NK cell culture medium, NK cell as well as culture method and application of NK cell

Abstract

The invention relates to the technical field of immune cell preparation, and particularly discloses an NK cell culture medium, an NK cell and a culture method and application of the NK cell. The NK cell culture medium comprises cell factors IL2, IL21 and TGF beta; the system further comprises IL4. The NK cell obtaining method comprises the step of contacting cells with the culture medium. The NK cells obtained by the invention have relatively high cell proliferation ability, and the proliferation ability, motility and in-vivo effectiveness of the cells after cryopreservation recovery are high.

Description

Technical Field

[0001] This application relates to the technical field of immune cell preparation, specifically to an NK cell culture medium, NK cells, their culture methods, and applications. Background Technology

[0002] NK cells, also known as natural killer cells, participate in bone marrow transplantation and graft-versus-host disease responses. They play a crucial role in immune regulation by secreting lymphokines such as interferon-gamma (IFN-γ) and interleukin-2 (IL-2), forming an important component of the body's immune surveillance system. With the increasing effectiveness of adoptive cell therapy (ACT) in cancer treatment, NK cell therapy has also gained attention. NK cell therapy involves activating or modifying isolated NK cells in vitro to enhance their cytotoxic activity before reinfusing them into the patient. NK cells can be obtained from umbilical cord blood, NK cell lines, peripheral blood, ES cell lines, and iPS cell lines. Among these, umbilical cord blood and peripheral blood, being primary human samples, are closer to human physiological characteristics, offering higher safety and better meeting clinical application expectations.

[0003] However, the proportion of NK cells derived from these two sources is limited, especially since NK cells in peripheral blood account for only about 5%-15% of white blood cells. Therefore, the culture protocol is crucial to meeting clinical needs. Currently, NK cell therapy products face the dual technical challenges of insufficient NK cell purity and expansion. Conventionally used NK cell culture cytokine combinations, such as IL-2 and IL-21, result in significantly reduced NK cell viability after cryopreservation, affecting NK cell function. Furthermore, NK cell function declines severely with prolonged in vitro culture. If the required number of NK cells for adoptive cell therapy is to be met, in vivo efficacy will significantly decrease; conversely, if the required number of therapeutic cells is to be met, the required number of therapeutic cells will be insufficient. Therefore, new NK cell preparation processes are essential. (Invention Content) This application provides an NK cell culture medium, NK cells, a culture method thereof, and an application thereof. The NK cells are cultured using a special combination of factors, and the resulting NK cells, after being frozen and thawed, show significantly improved cell viability and factor-free proliferation capacity. In addition, when the frozen and thawed NK cells are used in vivo, they have better amplification capacity and sustained drug efficacy, thereby achieving better therapeutic effects.

[0004] This application involves the following: 1. An NK cell culture medium comprising the cytokines IL2, IL21, and TGFβ; Preferably, it contains the cytokines IL4, IL2, IL21 and 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.

[0005] 2. The culture medium according to item 1, wherein the content of IL4 in the culture medium is 0 - 250 ng / ml, 0.5 - 250 ng / ml, 1 - 250 ng / ml, 2 - 250 ng / ml, 3 - 250 ng / ml, 4 - 250 ng / ml, 5 - 250 ng / ml, 6 - 250 ng / ml, 7 - 250 ng / ml, 8 - 250 ng / ml, 9 - 250 ng / ml, 10 - 250 ng / ml, 50 - 250 ng / ml, 200 - 250 ng / ml, 0 - 220 ng / ml, 0.5 - 220 ng / ml, 1 - 220 ng / ml, 2 - 220 ng / ml, 3 - 220 ng / ml, 4 - 220 ng / ml, 5 - 220 ng / ml, 6 - 220 ng / ml, 7 - 220 ng / ml, 8 - 220 ng / ml, 9 - 220 ng / ml, 10 - 220 ng / ml, 50 - 220 ng / ml, 200 - 220 ng / ml, 0 - 200 ng / ml, 0.5 - 200 ng / ml, 1 - 200 ng / ml, 2 - 200 ng / ml, 3 - 200 ng / ml, 4 - 200 ng / ml, 5 - 200 ng / ml, 6 - 200 ng / ml, 7 - 200 ng / ml, 8 - 200 ng / ml, 9 - 200 ng / ml, 10 - 200 ng / ml, 0 - 150 ng / ml, 0.5 - 150 ng / ml, 1 - 150 ng / ml, 2 - 150 ng / ml, 3 - 150 ng / ml, 4 - 150 ng / ml, 5 - 150 ng / ml, 6 - 150 ng / ml, 7 - 150 ng / ml, 8 - 150 ng / ml, 9 - 150 ng / ml, 10 - 150 ng / ml, 50 - 150 ng / ml, 0 - 100 ng / ml, 0.5 - 100 ng / ml, 1 - 100 ng / ml, 2 - 100 ng / ml, 3 - 100 ng / ml, 4 - 100 ng / ml, 5 - 100 ng / ml, 6 - 100 ng / ml, 7 - 100 ng / ml, 8 - 100 ng / ml, 9 - 100 ng / ml, 10 - 100 ng / ml, 50 - 100 ng / ml, 0 - 50 ng / ml, 0.5 - 50 ng / ml, 1 - 50 ng / ml, 2 - 50 ng / ml, 3 - 50 ng / ml, 4 - 50 ng / ml, 5 - 50 ng / ml, 6 - 50 ng / ml, 7 - 50 ng / ml, 8 - 50 ng / ml, 9 - 50 ng / ml, 10 - 50 ng / ml, 0 - 40 ng / ml, 0.5 - 40 ng / ml, 1 - 40 ng / ml, 2 - 40 ng / ml, 3 - 40 ng / ml, 4 - 40 ng / ml, 5 - 40 ng / ml, 6 - 40 ng / ml, 7 - 40 ng / ml, 8 - 40 ng / ml, 9 - 40 ng / ml, 10 - 40 ng / ml, 0 - 30 ng / ml, 0.5 - 30 ng / ml, 1 - 30 ng / ml, 2 - 30 ng / ml, 3 - 30 ng / ml, 4 - 30 ng / ml, 5 - 30 ng / ml, 6 - 30 ng / ml, 7 - 30 ng / ml, ⑧ - 30 ng / ml, 9 - 30 ng / ml, 10 - 30 ng / ml, 0 - 20 ng / ml, 0.5 - 20 ng / ml, 1 - 20 ng / ml, 2 - 20 ng / ml, 3 - 20 ng / ml, 4 - 20 ng / ml, 5 - 20 ng / ml, 6 - 20 ng / ml, 7 - 20 ng / ml, 8 - 20 ng / ml, 9 - 20 ng / ml, 10 - 20 ng / ml, 0 - 15 ng / ml, 0.5 - 15 ng / ml, 1 - 15 ng / ml, 2 - 15 ng / ml, 3 - 15 ng / ml, 4 - 15 ng / ml, 5 - 15 ng / ml, 6 - 15 ng / ml, 7 - 15 ng / ml, 8 - 15 ng / ml, 9 - 15 ng / ml, 10 - 15 ng / ml, 0 - 13 ng / ml, 0.5 - 13 ng / ml, 1 - 13 ng / ml, 2 - 13 ng / ml, 3 - 13 ng / ml, 4 - 13 ng / ml, 5 - 13 ng / ml, 6 - 13 ng / ml, 7 - 13 ng / ml, 8 - 13 ng / ml, 9 - 13 ng / ml or 10 - 13 ng / ml;. Preferably, the content of the IL4 in the medium is 1 - 100 ng / ml, 1 - 50 ng / ml, 1 - 40 ng / ml, 5 - 50 ng / ml, 5 - 10 ng / ml, 1 - 30 ng / ml, 1 - 20 ng / ml, 1 - 10 ng / ml or 1 - 5 ng / ml.

[0006] It should be noted that there is an "⑧" in the first line which seems to be an incorrect character. It is retained as it is in the translation according to the requirements.3. The culture medium according to item 1 or 2, wherein the content of TGFβ in the culture medium is 0.1 - 100 ng / ml, 0.1 - 100 ng / ml, 0.1 - 80 ng / ml, 0.1 - 60 ng / ml, 0.1 - 50 ng / ml, 0.1 - 40 ng / ml, 0.1 - 30 ng / ml, 0.1 - 20 ng / ml, 0.1 - 19 ng / ml, 0.1 - 18 ng / ml, 0.1 - 17 ng / ml, 0.1 - 16 ng / ml, 0.1 - 15 ng / ml, 0.1 - 14 ng / ml, 0.1 - 13 ng / ml, 0.1 - 12 ng / ml, 0.1 - 11 ng / ml, 0.1 - 10 ng / ml, 0.1 - 9 ng / ml, 0.1 - 8 ng / ml, 0.1 - 7 ng / ml, 0.1 - 6 ng / ml, 0.1 - 5 ng / ml, 0.1 - 4 ng / ml, 0.1 - 3 ng / ml, 0.1 - 2 ng / ml, 0.1 - 1 ng / ml, 0.3 - 100 ng / ml, 0.3 - 80 ng / ml, 0.3 - 60 ng / ml, 0.3 - 50 ng / ml, 0.3 - 40 ng / ml, 0.3 - 30 ng / ml, 0.3 - 20 ng / ml, 0.3 - 19 ng / ml, 0.3 - 18 ng / ml, 0.3 - 17 ng / ml, 0.3 - 16 ng / ml, 0.3 - 15 ng / ml, 0.3 - 14 ng / ml, 0.3 - 13 ng / ml, 0.3 - 12 ng / ml, 0.3 - 11 ng / ml, 0.3 - 10 ng / ml, 0.3 - 9 ng / ml, 0.3 - 8 ng / ml, 0.3 - 7 ng / ml, 0.3 - 6 ng / ml, 0.3 - 5 ng / ml, 0.3 - 4 ng / ml, 0.3 - 3 ng / ml, 0.3 - 2 ng / ml, 0.3 - 1 ng / ml, 0.5 - 100 ng / ml, 0.5 - 80 ng / ml, 0.5 - 60 ng / ml, 0.5 - 50 ng / ml, 0.5 - 40 ng / ml, 0.5 - 30 ng / ml, 0.5 - 20 ng / ml, 0.5 - 19 ng / ml, 0.5 - 18 ng / ml, 0.5 - 17 ng / ml, 0.5 - 16 ng / ml, 0.5 - 15 ng / ml, 0.5 - 14 ng / ml, 0.5 - 13 ng / ml, 0.5 - 12 ng / ml, 0.5 - 11 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, 0.7 - 100 ng / ml, 0.7 - 80 ng / ml, 0.7 - 60 ng / ml, 0.7 - 50 ng / ml, 0.7 - 40 ng / ml, 0.7 - 30 ng / ml, 0.7 - 20 ng / ml, 0.7 - 19 ng / ml, 0.7 - 18 ng / ml, 0.7 - 17 ng / ml, 0.7 - 16 ng / ml, 0.7 - 15 ng / ml, 0.7 - 14 ng / ml, 0.7 - 13 ng / ml, 0.7 - 12 ng / ml, 0.7 - 11 ng / ml, 0.7 - 10 ng / ml, 0.7 - 9 ng / ml, 0.7 - 8 ng / ml, 0.7 - 7 ng / ml, 0.7 - 6 ng / ml, 0.7 - 5 ng / ml, 0.7 - 4 ng / ml, 0.7 - 3 ng / ml, 0.7 - 2 ng / ml or 0.7 - 1 ng / ml;. Preferably, the content of TGFβ in the medium is 0.1 - 50 ng / ml, 0.1 - 30 ng / ml, 0.1 - 10 ng / ml, 0.1 - 5 ng / ml, 0.5 - 50 ng / ml, 0.5 - 40 ng / ml, 0.5 - 30 ng / ml, 0.5 - 20 ng / ml, 0.5 - 19 ng / ml, 0.5 - 18 ng / ml, 0.5 - 17 ng / ml, 0.5 - 16 ng / ml, 0.5 - 15 ng / ml, 0.5 - 14 ng / ml, 0.5 - 13 ng / ml, 0.5 - 12 ng / ml, 0.5 - 11 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 or 0.5 - 1 ng / ml.

