD3 antibody and / or CD28 antibody, a virus, nanoparticle or vesicle loaded with CD3 antibody and / or CD28 antibody, or any combination thereof; Preferably, the activation time is 5 min-48 h, 5-60 min, 1-6 h, 1-12 h, 1-24 h, 1-36 h, 6-12 h, 6-24 h, 6-36 h, 6-48 h, 12-24 h, 12-36 h, 12-48 h, or 24-48 h.

15. A hybrid T cell, obtained by the method according to any one of claims 9-14.

16. A method for culturing universal T cells, comprising culturing universal T cells using the culture medium according to any one of claims 1-8; Preferably, the culture time is 2-25 or 7-21 days; Preferably, the universal T cells are CD4 and CD8 double-negative CAR-T cells; More preferably, the universal T cell is a universal CAR-T cell (U CAR-T cell).

17. A universal T cell culture, obtained by culturing using the method of claim 16.

18. Use of the hybrid T cells of claim 15 or the universal T cell culture of claim 17 for the preparation of medicaments for the prevention and / or treatment of diseases.

19. A pharmaceutical composition comprising the hybrid T cells of claim 15 and / or the universal T cell culture of claim 17.

20. A method of treating a disease, comprising administering to a subject in need an effective amount of the hybrid T cells of claim 15 and / or the universal T cell culture of claim 17, and / or its cryopreservation and resuscitation product.

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