[0007] 4. The culture medium according to any one of items 1-3, wherein the IL2 content in the culture medium is 50-5000 IU / ml, 50-4000 IU / ml, 50-3000 IU / ml, 50-2000 IU / ml, 50-1800 IU / ml, 50-1600 IU / ml, 50-1500 IU / ml, 50-1400 IU / ml, 50-1300 IU / ml, 50-1200 IU / ml, 50-1100 IU / ml, 50-1000 IU / ml, 50-900 IU / ml, 50-800 IU / ml, 50-700 IU / ml, 50-600 IU / ml, 50-500 IU / ml. 50-400IU / ml, 50-300IU / ml, 50-200IU / ml, 50-100IU / ml, 100-5000IU / ml, 100-4000IU / ml, 100-3000IU / ml, 100-2000IU / ml, 100-1800IU / ml, 100-16 00IU / ml, 100-1400IU / ml, 100-1300IU / ml, 100-1200IU / ml, 100-1100IU / m l, 100-1000IU / ml, 100-900IU / ml, 100-800IU / ml, 100-700IU / ml, 100-600I U / ml, 100-500IU / ml, 100-400IU / ml, 100-300IU / ml, 100-200IU / ml, 200-5000IU / ml, 200-4000IU / ml, 200-3000IU / ml, 200-2000IU / ml, 200-1800IU / ml, 200-1600IU / ml, 200-1500IU / ml, 200-1400IU / ml, 200-1300IU / ml, 200-1200IU / ml, 200-1100IU / ml, 200-1000IU / ml, 200-900IU / ml, 200-800IU / ml, 200-700IU / ml, 200-600IU / ml, 200-500IU / ml, 200-400IU / ml, 200-300IU / ml, 250-5000IU / ml, 250-4000IU / ml, 250-3000IU / ml, 250-2000IU / ml, 250-1800IU / ml, 250-1600IU / ml, 250-1500IU / ml, 250-1400IU / ml, 250-13 00IU / ml, 250-1200IU / ml, 250-1100IU / ml, 250-1000IU / ml, 250-900IU / ml,250-800IU / ml, 250-700IU / ml, 250-600IU / ml, 250-500IU / ml, 250-400IU / ml, 250 -300IU / ml, 300-5000IU / ml, 300-4000IU / ml, 300-3000IU / ml, 300-2000IU / ml, 30 0-1800IU / ml, 300-1600IU / ml, 300-1500IU / ml, 300-1400IU / ml, 300-1300IU / ml, 300-1200IU / ml, 300-1100IU / ml, 300-1000IU / ml, 300-900IU / ml, 300-800IU / ml, 300-700IU / ml, 300-600IU / ml, 300-500IU / ml, 300-400IU / ml, 400-5000IU / ml, 40 0-4000IU / ml, 400-3000IU / ml, 400-2000IU / ml, 400-1800IU / ml, 400-1600IU / ml, 400-1500IU / ml, 400-1400IU / ml, 400-1300IU / ml, 400-1200IU / ml, 400-1100IU / m l, 400-1000IU / ml, 400-900IU / ml, 400-800IU / ml, 400-700IU / ml or 400-600IU / ml;, Preferably, the IL2 content in the culture medium is 50-1000 IU / ml, 500-1000 IU / ml, 50-250 IU / ml, 50-500 IU / ml, 100-900 IU / ml, 200-800 IU / ml, 250-1000 IU / ml, 300-700 IU / ml, 300-600 IU / ml, or 400-600 IU / ml.

[0008] 5. The culture medium according to any one of items 1-4, wherein the content of IL21 in the culture medium is 1-1500 ng / ml, 1-1250 ng / ml, 1-1000 ng / ml, 1-750 ng / ml, 1-500 ng / ml, 1-400 ng / ml, 1-300 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, 1-15 ng / ml, 1-10 ng / ml, 1-8 ng / ml, 1-6 ng / ml, 1-5 ng / ml, 1-4 ng / ml, 1-3 ng / ml, 5-1500 ng / ml, 5-1250 ng / ml, 5-1000 ng / ml, 5-750 ng / ml, 5-500 ng / ml, 5-400 ng / ml, 5-300 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, 5-15 ng / ml, 5-10 ng / ml, 5-8 ng / ml, 5-6 ng / ml, 10-1500 ng / ml, 10-1250 ng / ml, 10-1000 ng / ml, 10-750 ng / ml, 10-500 ng / ml, 10-400 ng / ml, 10-300 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, 10-15 ng / ml, 15-1500 ng / ml, 15-1250 ng / ml, 15-100 ng / ml, 15-750 ng / ml, 15-500 ng / ml, 15-400 ng / ml, 15-300 ng / ml, 15-250 ng / ml, 15-200 ng / ml, 15-150 ng / ml, 15-100 ng / ml, 15-50 ng / ml, 15-40 ng / ml, 15-30 ng / ml, 15-29 ng / ml,15-28ng / ml, 15-27ng / ml, 15-26ng / ml, 15-25ng / ml, 15-20ng / ml, 20-1500ng / ml, 20-1250ng / ml, 20-1000ng / ml, 20-750ng / ml, 20-500ng / ml, 20-400n g / ml, 20-300ng / ml, 20-250ng / ml, 20-200ng / ml, 20-150ng / ml, 20-100ng / ml, 20-50ng / ml, 20-40ng / ml, 20-30ng / ml, 20-29ng / ml, 20-28ng / ml, 20-27n g / ml, 20-26ng / ml, 20-25ng / ml, 22-1500ng / ml, 22-1250ng / ml, 22-1000ng / ml, 22-750ng / ml, 22-500ng / ml, 22-400ng / ml, 22-300ng / ml, 22-250ng / ml , 22-200ng / ml, 22-150ng / ml, 22-100ng / ml, 22-50ng / ml, 22-40ng / ml, 22-30ng / ml, 22-29ng / ml, 22-28ng / ml, 22-27ng / ml, 22-26ng / ml or 22-25ng / ml;, Preferably, the IL21 content in the culture medium is 2.5-1250 ng / ml, 2.5-250 ng / ml, 2.5-25 ng / ml, 2.5-5 ng / ml, 5-900 ng / ml, 5-700 ng / ml, 5-250 ng / ml, 5-25 ng / ml, 10-400 ng / ml, 10-200 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, or 10-25 ng / ml.

[0009] 6. The culture medium according to any one of items 1-5 further comprises any one or more cytokines as shown below: 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), TNF-α; Preferably, the culture medium also contains IL5.

[0010] 7. The culture medium according to item 6, wherein the IL15 content in the culture medium is 1-100 ng / ml, 1-100 ng / ml, 1-80 ng / ml, 2-80 ng / ml, 1-70 ng / ml, 1-60 ng / ml, 1-50 ng / ml, 1-40 ng / ml, 1-30 ng / ml, 1-20 ng / ml, 1-18 ng / ml, 1-16 ng / ml, 1-15 ng / ml, 1-14 ng / ml, 1-13 ng / ml, 1-12 ng / ml, 1- 11ng / ml, 1-10ng / ml, 1-8ng / ml, 1-6ng / ml, 1-5ng / ml, 3-100ng / ml, 3-80ng / ml, 3-70ng / ml, 3-60ng / ml, 3-50ng / ml , 3-40ng / ml, 3-30ng / ml, 3-20ng / ml, 3-18ng / ml, 3-16ng / ml, 3-15ng / ml, 3-14ng / ml, 3-13ng / ml, 3-12ng / ml, 3-11n g / ml, 3-10ng / ml, 3-8ng / ml, 3-6ng / ml, 3-5ng / ml, 5-100ng / ml, 5-80ng / ml, 5-70ng / ml, 5-60ng / ml, 5-50ng / ml, 5- 40ng / ml, 5-30ng / ml, 5-20ng / ml, 5-18ng / ml, 5-16ng / ml, 5-15ng / ml, 5-14ng / ml, 5-13ng / ml, 5-12ng / ml, 5-11ng / m l, 5-10ng / ml, 5-8ng / ml, 5-6ng / ml, 8-100ng / ml, 8-80ng / ml, 8-70ng / ml, 8-60ng / ml, 8-50ng / ml, 8-40ng / ml, 8-30 ng / ml, 8-20ng / ml, 8-18ng / ml, 8-16ng / ml, 8-15ng / ml, 8-14ng / ml, 8-13ng / ml, 8-12ng / ml, 8-11ng / ml or 8-10ng / ml; Preferably, the IL15 content in the culture medium is 2-80 ng / ml, 5-50 ng / ml, or 5-20 ng / ml.

[0011] 8. The culture medium according to any one of items 1-7, comprising a basal culture medium for cell culture; The basal culture medium is a general basal culture medium used for cell culture; The general basal culture medium is either serum-free or serum-containing; Preferably, the serum-free culture medium is selected from any one or more of the following: OpTmizer™ CTS™, Immunocult™ XF, CellGro™, TexMacs™, Stemline™, Xvivo15™, PrimeXV, ImmunoCult™-XF medium, SCGM medium, X-VIVO series serum-free immune cell culture medium, X-VIV010 serum-free immune aOHQiS medium, X-VIV015 serum-free immune cell culture medium, X-VIVO 20 serum-free cell culture medium, OptiVitro® T cell serum-free culture medium, lymphocyte HIPP-T009 serum-free culture medium, lymphocyte serum-free culture medium KBM581, SuperCulture® L500 lymphocyte serum-free culture medium, as well as StemXVivo, CTS NK-Xpander medium, NK MACS Medium / NK cell culture medium, OptiVitro® NK cell expansion kit, DKW37-NKP series culture medium, and NK MACS® series culture medium. Preferably, the culture medium may also be 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.

[0012] 9. A method for obtaining NK cells, comprising culturing peripheral blood mononuclear cells (PBMCs), umbilical cord blood mononuclear cells (UCBs), NK cells, or any combination thereof using the culture medium described in any one of items 1-8; Preferably, the culture time is 7-30 days; More preferably, the culture time is 14-28 days.

[0013] 10. The method according to claim 9, wherein the NK cell is a natural NK cell and / or a genetically modified NK cell; Preferably, the genetically modified NK cells contain a gene fragment encoding a chimeric antigen receptor CAR; Preferably, the gene-modified NK cells are CAR-NK cells.

[0014] 11. According to the method of item 10, 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; 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 CD123 and CD5.

[0015] 12. According to the method of item 11, when the target molecule is CD123, the amino acid sequence of the chimeric antigen receptor CAR includes 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 CD5, 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.

[0016] 13. The method according to any one of items 9-12 further includes the step of activating NK cells prior to culture; Preferably, the step of activating NK cells includes contacting the NK cells with an activating agent; preferably, the activating agent is selected from any one or more of Herceptin and CD28; 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.

[0017] 14. An NK cell obtained by the method described in any one of items 9-13.

[0018] 15. Use of the NK cells described in item 14 for the preparation of drugs, kits, and the prevention and / or treatment of diseases.

[0019] 16. A pharmaceutical composition comprising the NK cells described in claim 14 and / or their cryopreserved and resuscitated products.

[0020] 17. An NK cell kit comprising the culture medium described in any one of items 1-8; Preferably, the NK cell kit further includes an activator; Preferably, the activator is selected from any one or more of Herceptin and CD28.

[0021] Invention Effects 1. The study in this application found that using the cytokine combination SuperNK-Cyto, consisting of IL2+IL21+IL-4+TGFβ1, in the culture medium of NK cells can promote the proliferation of NK cells, improve the survival rate of cells after cryopreservation and thawing, and enhance the in vivo anti-tumor function and survival of CAR-NK cells.

[0022] 2. Before cell culture, this application provides a novel NK cell culture protocol: cells are incubated with magnetic nanobeads loaded with CD56 antibody to obtain a CD56-positive NK cell subset, and NK cells are enriched; NK cells are activated with Herceptin (trastuzumab for injection) and CD28; cell gene modification is then performed; cells are cultured in a medium containing the above cytokines, and cells are collected.

[0023] 3. The research in this application found that, based on the combination of TGFβ1+IL2+IL21+IL4 cytokines, other cytokines, such as IL15, IL18, and IL12, can be further added to obtain cells with strong expansion capacity, high cell survival rate after cryopreservation and thawing, and strong in vivo anti-tumor function and survival ability of CAR-NK cells. Attached Figure Description

[0024] Figure 1 The in vitro proliferation verification of CAR-NK cells prepared using different culture protocols is shown.

[0025] Figure 2 The results show the survival rates of CAR-NK cells prepared using different culture protocols after cryopreservation and subsequent recovery.

[0026] Figure 3 The functional validation of CAR-NK cells prepared using different culture protocols is shown.

[0027] Figure 4 The in vivo sustainability of CAR-NK cells prepared using different culture protocols is demonstrated.

[0028] Figure 5 The in vitro proliferation validation of CD5 CAR-NK cells prepared using different culture protocols is shown.

[0029] Figure 6 The results show the survival rates of CD5 CAR-NK cells prepared using different culture protocols after cryopreservation and subsequent recovery.

[0030] Figure 7 The functional validation of CD5 CAR-NK cells prepared using different culture protocols is shown.

[0031] Figure 8 This paper demonstrates the in vitro proliferation validation of peripheral blood-derived NK cells prepared using different culture protocols.

[0032] Figure 9 The in vitro proliferation validation of umbilical cord blood-derived NK cells prepared using different culture protocols is shown.

[0033] Figure 10 This study demonstrates how IL15 promotes CAR-NK proliferation in SuperNK-Cyto factor combination cultures.

[0034] Figure 11 The in vitro proliferation validation of CD5 CAR-NK cells prepared with different concentration combinations of factors is shown; among them, Figure 11 A represents the fold increase of SuperCAR-NK and CAR-NK cells cultured up to day 14. Figure 11 B represents the multiplication rate of SuperCAR-NK and CAR-NK cells cultured for 28 days.

[0035] Figure 12 The in vitro proliferation validation of CD5 CAR-NK cells prepared with different basal media is shown; among them, Figure 12 A uses Medium-1 as the basal culture medium; Figure 12 B uses Medium-2 as the basal culture medium; Figure 12 C uses Medium-3 as the basal culture medium.

[0036] Figure 13 Proliferation of different variants of SuperNK-Cyto. Detailed Implementation

[0037] 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.

[0038] 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.

[0039] 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".

[0040] 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.

[0041] As used in this article, the term “approximately XY” has the same meaning as “approximately X to approximately Y”.

[0042] 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”.

[0043] 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).

[0044] 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.

[0045] 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.

[0046] 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 IL15, participates in regulating B cell proliferation, and synergistically promotes bone marrow progenitor cell proliferation and NK cell proliferation, differentiation, and cytotoxic activity with IL15. 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 in this application, referring to a cytokine belonging to the chemokine family. As used herein, the terms “TGF-β1”, “TGFβ1”, and “transforming growth factor β1” are used interchangeably in this application. TGF-β is a polypeptide member of the transforming growth factor β superfamily and possesses various cellular functions, including regulating cell growth, proliferation, differentiation, and apoptosis. As used herein, the terms “antiIFN-γ” and “interferon-γ antibody” are used interchangeably in this application.

[0047] PBMCs (Peripheral blood mononuclear cells, referred to as mononuclear cells in this example) are cells with a single nucleus in peripheral blood, including lymphocytes and monocytes. Leukocytes (white blood cells), abbreviated as WBCs, are colorless, spherical, nucleated, heterogeneous blood cells, including granulocytes, monocytes, and lymphocytes. Cell populations containing NK cells can be enriched NK cells, or leukocytes containing NK cells, PBMCs, or cell populations derived from umbilical cord blood; these cell populations can be fresh or cryopreserved.

[0048] 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.

[0049] 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.

[0050] The term "cell cryopreservation" refers to a technique that places cells in a "dormant" state at ultra-low temperatures, allowing them to be thawed when needed.

[0051] The term "cell resuscitation" refers to the process of thawing frozen cells.

[0052] 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.

[0053] 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, having similar functions, being within a similar size 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 an NK cell population.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] This application provides an NK cell culture medium containing cytokines IL2, IL21, and TGFβ. In some embodiments, the NK cell culture medium contains cytokines IL4, IL2, IL21, and TGFβ.

[0060] 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.

[0061] In some implementations, the IL4 concentration in the culture medium is 0-250 ng / ml or 1-250 ng / ml; in some embodiments, the IL4 concentration in the culture medium is 0-13 ng / ml, 0-15 ng / ml, 0-20 ng / ml, 0-30 ng / ml, 0-40 ng / ml, 0-50 ng / ml, 0-100 ng / ml, 0-150 ng / ml, 0-200 ng / ml, 0-220 ng / ml, 0-250 ng / ml, 0.5-13 ng / ml, 0.5-15 ng / ml, 0.5-20 ng / ml, 0.5-30 ng / ml, 0.5-40 ng / ml, 0.5-50 ng / ml. ml, 0.5-100ng / ml, 0.5-150ng / ml, 0.5-200ng / ml, 0.5-220ng / ml, 0.5-250ng / ml, 1-2ng / ml, 1-3ng / ml, 1-4ng / ml, 1-5ng / ml, 1-6ng / ml, 1-7ng / ml, 1-8 ng / ml, 1-9ng / ml, 1-10ng / ml, 1-13ng / ml, 1-15ng / ml, 1-20ng / ml, 1-30ng / ml, 1-40ng / ml, 1-50ng / ml, 1-60ng / ml, 1-70ng / ml, 1-80ng / ml, 1-90ng / ml, 1-100ng / ml, 1-150ng / ml, 1-200ng / ml, 1-220ng / 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, 2-13ng / ml, 2-15ng / ml, 2-20ng / ml, 2-30ng / ml, 2-40ng / ml, 2-50ng / ml, 2-70ng / ml, 2-80ng / ml, 2-100ng / ml, 2-150ng / ml, 2-200ng / ml, 2-220ng / m l, 2-250ng / ml, 3-5ng / ml, 3-10ng / ml, 3-13ng / ml, 3-15ng / ml, 3-20ng / ml, 3-30ng / ml, 3-40ng / ml, 3-50ng / ml, 3-100ng / ml, 3-150ng / ml, 3-200ng / ml, 3-220ng / ml, 3-250ng / ml, 4-10ng / ml, 4-13ng / ml, 4-15ng / ml, 4-20ng / ml, 4-30ng / ml, 4-40ng / ml, 4-50ng / ml, 4-100ng / ml, 4-150ng / ml, 4-200ng / ml,4-220ng / ml、4-250ng / ml、5-10ng / ml、5-13ng / ml、5-15ng / ml、5-20ng / ml、5-25ng / ml、5-30ng / ml、5-30ng / ml、5-40ng / ml、5-50ng / ml、5-100ng / ml、5-150ng / ml、5-200ng / ml、5-220ng / ml、5-250ng / ml、6-10ng / ml、6-13ng / ml、6-15ng / ml、6-20ng / ml、6-30ng / ml、6-40ng / ml、6-50ng / ml、6-100ng / ml、6-150ng / ml、6-200ng / ml、6-220ng / ml、6-250ng / ml、7-10ng / ml、7-13ng / ml、7-15ng / ml、7-20ng / ml、7-30ng / ml、7-40ng / ml、7-50ng / ml、7-100ng / ml、7-150ng / ml、7-200ng / ml、7-220ng / ml、7-250ng / ml、8-10ng / ml、8-13ng / ml、8-15ng / ml、8-20ng / ml、8-30ng / ml、8-40ng / ml、8-50ng / ml、8-100ng / ml、8-150ng / ml、8-200ng / ml、8-220ng / ml、8-250ng / ml、9-10ng / ml、9-13ng / ml 9-15ng / ml、9-20ng / ml、9-30ng / ml、9-40ng / ml、9-50ng / ml、9-100ng / ml、9-150ng / ml、9-200ng / ml、9-220ng / ml、9-250ng / ml、10-13ng / ml、10-15ng / ml、10-20ng / ml、10-30ng / ml、10-40ng / ml、10-50ng / ml、10-100ng / ml、10-150ng / ml、10-200ng / ml、10-220ng / ml、10-250ng / ml、15-100ng / ml、15-250ng / ml、20-100ng / ml、20-250ng / ml、30-100ng / ml、30-250ng / ml、40-80ng / ml、40-250ng / ml、50-100ng / ml、50-150ng / ml、50-220ng / ml、50-250ng / ml、60-250ng / ml、90-250ng / ml、200-220ng / ml、The concentration range is any within the range of 200-250 ng / ml or 1-250 ng / ml; in one specific embodiment, the concentration of IL4 in the culture medium is any within the range of 0 ng / ml, 0.5 ng / ml, 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, 16 ng / ml, 17 ng / ml, 18 ng / ml, 19 ng / ml, 20 ng / ml, 50 ng / ml, 100 ng / ml, 250 ng / ml or 1-250 ng / ml.

[0062] In some embodiments, the TGFβ concentration in the culture medium is 0.1-100 ng / ml; in other embodiments, the TGFβ concentration in the culture medium is 0.1-0.5 ng / ml, 0.1-1 ng / ml, 0.1-2 ng / ml, 0.1-3 ng / ml, 0.1-4 ng / ml, 0.1-5 ng / ml, 0.1-6 ng / ml, 0.1-7 ng / ml, 0.1-8 ng / ml, 0.1-9 ng / ml, 0.1-10 ng / ml, 0.1-11 ng / ml, 0.1-12 ng / ml, 0.1-13 ng / ml, 0.1-14 ng / ml, 0.1-15 ng / ml, 0.1-16 ng / ml. ng / ml, 0.1-17ng / ml, 0.1-18ng / ml, 0.1-19ng / ml, 0.1-20ng / ml, 0.1-30ng / ml, 0.1-40ng / ml, 0.1-50ng / ml, 0.1-60ng / ml, 0.1-70ng / ml, 0.1-80ng / ml , 0.1-100ng / ml, 0.3-0.5ng / ml, 0.3-1ng / ml, 0.3-2ng / ml, 0.3-3ng / ml, 0.3-4ng / ml, 0.3-5ng / ml, 0.3-6ng / ml, 0.3-7ng / ml, 0.3-8ng / ml, 0.3-9ng / ml , 0.3-10ng / ml, 0.3-11ng / ml, 0.3-12ng / ml, 0.3-13ng / ml, 0.3-14ng / ml, 0.3-15ng / ml, 0.3-16ng / ml, 0.3-17ng / ml, 0.3-18ng / ml, 0.3-19ng / ml, 0.3- 20ng / ml, 0.3-30ng / ml, 0.3-40ng / ml, 0.3-50ng / ml, 0.3-60ng / ml, 0.3-70ng / ml, 0.3-80ng / ml, 0.3-100ng / ml, 0.5-1ng / ml, 0.5-2ng / ml, 0.5-3ng / ml , 0.5-4ng / ml, 0.5-5ng / ml, 0.5-6ng / ml, 0.5-7ng / ml, 0.5-8ng / ml, 0.5-10ng / ml, 0.5-11ng / ml, 0.5-12ng / ml, 0.5-13ng / ml, 0.5-14ng / ml, 0.5-15ng / ml, 0.5-16ng / ml, 0.5-17ng / ml, 0.5-18ng / ml, 0.5-19ng / ml, 0.5-20ng / 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, 0.7-1ng / ml, 0.7-2ng / ml, 0.7-3ng / ml, 0.7-4ng / ml, 0.7-5ng / ml, 0.7-6ng / ml, 0.7-7ng / ml, 0.7-8ng / ml, 0.7-9ng / ml, 0.7-10ng / ml, 0.7-11ng / ml, 0.7-12ng / ml, 0.7-13ng / ml, 0.7-14ng / ml, 0.7-15ng / ml, 0.7-16ng / ml, 0.7-17ng / ml, 0.7-18ng / ml, 0.7- 19ng / ml, 0.7-20ng / ml, 0.7-30ng / ml, 0.7-40ng / ml, 0.7-50ng / ml, 0.7-60ng / ml, 0.7-70ng / ml, 0.7-80ng / ml, 0.7-100ng / 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, 1-50ng / ml, 1-100ng / ml, 10-100ng / ml, 1.5-100ng / ml, 2-100ng / ml, 2.5 -100ng / ml, 3-100ng / ml, 4-100ng / ml, 5-100ng / ml, 8-100ng / ml, 10-100ng / ml, 20-100ng / ml, 25-100ng / ml, 30-100ng / ml, 40-100ng / ml, 50-100ng / ml, 60-100ng / ml, 70-100ng / ml, 90-100ng / ml, 2-50ng / ml, 5-50ng / ml, 1-90ng / ml, 2-90ng / ml, 1-80ng / ml, 2-80ng / ml, 1-70ng / ml, 2-70ng / ml1-60n The concentration range is any one of the following: g / ml, 1-40 ng / ml, 1-30 ng / ml, 1-20 ng / ml, 1-15 ng / ml, or 1-25 ng / ml. In one specific embodiment, the concentration of TGFβ in the culture medium is any one of the following: 0.1 ng / ml, 0.5 ng / ml, 1 ng / ml, 1.5 ng / ml, 2 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, or any concentration within the range of 0.1-100 ng / ml.

[0063] In some embodiments, the concentration of IL21 in the culture medium is 1-1500 ng / ml; in other embodiments, the concentration of IL21 in the culture medium is 1-1500 ng / ml, 1-1250 ng / ml, 1-1000 ng / ml, 1-750 ng / ml, 1-500 ng / ml, 1-400 ng / ml, 1-300 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. g / ml, 1-25ng / ml, 1-20ng / ml, 1-15ng / ml, 1-10ng / ml, 1-8ng / ml, 1-6ng / ml, 1-5ng / ml, 1-4ng / ml, 1-3ng / ml, 5-1500ng / ml, 5-1250ng / ml, 5-1000ng / m l, 5-750ng / ml, 5-500ng / ml, 5-400ng / ml, 5-300ng / ml, 5-250ng / ml, 5-200ng / ml, 5-150ng / ml, 5-100ng / ml, 5-80ng / ml, 5-50ng / ml, 5-40ng / ml, 5-30 ng / ml, 5-29ng / ml, 5-28ng / ml, 5-27ng / ml, 5-26ng / ml, 5-25ng / ml, 5-20ng / ml, 5-15ng / ml, 5-10ng / ml, 5-8ng / ml, 5-6ng / ml, 10-1500ng / ml, 10-125 0ng / ml, 10-1000ng / ml, 10-800ng / ml, 10-750ng / ml, 10-500ng / ml, 10-400ng / ml, 10-300ng / ml, 10-250ng / ml, 10-200ng / ml, 10-150ng / ml, 10-100ng / ml, 10-80ng / ml, 10-50ng / ml, 10-40ng / ml, 10-30ng / ml, 10-29ng / ml, 10-28ng / ml, 10-27ng / ml, 10-26ng / ml, 10-25ng / ml, 10-20ng / ml, 10-15ng / ml , 15-1500ng / ml, 15-1250ng / ml, 15-1000ng / ml, 15-750ng / ml, 15-500ng / ml, 15-400ng / ml, 15-300ng / ml, 15-250ng / ml, 15-200ng / ml, 15-150ng / ml,15-100ng / ml, 15-50ng / ml, 15-40ng / ml, 15-30ng / ml, 15-29ng / ml, 15-28ng / ml, 15-27ng / ml, 15-26ng / ml, 15-25ng / ml, 15-20ng / ml, 20-1500ng / ml, 20-1250ng / ml, 20-1000ng / ml, 20-750ng / ml, 20-500ng / ml, 20-400ng / ml, 20-300ng / ml, 20-2 50ng / ml, 20-200ng / ml, 20-150ng / ml, 20-100ng / ml, 20-50ng / ml, 20-40ng / ml, 20-30ng / ml, 20-29ng / ml, 20-28ng / ml, 20-27ng / ml, 20-2 6ng / ml, 20-25ng / ml, 22-1500ng / ml, 22-1250ng / ml, 22-1000ng / ml, 22-750ng / ml, 22-500ng / ml, 22-400ng / ml, 22-300ng / ml, 22-250ng / ml, 22-200ng / ml, 22-150ng / ml, 22-100ng / ml, 22-50ng / ml, 22-40ng / ml, 22-30ng / ml, 22-29ng / ml, 22-28ng / ml, 22-27ng / ml, 22-26ng / ml, 22-25ng / ml, 25-30ng / ml, 25-40ng / ml, 25-50ng / ml, 25-80ng / ml, 25-100ng / ml, 25-250ng / ml, 25-1250ng / ml, 30-100ng / ml, 30-250 ng / ml, 30-1250 / ml, 40-80ng / ml, 50-100ng / ml, 50-250ng / ml, 50-1250ng / ml, 50-70ng / ml, 70-90ng / ml, 80-100ng / ml, 80-1250ng / ml, 90 The concentrations are within any range of -100 ng / ml, 90-1250 ng / ml, 100-900 ng / ml, 250-1250 ng / ml, or 5-1000 ng / ml; in one specific embodiment, the concentration of IL21 in the culture medium is 1 ng / ml, 2 ng / ml, 2.5 ng / ml, 3 ng / ml, 4 ng / ml, 5 ng / ml, 8 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 21 ng / ml, 22 ng / ml, 23 ng / ml, 24 ng / ml, 25 ng / ml, 26 ng / ml, 27 ng / ml.Any content within the range of 28 ng / ml, 29 ng / ml, 30 ng / ml, 45 ng / ml, 50 ng / ml, 55 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, 100 ng / ml, 150 ng / ml, 200 ng / ml, 210 ng / ml, 220 ng / ml, 230 ng / ml, 240 ng / ml, 250 ng / ml, 260 ng / ml, 270 ng / ml, 280 ng / ml, 290 ng / ml, 300 ng / ml, 400 ng / ml, 450 ng / ml, 480 ng / ml, 500 ng / ml, 530 ng / ml, 550 ng / ml, 580 ng / ml, 600 ng / ml, 700 ng / ml, 800 ng / ml, 900 ng / ml, 1000 ng / ml, 1100 ng / ml, 1150 ng / ml, 1200 ng / ml, 1250 ng / ml, 1300 ng / ml, 1400 ng / ml, 1500 ng / ml or any content within the range of 1 - 1500 ng / ml.

[0064] In some embodiments, the IL2 content in the culture medium is 50-5000 IU / ml; in some embodiments, the IL2 content in the composition is 50-5000 IU / ml, 50-4000 IU / ml, 50-3000 IU / ml, 50-2000 IU / ml, 50-1800 IU / ml, 50-1600 IU / ml, 50-1500 IU / ml, 50-1400 IU / ml, 50-1300 IU / ml, 50-1200 IU / ml, 50-1100 IU / ml, 50-1000 IU / ml, 50-900 IU / ml, 50-800 IU / ml, or 50-700 IU / ml. , 50-600IU / ml, 50-500IU / ml, 50-400IU / ml, 50-300IU / ml, 50-200IU / ml, 50-100IU / ml, 100-5000IU / ml, 100-4000IU / ml, 100-3000IU / ml, 100-2000I U / ml, 100-1800IU / ml, 100-1600IU / ml, 100-1400IU / ml, 100-1300IU / ml, 100-1200IU / ml, 100-1100IU / ml, 100-1000IU / ml, 100-900IU / ml, 100-800I U / ml, 100-700IU / ml, 100-600IU / ml, 100-500IU / ml, 100-400IU / ml, 100-300IU / ml, 100-200IU / ml, 200-5000IU / ml, 200-4000IU / ml, 200-3000IU / ml , 200-2000IU / ml, 200-1800IU / ml, 200-1600IU / ml, 200-1500IU / ml, 200-1 400IU / ml, 200-1300IU / ml, 200-1200IU / ml, 200-1100IU / ml, 200-1000IU / ml, 200-900IU / ml, 200-800IU / ml, 200-700IU / ml, 200-600IU / ml, 200-500IU / ml, 200-400IU / ml, 200-300IU / ml, 250-5000IU / ml, 250-4000IU / ml, 25 0-3000IU / ml, 250-2000IU / ml, 250-1800IU / ml, 250-1600IU / ml, 250-1500IU / ml, 250-1400IU / ml, 250-1300IU / ml, 250-1200IU / ml, 250-1100IU / ml,250-1000IU / ml, 250-900IU / ml, 250-800IU / ml, 250-700IU / ml, 250-600IU / ml, 250-500IU / ml, 250-400IU / ml, 250-300IU / ml, 300-5000IU / ml, 300-4 000IU / ml, 300-3000IU / ml, 300-2000IU / ml, 300-1800IU / ml, 300-1600IU / ml, 300-1500IU / ml, 300-1400IU / ml, 300-1300IU / ml, 300-1200IU / ml, 300- 1100IU / ml, 300-1000IU / ml, 300-900IU / ml, 300-800IU / ml, 300-700IU / ml, 300-600IU / ml, 300-500IU / ml, 300-400IU / ml, 400-5000IU / ml, 400-4000 IU / ml, 400-3000IU / ml, 400-2000IU / ml, 400-1800IU / ml, 400-1600IU / ml, 400-1500IU / ml, 400-1400IU / ml, 400-1300IU / ml, 400-1200IU / ml, 400-110 The concentration range is any of the following: 0 IU / ml, 400-1000 IU / ml, 400-900 IU / ml, 400-800 IU / ml, 400-700 IU / ml, 400-600 IU / ml, 500-1000 IU / ml, 800-1000 IU / ml, 10-100 IU / ml, 500-700 IU / ml, 700-900 IU / ml, 800-1000 IU / ml, or 50-5000 IU / ml; in one specific embodiment, the IL2 concentration in the culture medium is 50 IU / ml, 100 IU / ml, 200 IU / ml, 210 IU / ml, 220 IU / ml, or 230 IU / ml. / ml, 240IU / ml, 250IU / ml, 260IU / ml, 270IU / ml, 280IU / ml, 290IU / ml, 300IU / ml, 320IU / ml, 350IU / ml, 340IU / ml, 360IU / ml, 400IU / ml, 450IU / ml, 480I U / ml, 500IU / ml, 520IU / ml, 550IU / ml, 580IU / ml, 600IU / ml, 620IU / ml, 650IU / ml, 750IU / ml, 800IU / ml, 900IU / ml, 1000IU / ml, 1500IU / ml, 2000IU / ml,Any concentration within the range of 2500 IU / ml, 3000 IU / ml, 3500 IU / ml, 4000 IU / ml, 4500 IU / ml, 5000 IU / ml, or 50-5000 IU / ml.

[0065] In one specific embodiment, the cytokines are 500 IU / mL IL2, 25 ng / mL IL21, 10 ng / mL IL4, and 1 ng / mL TGFβ1.

[0066] In some embodiments, the culture medium further includes any one or more of the following cytokines: 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, IL-37, anti-IFNγ (interferon-gamma antibody), TNFα.

[0067] In some embodiments, the culture medium also includes IL15.

[0068] In some implementations, the concentration of IL15 in the culture medium is 0-100 ng / ml;In some embodiments, the content of IL15 in the medium is in any content range within the range of 1 - 100 ng / ml, 1 - 80 ng / ml, 2 - 80 ng / ml, 1 - 70 ng / ml, 1 - 60 ng / ml, 1 - 50 ng / ml, 1 - 40 ng / ml, 1 - 30 ng / ml, 1 - 20 ng / ml, 1 - 18 ng / ml, 1 - 16 ng / ml, 1 - 15 ng / ml, 1 - 14 ng / ml, 1 - 13 ng / ml, 1 - 12 ng / ml, 1 - 11 ng / ml, 1 - 10 ng / ml, 1 - 8 ng / ml, 1 - 6 ng / ml, 1 - 5 ng / ml, 3 - 100 ng / ml, 3 - 80 ng / ml, 3 - 70 ng / ml, 3 - 60 ng / ml, 3 - 50 ng / ml, 3 - 40 ng / ml, 3 - 30 ng / ml, 3 - 20 ng / ml, 3 - 18 ng / ml, 3 - 16 ng / ml, 3 - 15 ng / ml, 3 - 14 ng / ml, 3 - 13 ng / ml, 3 - 12 ng / ml, 3 - 11 ng / ml, 3 - 10 ng / ml, 3 - 8 ng / ml, 3 - 6 ng / ml, 3 - 5 ng / ml, 5 - 100 ng / ml, 5 - 80 ng / ml, 5 - 70 ng / ml, 5 - 60 ng / ml, 5 - 50 ng / ml, 5 - 40 ng / ml, 5 - 30 ng / ml, 5 - 20 ng / ml, 5 - 18 ng / ml, 5 - 16 ng / ml, 5 - 15 ng / ml, 5 - 14 ng / ml, 5 - 13 ng / ml, 5 - 12 ng / ml, 5 - 11 ng / ml, 5 - 10 ng / ml, 5 - 8 ng / ml, 5 - 6 ng / ml, 8 - 100 ng / ml, 8 - 80 ng / ml, 8 - 70 ng / ml, 8 - 60 ng / ml, 8 - 50 ng / ml, 8 - 40 ng / ml, 8 - 30 ng / ml, 8 - 20 ng / ml, 8 - 18 ng / ml, 8 - 16 ng / ml, 8 - 15 ng / ml, 8 - 14 ng / ml, 8 - 13 ng / ml, 8 - 12 ng / ml, 8 - 11 ng / ml, 8 - 10 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, 20 - 80 ng / ml, 20 - 70 ng / ml, 30 - 80 ng / ml, 30 - 70 ng / ml, 40 - 60 ng / ml, 25 - 100 ng / ml, 15 - 25 ng / ml, 25 - 40 ng / ml, 60 - 80 ng / ml, 80 - 100 ng / ml or 0 - 100 ng / ml;In one specific embodiment, the IL15 concentration in the culture medium is any concentration within the range of 0 ng / ml, 1 ng / ml, 2 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, 12 ng / ml, 15 ng / ml, 18 ng / ml, 20 ng / ml, 22 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 0-100 ng / ml.

[0069] 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, 80-200 ng / ml, 100-200 ng / ml, 150-200 ng / ml, 50-150 ng / ml, 70-140 ng / ml, 80-130 ng / ml, 90-120 ng / ml, 5-150 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. The concentration range is 140-160 ng / ml, 160-180 ng / ml, 180-200 ng / ml, or 0-200 ng / ml; in one specific embodiment, the concentration of antiIFN-γ in the 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.

[0070] In one specific embodiment, the culture medium includes any one of the following combinations: A combination of 500 IU / ml IL2, 1 ng / ml TGFβ1, 25 ng / ml IL21 and 10 ng / ml IL4; A combination of 500 IU / ml IL2, 10 ng / ml IL4, 1 ng / ml TGFβ1, 25 ng / ml IL21 and 100 ng / ml antiIFN-γ; A combination of 100 ng / ml IL21, 100 ng / ml IL4, 10 ng / ml TGFβ1, 500 IU / ml IL2 and 100 ng / ml anti IFN-γ; A combination of 50 IU / ml IL2, 50 ng / ml TGFβ1, 5 ng / ml IL21 and 250 ng / ml IL4; A combination of 1000 IU / ml IL2, 0.1 ng / ml TGFβ1, 1000 ng / ml IL21, and 1 ng / ml IL4; A combination of 400 IU / ml IL2, 0.5 ng / ml TGFβ1, 100 ng / ml IL21, and 1 ng / ml IL4; A combination of 600 IU / ml IL2, 5 ng / ml TGFβ1, 10 ng / ml IL21 and 20 ng / ml IL4; A combination of 500 IU / ml IL2, 1 ng / ml TGFβ1, 25 ng / ml IL21, 10 ng / ml IL4 and 5 ng / ml IL15; A combination of 500 IU / ml IL2, 1 ng / ml TGFβ1, 25 ng / ml IL21, 10 ng / ml IL4 and 15 ng / ml IL15; A combination of 500 IU / ml IL2, 1 ng / ml TGFβ1, 25 ng / ml IL21, 10 ng / ml IL4 and 20 ng / ml IL15; A combination of 50 IU / ml IL2, 0.5 ng / ml TGFβ1, 2.5 ng / ml IL21, and 1 ng / ml IL4; A combination of 250 IU / ml IL2, 5 ng / ml TGFβ1, 5 ng / ml IL21 and 5 ng / ml IL4; A combination of 1000 IU / ml IL2, 10 ng / ml TGFβ1, 250 ng / ml IL21 and 50 ng / ml IL4; A combination of 5000 IU / ml IL2, 100 ng / ml TGFβ1, 1250 ng / ml IL21 and 200 ng / ml IL4; A combination of 500 IU / ml IL2, 1 ng / ml TGFβ1, and 25 ng / ml IL21; Alternatively, a combination of 500 IU / ml IL2, 1 ng / ml TGFβ1, 25 ng / ml IL21, 10 ng / ml IL4, and 50 ng / ml IL15.

[0071] 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.

[0072] "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.

[0073] 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.

[0074] 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 culture 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 culture medium is a medium suitable for immune cell culture; in some embodiments, the medium suitable for immune cell culture is selected from any one or more of the following: Minimal Essential Media, α-MEM, RPMI 1640, AIM-V, DMEM, F-12, X-vivo 15 (Lonza), X-Vivo 20, OpTmizer, and IMDM or Gibco Cell Therapy Systems (CTS) family of products.In some embodiments, the basal culture medium may also be any one or more selected from STEMCELL's ImmunoCult™-XF, CellGenix's SCGM medium, Lonza's X-VIVO series of serum-free immune cell culture media (such as X-VIV010 serum-free immune aOHQiS medium, X-VIV015 serum-free immune cell culture medium, X-VIVO 20 serum-free cell culture medium), Thermo Fisher Scientific's CTS NK-Xpander medium, Miltenyi's NK MACS Medium / NK cell culture medium, ExCell Bio's OptiVitro® NK cell expansion basal kit, Dakowei's DKW37-NKP series of culture media, and Miltenyi's NK MACS® series of culture media.

[0075] In some implementations, the universal basal culture medium is prepared in advance. 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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).

[0083] This application provides a method for obtaining NK cells, including culturing peripheral blood mononuclear cells (PBMCs), umbilical cord blood mononuclear cells (UCBs), NK cells, or any combination thereof using the culture medium.

[0084] The culture medium for culturing NK cells is as described above and will not be repeated here.

[0085] In some embodiments, the NK cells are natural NK cells and / or genetically modified NK cells.

[0086] The term "genetically modified NK cells" in this application means NK cells that express exogenous or endogenous genes and are suitable for use in adoptive cell therapy. In some embodiments, the NK cells are natural NK cells and / or modified NK cells; in some embodiments, the modification is selected from any one or more of chemical, physical, and biological modifications. In some embodiments, the gene-modified NK cells include NK cells transduced with a target gene, such as CAR-NK cells (NK cells modified with chimeric antigen receptor CAR) containing a gene fragment encoding a chimeric antigen receptor CAR, enhanced NK cells expressing CD16, NK cells expressing the IL15 / IL15R fusion protein, and other gene-modified NK cells used in adoptive cell therapy.

[0087] 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.

[0088] The terms "extracellular antigen-binding domain" and "extracellular antigen-binding region" are used interchangeably in this application and refer 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.

[0089] 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.

[0090] In some implementations, the target molecule is selected from any one or more of the following: CD123, CD5.

[0091] In some embodiments, when the target molecule is CD123, 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 one 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.

[0092] In some embodiments, when the target molecule is CD5, 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.

[0093] In some embodiments, the CAR-NK cells are CD123 CAR-NK cells, CD5 CAR-NK cells, or any combination of the above-listed cells. In some embodiments, gene transduction of activated NK cells with a vector containing the CAR structural gene or target gene, such as a plasmid, lentivirus, or a VLP vector with a modified viral envelope (such as the vector described in patent 202310887156.5), can yield gene-modified NK cells.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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), scFv, Leucine ZipFv linked to antibody, Streptavidin 2 (mSA2) biotin-binding domain, VIPER CAR inducible structure, and biotin-biding immune receptor (BBIR) systems and logic gate regulatory systems bound to SynNotch receptors.

[0098] In some implementations, the method further includes the step of activating NK cells before culture.

[0099] It should be noted that, in this application, there are no further restrictions on the order of cell activation and cell culture. In some embodiments, cell activation and cell culture are performed simultaneously; in other embodiments, cell culture is started after cell activation.

[0100] As explained above, the NK cells can be natural or modified. In some embodiments, a step of modifying the NK cells is also included. In this application, cell activation and cell modification can be performed simultaneously or separately. For example, in some embodiments, cell activation is performed before cell modification; in other embodiments, cell activation and cell modification are performed simultaneously. When 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 cell activation and cell modification are performed simultaneously, the total activation and modification time is 24-48 hours. In some embodiments, the total activation and modification time is any time range within 28-44 hours, 32-40 hours, 30-40 hours, 35-48 hours, and 24-48 hours. In a specific embodiment, the total activation and modification time is any time range within 24 hours, 28 hours, 30 hours, 36 hours, 40 hours, 48 ​​hours, and 24-48 hours.

[0101] In some embodiments, the step of activating NK cells includes contacting the NK cells with an activating agent. In some embodiments, the activating agent is selected from any one or more of Herceptin and CD28.

[0102] In some implementations, methods for obtaining NK cells include: Cell enrichment and activation: NK cells were treated with an activator and then cultured in the culture medium described above. Gene modification: Transfecting / transducing NK cells with CAR structural genes or target genes to obtain gene-modified NK cells; Continue culturing: The gene-modified NK cells are cultured on the culture medium.

[0103] In some embodiments, the NK cells are derived from peripheral blood mononuclear cells (PBMCs), umbilical cord blood mononuclear cells (UCBs), NK cells, or any combination thereof. In some embodiments, the method for obtaining NK cells includes: contacting peripheral blood mononuclear cells (PBMCs) or umbilical cord blood mononuclear cells (UCBs) with a CD56 antibody to obtain a subset of NK cells expressing CD56 positivity, and enriching the NK cells.

[0104] In some implementations, the culture time during continued culture is 7-30 days; in some implementations, the culture time is any time range within the range of 7-30 days, 7-28 days, 14-28 days, 18-25 days, or 7-30 days; in one embodiment, the culture time is any time range within the range of 7 days, 10 days, 12 days, 14 days, 18 days, 21 days, 24 days, 28 days, 30 days, or 7-30 days.

[0105] In some implementations, the cryopreservation method includes the step of subjecting the NK / CAR-NK cells to dormancy in an ultra-low temperature environment.

[0106] 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.

[0107] 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.

[0108] 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, OptiVitro®, Gibco Recovery, and NutriFreez®.

[0109] 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.

[0110] 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.

[0111] In some embodiments, the volume percentages of the components in the cryopreservation solution are as follows: 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.

[0112] 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.

[0113] In some implementations, the resuscitation method includes the following steps: thawing the frozen immune cells at a temperature above -20°C.

[0114] 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.

[0115] This application provides an NK cell obtained by culturing using the method described above.

[0116] This application provides the above-mentioned uses of NK cells for the preparation of drugs and the prevention and / or treatment of diseases.

[0117] This application provides a pharmaceutical composition comprising the above-mentioned NK cells and / or their cryopreserved and revived products.

[0118] This application provides an NK cell kit comprising the aforementioned culture medium. In some embodiments, it further comprises an activator; in some embodiments, the activator is Herceptin; in some embodiments, the activator is CD28; in some embodiments, the activator is both Herceptin and CD28.

[0119] This application provides a treatment method for a disease, comprising administering to a subject in need an effective amount of the above-mentioned NK cells and / or their cryopreserved and thawed products, and / or the above-mentioned pharmaceutical composition.

[0120] Example 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.

[0121] Example Example 1. Factor combinations of TGFβ1 can promote the fold increase of CAR-NK. 1. Prepare a coating solution containing 2.5 mg / ml Herceptin (Roche) and 1 μg / ml CD28 (MACS, 170-076-117), and take 0.5~1 ml to evenly spread on the bottom of the wells of a 6-well plate to coat the culture plate (2h~24h).

[0122] 2. Preparation of NK cells: Blood samples or PBMCs obtained from them are incubated with CD56 antibody-loaded magnetic nanobeads (CliniMACS® CD56 Reagent, 200-070-128). The CD56 nanobeads bind to the CD56 antigen on the surface of NK cells, and the CD56-positive NK cell subset is adsorbed by a magnetic sorting column, enriching the NK cells to obtain the prepared NK cells. These NK cells can be cryopreserved using the cryopreservation solution described in this application.

[0123] 3. Activation Culture: The NK cells recruited in step 2, or the NK cells thawed after cryopreservation, were resuspended in medium supplemented with IL-2 and IL-21 (named TraNK-Cyto) (OptiVitro® NK, NE000-N032) or medium supplemented with a combination of IL-2, IL-21, IL-4, and TGF-β1 cytokines (named SuperNK-Cyto) (OptiVitro® NK, NE000-N032) at a concentration of 1×10⁶ cells per well. 6 The cells were seeded onto the 6-well culture plates coated in step 1, with a total cell suspension volume of 2 ml per well. After activation and culture for 12-24 hours, the cells were ready for use.

[0124] 4. After activating and culturing NK cells for 12-24 hours in step 3, co-incubate lentivirus (HIV-1, human immunodeficiency virus type I) containing the CD123 CAR structural gene vector with the activated NK cells for 12-24 hours to obtain genetically modified CD123 CAR-NK cells. The obtained CD123 CAR-NK cells are further cultured for 28 days in a medium (OptiVitro®NK, NE000-N032) supplemented with IL-2 and IL-21 (TraNK-Cyto) or a combination of IL-2, IL-21, IL-4, and TGF-β1 cytokines (named SuperNK-Cyto). The cells are then harvested and cryopreserved for subsequent experiments.

[0125] The CD123 CAR structure is: CD123scFv-8h-8TM-BBz; its amino acid sequence is shown in SEQ ID NO:1.

[0126] The cytokine information and dosage involved in this embodiment are as follows: IL-2: 500 IU / ml (Human interleukin-2 for injection, Shandong Quanzhou Port Pharmaceutical Co., Ltd.); IL-21: 25ng / ml (Recombinant Human IL-21, AF-200-21); IL-4: 10ng / ml (Recombinant Human IL-4, GMP-101-04); TGF-β1: 1ng / ml (Recombinant Human TGF-β1, 105-49).

[0127] Following the above preparation protocol, cells cultured using the IL-2 and IL-21 cytokine combination (TraNK-Cyto) were directly labeled with CAR-NK, while cells cultured using the IL-2, IL-21, IL-4, and TGF-β1 cytokine combination (SuperNK-Cyto) were labeled with SuperCAR-NK. Both CAR-NK and SuperCAR-NK are NK cells expressing CAR structures targeting CD123.

[0128] During cell culture, CD123 CAR-NK cells (CAR-NK) and CD123 SuperCAR-NK cells (SuperCAR-NK) were counted separately, and the results are as follows: Figure 1As shown in the results, CD123 SuperCAR-NK cells (SuperCAR-NK) cultured with the SuperNK-Cyto factor combination exhibit superior proliferation capacity compared to CD123 CAR-NK cells (CAR-NK) cultured with the conventional TraNK-Cyto factor combination, with a proliferation difference of approximately 10-fold. In other words, the SuperNK-Cyto factor combination of this application can promote the proliferation capacity of NK cells and genetically modified CAR-NK cells.

[0129] Cryopreservation of cells can damage them. When CAR-NK products are used as industrial products, the cryopreservation and cold chain transportation link is essential. Here, cryopreserved cells are used to simulate the form of industrial products. Of course, in some embodiments, for short distances or for rapid transportation or short-term fresh preservation such as 24h or 48h with the development of technology, cryopreservation may not be necessary, and freshly harvested CAR-NK cells can be used directly for subsequent experiments.

[0130] Frozen CD123 CAR-NK cells (CAR-NK) and CD123 SuperCAR-NK cells (SuperCAR-NK) were rapidly thawed in a 37°C water bath and then cultured in a medium without any added cytokines for 24 hours. Cell viability was assessed using AOPI, and the results are as follows: Figure 2 As shown. After cryopreservation and thawing, SuperCAR-NK cells prepared using the SuperNK-Cyto factor combination of this application showed a more significant advantage in post-thawing viability, **P=0.0036. That is, NK cells and genetically modified CAR-NK cells cultured using the SuperNK-Cyto factor combination scheme of this application are more cryogenic, which helps maintain NK activity and improve in vivo efficacy.

[0131] Example 2: In vivo functional verification of SuperCAR-NK Six- to eight-week-old female NCG mice were inoculated via tail vein (iv) with CD123-expressing THP-1-Luc-GFP (human acute myeloid leukemia cells) tumor cells at a dose of 1.0E+06 / mouse to establish an acute myeloid leukemia model, with the mice bearing tumors for one day. Frozen CD123 CAR-NK cells and CD123SuperCAR-NK cells prepared in Example 1 were collected on day 14 and day 21 of culture, respectively, and revived. These cells were then intravenously infused into the aforementioned model mice at a dose of 5.0E+06 CAR+ / mouse (day 0). In vivo imaging was performed weekly to compare the in vivo antitumor effect of CAR-NK cells. Blood samples were collected from mice, and the survival of CAR-NK cells in the blood was detected using RT-PCR.

[0132] The results are as follows Figure 3 and Figure 4As shown, Figure 3 This is a fluorescence imaging image of a mouse. Figure 4 The x-axis represents the copy number of gene-modified NK cells in tumor-bearing mice on day 14 after infusion of gene-modified NK cells. The x-axis represents the CAR-NK cell types with different preparation methods and preparation times, and the y-axis represents the copy number of CAR-NK cells in mouse blood. CAR-NK cells targeting CD123, cultured for 14 days with the IL-2 and IL-21 cytokine combination (TraNK-Cyto), were denoted as CAR-NK-D14; CAR-NK cells targeting CD123, cultured for 21 days with the IL-2 and IL-21 cytokine combination (TraNK-Cyto), were denoted as CAR-NK-D21; CAR-NK cells targeting CD123, cultured for 14 days with the IL-2, IL-21, IL-4, and TGF-β1 cytokine combination (SuperNK-Cyto), were denoted as SuperCAR-NK-D14; CAR-NK cells targeting CD123, cultured for 21 days with the IL-2, IL-21, IL-4, and TGF-β1 cytokine combination (SuperNK-Cyto), were denoted as SuperCAR-NK-D21; and unmodified NK cells were denoted as Control-NK. Results are as follows: Figure 3 The results show that, regardless of whether the culture period is 14 days or 21 days, CD123 SuperCAR-NK cells cultured using the SuperNK-Cyto factor combination regimen have better in vivo efficacy. The CD123 CAR-NK group cultured using the traditional TraNK-Cyto factor combination regimen has a certain anti-tumor effect after short-term in vitro culture (14 days), but the anti-tumor ability of CD123 CAR-NK is significantly reduced as the in vitro culture period is extended (21 days). In contrast, CD123 SuperCAR-NK cells cultured using the SuperNK-Cyto factor combination regimen of this application maintain effective anti-tumor ability even after long-term in vitro culture (21 days).

[0133] In the above in vivo experiments, blood samples were collected weekly from different experimental groups of mice, and the survival of CAR-NK cells in the blood was detected using RT-PCR. The results are as follows: Figure 4 As shown, Figure 4The x-axis represents the copy number of gene-modified NK cells in tumor-bearing mice on day 14 after infusion of gene-modified NK cells. The y-axis represents the CAR-NK cell type with different preparation methods and preparation times, and the y-axis represents the copy number of CAR-NK cells in mouse blood. Regardless of whether the culture period was 14 or 21 days, CD123 SuperCAR-NK cells cultured using the SuperNK-Cyto factor combination scheme of this application exhibited superior in vivo sustainability in mice with acute myeloid leukemia, helping to maintain NK cell activity and improve in vivo efficacy. In contrast, the CD123 CAR-NK cells cultured using the traditional TraNK-Cyto factor combination showed a significant decrease in in vivo sustainability after prolonged in vitro culture, and were significantly lower than those cultured using the SuperNK-Cyto factor combination scheme of this application (**P=0.0079).

[0134] These results demonstrate that the gene-modified NK cells prepared using the SuperNK-Cyto factor combination scheme of this application exhibit superior in vivo sustainability and efficacy. Furthermore, the gene-modified NK cells prepared using the SuperNK-Cyto factor combination scheme of this application maintain high functional activity and persistence even after long-term in vitro culture, fully meeting the dual requirements of adoptive cell therapy for both the quantity and efficacy of NK cells.

[0135] Example 3: Applicability of different CAR combinations to the SuperNK-Cyto factor combination scheme CAR-NK cells were prepared, collected, and cryopreserved according to the culture protocol of Example 1. The CAR was a combination of CARs targeting CD5, with the CD5 CAR structure being CD5scFv-8h-8TM-BBz-2A-mbIL15, and its sequence is shown in SEQ ID NO:2.

[0136] During cell culture, CD5 CAR-NK cells and CD5 SuperCAR-NK cells were counted separately, and the results are as follows: Figure 5 As shown in the results, CD5 SuperCAR-NK cells (SuperCAR-NK) cultured with the SuperNK-Cyto cytokine combination (IL-2, IL-21, IL-4, TGF-β1) exhibited superior proliferation capacity compared to CD5 CAR-NK cells (CAR-NK) cultured with the conventional TraNK-Cyto cytokine combination (IL-2, IL-21), with a proliferation difference of 23-fold. In other words, the SuperNK-Cyto factor combination regimen of this application can promote the proliferation capacity of NK cells and is applicable to CAR-NK cells with various structures and forms of gene modifications.

[0137] Frozen CD5 CAR-NK cells (CAR-NK) and CD5 SuperCAR-NK cells (SuperCAR-NK) were rapidly thawed in a 37°C water bath and then seeded into culture medium (OptiVitro® NK, NE000-N032) without any cytokines and cultured for 24 h. Cell viability was assessed using AOPI. Results are as follows: Figure 6 As shown, after cryopreservation and thawing, CD5 SuperCAR-NK cells prepared using the SuperNK-Cyto factor combination of this application exhibit a more significant survival rate advantage after thawing (**P=0.0029). That is, the gene-modified NK cells cultured using the SuperNK-Cyto factor combination scheme of this application are more cryogenic, helping to maintain NK activity and improve in vivo efficacy, and are applicable to various structural and form-modified CAR-NK cells.

[0138] Example 4: Functional CD5 CAR-NK cells prepared using the SuperNK-Cyto factor combination regimen CAR-NK cells were prepared, collected, and cryopreserved according to the culture protocol of Example 1. The CARs used were a combination of CARs targeting CD5. NALM6-Luc-GFP cells (human acute lymphoblastic leukemia cells) expressing CD5 were used as target cells to verify the function of CD5 CAR-NK cells prepared using different protocols. CAR-NK cells and target cells were co-incubated for 24 hours at an effector-to-target ratio of 1:2. The killing ability of NK cells cultured under different protocols against CD5-positive tumor cells was calculated using the cell killing rate (%) = (Luc value of control group - Luc value of experimental group) / Luc value of control group × 100%. The control group consisted of NK cells not transfected with CAR. Results are as follows: Figure 7As shown, genetically modified NK cells cultured using the IL-2 and IL-21 cytokine combination (TraNK-Cyto) are represented by TraNK-Cyto, and genetically modified NK cells cultured using the IL-2, IL-21, IL-4, and TGF-β1 cytokine combination (SuperNK-Cyto) are represented by SuperNK-Cyto. Each protocol has subgroups based on different culture times of 11 days and 19 days. The results showed that CD5 SuperCAR-NK cells cultured for 11 days using the SuperNK-Cyto factor combination scheme of this application exhibited stronger killing function against CD5-positive tumor cells (killing rate of 89.85%), which was significantly superior to the traditional TraNK-Cyto factor combination scheme cultured for the same gene-modified NK cells for 11 days (killing rate of 65.20%). Furthermore, with the extension of the culture period, the killing ability of the traditional TraNK-Cyto factor combination decreased significantly (killing rate of 33.60% (CAR-NK-19d in the TraNK-Cyto group), a decrease of 31.60%), while the SuperNK-Cyto factor combination scheme maintained a high killing function even with the culture period extended to 19 days (killing rate of 75.35% (CAR-NK-19d in the SuperNK-Cyto group), a decrease of only 14.50%). The SuperNK-Cyto factor combination scheme helps maintain the activity of gene-modified NK cells and improves the effectiveness against tumors.

[0139] Example 5: The SuperNK-Cyto factor combination regimen is applicable to NK cells from different sources. NK cells were cultured and prepared using the method described in Example 1, wherein the NK cells were derived from peripheral blood mononuclear cells (PBMCs) and umbilical cord blood mononuclear cells (UCBs), respectively.

[0140] The method for preparing NK cells using peripheral blood mononuclear cells (PBMCs) and umbilical cord blood mononuclear cells (UCBs) is the same as step 2 of Example 1, wherein the sample recruited for preparing NK cells using umbilical cord blood mononuclear cells (UCBs) is umbilical cord blood.

[0141] The two NK cells cultured using the traditional TraNK-Cyto factor combination are PB-NK and UCB-NK, and the two NK cells prepared using the SuperNK-Cyto factor combination are SuperPB-NK and SuperUCB-NK.

[0142] During the culture process, various NK cells were counted, and the statistical results are as follows: Figure 8 and Figure 9 As shown, Figure 8The proliferation of NK cells derived from peripheral blood cultured under the TraNK-Cyto factor combination and SuperNK-Cyto factor combination protocols; Figure 9 The proliferation of NK cells derived from umbilical cord blood was observed under the TraNK-Cyto and SuperNK-Cyto factor combination regimens. The results show that the SuperNK-Cyto factor combination regimen of this application is applicable to the culture of NK cells from any source, including peripheral blood and umbilical cord blood. Culture using the SuperNK-Cyto factor combination regimen can improve the expansion capacity of NK cells, with the expansion fold of PBMC-derived cells increasing by 87-fold compared to traditional factor combination regimens. Even with NK cells derived from umbilical cord blood exhibiting a more "immature" phenotype, the SuperNK-Cyto factor combination regimen can still increase their expansion capacity by 3-fold.

[0143] It is evident that the SuperNK-Cyto factor combination regimen can significantly enhance the proliferative capacity of NK cells, which can be derived from multiple sources.

[0144] Example 6: The SuperNK-Cyto factor combination can be used as a basic combination for further optimization. In the culture protocol of Example 1, IL15 (Shanghai Puxin, GMP-101-15) was simultaneously added to the culture medium at a final concentration of 10 ng / ml in addition to the SuperNK-Cyto factor combination. The cultured cells were represented as SuperCAR-NK+IL15, cells cultured with the SuperNK-Cyto factor combination were represented as SuperCAR-NK, and cells cultured with the conventional TraNK-Cyto factor combination were represented as CAR-NK. Cell counts were performed on each group during cell culture, and the results are as follows: Figure 10 As shown in the results, for CD123-targeting CAR-NK products, the SuperNK-Cyto culture protocol can increase the proliferation rate of CAR-NK by 69 times compared to the traditional TraNK-Cyto culture protocol. Adding IL15 to SuperNK-Cyto can further increase the proliferation capacity of CAR-NK by 41 times.

[0145] The results indicate that the SuperNK-Cyto factor combination can be further optimized as a base combination for NK and genetically modified NK such as CAR-NK, such as by adding other related factors such as IL15.

[0146] The SuperNK-Cyto factor combination described in this application can also be used in kits for NK cells and genetically modified NK cells such as CAR-NK. These kits contain basal culture medium and cytokine components, wherein the cytokines are IL2, IL21, IL-4, and TGFβ1. In some embodiments, the kit contains basal culture medium, NK activation components, and cytokine components, wherein the NK activation components are Herceptin and CD28, and the cytokines are IL2, IL21, IL-4, and TGFβ1. In some embodiments, the kit contains basal culture medium, NK activation components, and cytokine components, wherein the cytokines are IL2, IL21, IL-4, TGFβ1, and IL15.

[0147] Example 7: Validation of Factor Concentrations in Different Factor Combinations CD5-SuperCAR-NK and conventional CD5CAR-NK prepared using a combination of IL2 and IL21 cytokines (TraNK-Cyto process protocol see: Example 1) were prepared according to the method of Example 1. The CD5CAR structure is shown in Example 3: CD5scFv-8h-8TM-BBz-2A-mbIL15, and its sequence is shown in SEQ ID NO:2. In this example, CD5-SuperCAR-NK prepared using the SuperNK-Cyto protocol of this application is referred to as SuperCAR-NK, and conventional CD5CAR-NK prepared using the TraNK-Cyto protocol is referred to as CAR-NK. In this example, the factor combinations of the SuperNK-Cyto protocol with IL21 at 2.5 ng / ml-1250 ng / ml, IL2 at 50 IU-5000 IU, IL4 at 1 ng / ml-200 ng / ml, and TGFβ1 at 0.5 ng / ml-100 ng / ml were validated at different concentrations. The concentration combinations are shown in Table 1 below.

[0148] Table 1

[0149] The cytokine sources are the same as in the previous examples.

[0150] During cell culture, CD5 CAR-NK cells (CAR-NK) and CD5 SuperCAR-NK cells (SuperCAR-NK) with different concentrations of factors were counted, and the results are as follows: Figure 11 As shown, Figure 11 A: SuperCAR-NK and CAR-NK proliferation rates after 14 days of culture; Figure 11The proliferation rates of SuperCAR-NK and CAR-NK cells cultured to day 28 were observed. Results showed that CD5+ proliferation was significantly increased when the SuperNK-Cyto factor combination was cultured at the following concentrations: IL21: 2.5 ng / ml-1250 ng / ml, IL2: 50 IU-5000 IU, IL4: 1 ng / ml-200 ng / ml, and TGFβ: 0.5 ng / ml-100 ng / ml. SuperCAR-NK cells (SuperCAR-NK) exhibit superior proliferation capacity compared to CD5CAR-NK cells (CAR-NK) cultured with the conventional TraNK-Cyto factor combination. The preferred concentrations are SuperCAR-NK-B, SuperCAR-NK-C, and SuperCAR-NK-D regimens. SuperCAR-NK-B regimen: IL21: 5 ng / ml, IL2: 250 IU, IL4: 5 ng / ml, TGFβ: 5 ng / ml; SuperCAR-NK-C regimen: IL21: 25 ng / ml, IL2: 500 IU, IL4: 10 ng / ml, TGFβ: 1 ng / ml; SuperCAR-NK-D regimen: IL21: 250 ng / ml, IL2: 1000 IU, IL4: 50 ng / ml, TGFβ: 10 ng / ml.

[0151] The results showed that the SuperCAR-NK regimen of this application promoted NK cell proliferation in the following doses: IL21: 2.5ng / ml-1250ng / ml, IL2: 50IU-5000IU, IL4: 1ng / ml-200ng / ml, and TGFβ: 0.5ng / ml-100ng / ml.

[0152] Example 8: Adapting the Cytokine Combinations of this Application to Different Basal Culture Media 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 the basal culture medium using three different culture media; we also verified possible culture media containing the cytokine combination described in this application. Specific culture media are shown in Table 2 below.

[0153] Table 2. 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.

[0154] The cytokine sources and concentrations were as shown in Example 1. CD5-SuperCAR-NK and CD5CAR-NK were prepared according to the protocol of Example 1 (see Example 7), and cell counts were performed on days 14 and 21 of culture to analyze the cell proliferation fold under different culture media. The results are as follows: Figure 12 As shown. Figure 12 A represents the fold increase in CD5-SuperCAR-NK and CD5CAR-NK cells cultured in Medium-1 as the basal medium. Figure 12 B represents the fold increase of CD5-SuperCAR-NK and CD5CAR-NK cells cultured in Medium-2 as the basal medium. Figure 12 C represents the fold increase of CD5-SuperCAR-NK and CD5CAR-NK cultured in Medium-3 as the basal medium. The results indicate that the SuperCAR-NK protocol of this application is applicable to any universal basal medium.

[0155] Example 9. Validation of the Proliferation Effect of Different Cytokine Combinations on CAR-NK Cells CD19-SuperCAR-NK and conventional CD19CAR-NK prepared using the combination of IL2 and IL21 cytokines (see Example 1 for the TraNK-Cyto process) were prepared according to the scheme of Example 1, wherein the CD19CAR sequence is shown in SEQ ID NO:3. In this example, the SuperNK-Cyto scheme of this application is referred to as SuperCAR-NK, and the TraNK-Cyto scheme is referred to as CAR-NK. In this example, the SuperCAR-NK scheme of the aforementioned embodiment was further optimized. The optimized group 2 is: based on the preparation of SuperCAR-NK in Example 1, IL4 cytokine was removed (i.e., the cytokines contained in the culture medium are only IL21: 25 ng / ml, IL2: 500 IU, TGFβ1: 1 ng / ml). Group 3: Based on the SuperCAR-NK preparation protocol of Example 1, steps 1-3 are the same as steps 1-3 of Example 1. In step 4: after activating and culturing NK cells for 12-24 hours in step 3, lentivirus (HIV-1, human immunodeficiency virus type I) containing the CD19 CAR structural gene vector is co-incubated with the activated NK cells for 12-24 hours to obtain genetically modified CD19 CAR-NK cells. The obtained CD19 CAR-NK cells are cultured in a medium (OptiVitro® NK, NE000-N032) containing a combination of IL2, IL21, and TGFβ1 cytokines (named SuperNK-Cyto) for 7 days, and then IL4 is added at 10 ng / ml for further culture (i.e., after the 7th day of culture, the medium is changed to: IL21: 25 ng / ml, IL2: 500 IU, IL4: 10 ng / ml, TGFβ1: 1 ng / ml). Subsequently, the proliferation fold of CD5-SuperCAR-NK and CD5CAR-NK is measured. The specific cytokines and their addition amounts for each SuperNK-Cyto regimen group are shown in Table 3 below.

[0156] Table 3

[0157] The results are as follows Figure 13 As shown, Figure 13 The proliferation of different SuperNK-Cyto variants was recorded, with the proliferation count being calculated at 21 days of culture.

[0158] The results showed that both Group 2 (IL21: 25 ng / ml, IL2: 500 IU, TGFβ1: 1 ng / ml) without IL4 and Group 3 (IL21: 25 ng / ml, IL2: 500 IU, IL4: 10 ng / ml (added after 7 days of culture), TGFβ1: 1 ng / ml) with IL4 added later in the culture period achieved superior proliferation compared to the traditional regimen. Specifically, Group 2 showed a 7.40-fold increase in proliferation compared to the CAR-NK group, and Group 3 showed a 22.2-fold increase in proliferation compared to the CAR-NK group. These results demonstrate that in the SuperNK-Cyto protocol, IL4 can be removed or added during culture depending on the specific culture regimen.

[0159] The sequences used in the above embodiments of this application are shown in the following sequence list. It should be understood that the following sequences are merely exemplary sequences for the embodiments of this application, and not any limitation thereof.

[0160]

[0161] 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. An NK cell culture medium comprising cytokines IL2, IL21 and TGFβ; Preferably, it contains the cytokines IL4, IL2, IL21 and 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 IL4 in the culture medium is 0-250 ng / ml, 0.5-250 ng / ml, 1-250 ng / ml, 2-250 ng / ml, 3-250 ng / ml, 4-250 ng / ml, 5-250 ng / ml, 6-250 ng / ml, 7-250 ng / ml, 8-250 ng / ml, 9-250 ng / ml, 10-250 ng / ml, 50-250 ng / ml, 200-250 ng / ml, 0-220 ng / ml, 0.5-220 ng / ml, 1-220 ng / ml, 2 -220ng / ml, 3-220ng / ml, 4-220ng / ml, 5-220ng / ml, 6-220ng / ml, 7-220ng / ml, 8-220ng / ml, 9-220ng / ml, 10-220ng / ml, 50-220ng / ml, 200 -220ng / ml, 0-200ng / ml, 0.5-200ng / ml, 1-200ng / ml, 2-200ng / ml, 3-200ng / ml, 4-200ng / ml, 5-200ng / ml, 6-200ng / ml, 7-200ng / ml, 8-20 0ng / ml, 9-200ng / ml, 10-200ng / ml, 0-150ng / ml, 0.5-150ng / ml, 1-150ng / ml, 2-150ng / ml, 3-150ng / ml, 4-150ng / ml, 5-150ng / ml, 6-150 ng / ml, 7-150ng / ml, 8-150ng / ml, 9-150ng / ml, 10-150ng / ml, 50-150ng / ml, 0-100ng / ml, 0.5-100ng / ml, 1-100ng / ml, 2-100ng / ml, 3-100n g / ml, 4-100ng / ml, 5-100ng / ml, 6-100ng / ml, 7-100ng / ml, 8-100ng / ml, 9-100ng / ml, 10-100ng / ml, 50-100ng / ml, 0-50ng / ml, 0.5-50ng / m l, 1-50ng / ml, 2-50ng / ml, 3-50ng / ml, 4-50ng / ml, 5-50ng / ml, 6-50ng / ml, 7-50ng / ml, 8-50ng / ml, 9-50ng / ml, 10-50ng / ml, 0-40ng / ml, 0.5 - 40 ng / ml, 1 - 40 ng / ml, 2 - 40 ng / ml, 3 - 40 ng / ml, 4 - 40 ng / ml, 5 - 40 ng / ml, 6 - 40 ng / ml, 7 - 40 ng / ml, 8 - 40 ng / ml, 9 - 40 ng / ml, 10 - 40 ng / ml, 0 - 30 ng / ml, 0.5 - 30 ng / ml, 1 - 30 ng / ml, 2 - 30 ng / ml, 3 - 30 ng / ml, 4 - 30 ng / ml, 5 - 30 ng / ml, 6 - 30 ng / ml, 7 - 30 ng / ml, 8 - 30 ng / ml, 9 - 30 ng / ml, 10 - 30 ng / ml, 0 - 20 ng / ml, 0.5 - 20 ng / ml, 1 - 20 ng / ml, 2 - 20 ng / ml, 3 - 20 ng / ml, 4 - 20 ng / ml, 5 - 20 ng / ml, 6 - 20 ng / ml, 7 - 20 ng / ml, 8 - 20 ng / ml, 9 - 20 ng / ml, 10 - 20 ng / ml, 0 - 15 ng / ml, 0.5 - 15 ng / ml, 1 - 15 ng / ml, 2 - 15 ng / ml, 3 - 15 ng / ml, 4 - 15 ng / ml, 5 - 15 ng / ml, 6 - 15 ng / ml, 7 - 15 ng / ml, 8 - 15 ng / ml, 9 - 15 ng / ml, 10 - 15 ng / ml, 0 - 13 ng / ml, 0.5 - 13 ng / ml, 1 - 13 ng / ml, 2 - 13 ng / ml, 3 - 13 ng / ml, 4 - 13 ng / ml, 5 - 13 ng / ml, 6 - 13 ng / ml, 7 - 13 ng / ml, 8 - 13 ng / ml, 9 - 13 ng / ml or 10 - 13 ng / ml;. Preferably, the IL4 content in the culture medium is 1-100 ng / ml, 1-50 ng / ml, 1-40 ng / ml, 5-50 ng / ml, 5-10 ng / ml, 1-30 ng / ml, 1-20 ng / ml, 1-10 ng / ml, or 1-5 ng / ml.

3. The culture medium according to claim 1 or 2, wherein the content of TGFβ in the culture medium is 0.1 - 100 ng / ml, 0.1 - 80 ng / ml, 0.1 - 60 ng / ml, 0.1 - 50 ng / ml, 0.1 - 40 ng / ml, 0.1 - 30 ng / ml, 0.1 - 20 ng / ml, 0.1 - 19 ng / ml, 0.1 - 18 ng / ml, 0.1 - 17 ng / ml, 0.1 - 16 ng / ml, 0.1 - 15 ng / ml, 0.1 - 14 ng / ml, 0.1 - 13 ng / ml, 0.1 - 12 ng / ml, 0.1 - 11 ng / ml, 0.1 - 10 ng / ml, 0.1 - 9 ng / ml, 0.1 - 8 ng / ml, 0.1 - 7 ng / ml, 0.1 - 6 ng / ml, 0.1 - 5 ng / ml, 0.1 - 4 ng / ml, 0.1 - 3 ng / ml, 0.1 - 2 ng / ml, 0.1 - 1 ng / ml, 0.3 - 100 ng / ml, 0.3 - 80 ng / ml, 0.3 - 60 ng / ml, 0.3 - 50 ng / ml, 0.3 - 40 ng / ml, 0.3 - 30 ng / ml, 0.3 - 20 ng / ml, 0.3 - 19 ng / ml, 0.3 - 18 ng / ml, 0.3 - 17 ng / ml, 0.3 - 16 ng / ml, 0.3 - 15 ng / ml, 0.3 - 14 ng / ml, 0.3 - 13 ng / ml, 0.3 - 12 ng / ml, 0.3 - 11 ng / ml, 0.3 - 10 ng / ml, 0.3 - 9 ng / ml, 0.3 - 8 ng / ml, 0.3 - 7 ng / ml, 0.3 - 6 ng / ml, 0.3 - 5 ng / ml, 0.3 - 4 ng / ml, 0.3 - 3 ng / ml, 0.3 - 2 ng / ml, 0.3 - 1 ng / ml, 0.5 - 100 ng / ml, 0.5 - 80 ng / ml, 0.5 - 60 ng / ml, 0.5 - 50 ng / ml, 0.5 - 40 ng / ml, 0.5 - 30 ng / ml, 0.5 - 20 ng / ml, 0.5 - 19 ng / ml, 0.5 - 18 ng / ml, 0.5 - 17 ng / ml, 0.5 - 16 ng / ml, 0.5 - 15 ng / ml, 0.5 - 14 ng / ml, 0.5 - 13 ng / ml, 0.5 - 12 ng / ml, 0.5 - 11 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, 0.7 - 100 ng / ml, 0.7 - 80 ng / ml, 0.7 - 60 ng / ml, 0.7 - 50 ng / ml, 0.7 - 40 ng / ml, 0.7 - 30 ng / ml, 0.7 - 20 ng / ml, 0.7 - 19 ng / ml, 0.7 - 18 ng / ml, 0.7 - 17 ng / ml, 0.7 - 16 ng / ml, 0.7 - 15 ng / ml, 0.7 - 14 ng / ml, 0.7 - 13 ng / ml, 0.7 - 12 ng / ml, 0.7 - 11 ng / ml, 0.7 - 10 ng / ml, 0.7 - 9 ng / ml, 0.7 - 8 ng / ml, 0.7 - 7 ng / ml, 0.7 - 6 ng / ml, 0.7 - 5 ng / ml, 0.7 - 4 ng / ml, 0.7 - 3 ng / ml, 0.7 - 2 ng / ml or 0.7 - 1 ng / ml;. Preferably, the TGFβ content in the culture medium is 0.1-50 ng / ml, 0.1-30 ng / ml, 0.1-10 ng / ml, 0.1-5 ng / ml, 0.5-50 ng / ml, 0.5-40 ng / ml, 0.5-30 ng / ml, 0.5-20 ng / ml, 0.5-19 ng / ml, 0.5-18 ng / ml, 0.5-17 ng / ml, 0.5-16 ng / ml, 0.5- 15ng / ml, 0.5-14ng / ml, 0.5-13ng / ml, 0.5-12ng / ml, 0.5-11ng / ml, 0.5-10ng / ml, 0.5-9ng / ml, 0.5- 8ng / ml, 0.5-7ng / ml, 0.5-6ng / ml, 0.5-5ng / ml, 0.5-4ng / ml, 0.5-3ng / ml, 0.5-2ng / ml or 0.5-1ng / ml.

4. The culture medium according to any one of claims 1-3, wherein the IL2 content in the culture medium is 50-5000 IU / ml, 50-4000 IU / ml, 50-3000 IU / ml, 50-2000 IU / ml, 50-1800 IU / ml, 50-1600 IU / ml, 50-1500 IU / ml, 50-1400 IU / ml, 50-1300 IU / ml, 50-1200 IU / ml, 50-1100 IU / ml, 50-1000 IU / ml, 50-900 IU / ml, 50-800 IU / ml, 50-700 IU / ml, 50-600 IU / ml, or 50-500 IU / ml. / ml, 50-400IU / ml, 50-300IU / ml, 50-200IU / ml, 50-100IU / ml, 100-5000IU / ml, 100-4000IU / ml, 100-3000IU / ml, 100-2000IU / ml, 100-1800IU / ml, 100 -1600IU / ml, 100-1400IU / ml, 100-1300IU / ml, 100-1200IU / ml, 100-1100IU / ml, 100-1000IU / ml, 100-900IU / ml, 100-800IU / ml, 100-700IU / ml, 100-6 00IU / ml, 100-500IU / ml, 100-400IU / ml, 100-300IU / ml, 100-200IU / ml, 20 0-5000IU / ml, 200-4000IU / ml, 200-3000IU / ml, 200-2000IU / ml, 200-1800I U / ml, 200-1600IU / ml, 200-1500IU / ml, 200-1400IU / ml, 200-1300IU / ml, 200-1200IU / ml, 200-1100IU / ml, 200-1000IU / ml, 200-900IU / ml, 200-800IU / ml, 200-700IU / ml, 200-600IU / ml, 200-500IU / ml, 200-400IU / ml, 200-300IU / ml, 250-5000IU / ml, 250-4000IU / ml, 250-3000IU / ml, 250-2000IU / ml, 250-1800IU / ml, 250-1600IU / ml, 250-1500IU / ml, 250-1400IU / ml, 250-13 00IU / ml, 250-1200IU / ml, 250-1100IU / ml, 250-1000IU / ml, 250-900IU / ml,250 - 800 IU / ml, 250 - 700 IU / ml, 250 - 600 IU / ml, 250 - 500 IU / ml, 250 - 400 IU / ml, 250 - 300 IU / ml, 300 - 5000 IU / ml, 300 - 4000 IU / ml, 300 - 3000 IU / ml, 300 - 2000 IU / ml, 300 - 1800 IU / ml, 300 - 1600 IU / ml, 300 - 1500 IU / ml, 300 - 1400 IU / ml, 300 - 1300 IU / ml, 300 - 1200 IU / ml, 300 - 1100 IU / ml, 300 - 1000 IU / ml, 300 - 900 IU / ml, 300 - 800 IU / ml, 300 - 700 IU / ml, 300 - 600 IU / ml, 300 - 500 IU / ml, 300 - 400 IU / ml, 400 - 5000 IU / ml, 400 - 4000 IU / ml, 400 - 3000 IU / ml, 400 - 2000 IU / ml, 400 - 1800 IU / ml, 400 - 1600 IU / ml, 400 - 1500 IU / ml, 400 - 1400 IU / ml, 400 - 1300 IU / ml, 400 - 1200 IU / ml, 400 - 1100 IU / ml, 400 - 1000 IU / ml, 400 - 900 IU / ml, 400 - 800 IU / ml, 400 - 700 IU / ml or 400 - 600 IU / ml; Preferably, the IL2 content in the culture medium is 50-1000 IU / ml, 500-1000 IU / ml, 50-250 IU / ml, 50-500 IU / ml, 100-900 IU / ml, 200-800 IU / ml, 250-1000 IU / ml, 300-700 IU / ml, 300-600 IU / ml, or 400-600 IU / ml.

5. The culture medium according to any one of claims 1-4, wherein the content of IL21 in the culture medium is 1-1500 ng / ml, 1-1250 ng / ml, 1-1000 ng / ml, 1-750 ng / ml, 1-500 ng / ml, 1-400 ng / ml, 1-300 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, or 1-20 ng / ml. / ml, 1-15ng / ml, 1-10ng / ml, 1-8ng / ml, 1-6ng / ml, 1-5ng / ml, 1-4ng / ml, 1-3ng / ml, 5-1500ng / ml, 5-1250ng / ml, 5-1000ng / ml, 5-750ng / ml, 5-500ng / ml, 5-400ng / ml, 5-300ng / ml, 5-250ng / ml, 5-200ng / ml, 5-150ng / ml, 5-100ng / ml, 5-50ng / ml, 5-40ng / ml, 5-30ng / ml, 5-29ng / ml, 5-28ng / ml, 5-27n g / ml, 5-26ng / ml, 5-25ng / ml, 5-20ng / ml, 5-15ng / ml, 5-10ng / ml, 5-8ng / ml, 5-6ng / ml, 10-1500ng / ml, 10-1250ng / ml, 10-1000ng / ml, 10-750ng / ml, 10-500ng / ml, 10-400ng / ml, 10-300ng / ml, 10-250ng / ml, 10-200ng / ml, 10-150ng / ml, 10-100ng / ml, 10-50ng / ml, 10-40ng / ml, 10-30ng / ml, 10-29ng / ml, 10-28ng / ml, 10-27ng / ml, 10-26ng / ml, 10-25ng / ml, 10-20ng / ml, 10-15ng / ml, 15-1500ng / ml, 15-1250ng / ml, 15-1000ng / ml, 15-750ng / ml, 15- 500ng / ml, 15-400ng / ml, 15-300ng / ml, 15-250ng / ml, 15-200ng / ml, 15-150ng / ml, 15-100ng / ml, 15-50ng / ml, 15-40ng / ml, 15-30ng / ml, 15-29ng / ml,15 - 28 ng / ml, 15 - 27 ng / ml, 15 - 26 ng / ml, 15 - 25 ng / ml, 15 - 20 ng / ml, 20 - 1500 ng / ml, 20 - 1250 ng / ml, 20 - 1000 ng / ml, 20 - 750 ng / ml, 20 - 500 ng / ml, 20 - 400 ng / ml, 20 - 300 ng / ml, 20 - 250 ng / ml, Preferably, the IL21 content in the culture medium is 2.5-1250 ng / ml, 2.5-250 ng / ml, 2.5-25 ng / ml, 2.5-5 ng / ml, 5-900 ng / ml, 5-700 ng / ml, 5-250 ng / ml, 5-25 ng / ml, 10-400 ng / ml, 10-200 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, or 10-25 ng / ml.

6. The culture medium according to any one of claims 1-5 further comprises any one or more cytokines as shown below: 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), TNF-α; Preferably, the culture medium also contains IL5.

7. The culture medium according to claim 6, wherein the IL15 content in the culture medium is 0-100 ng / ml, 1-100 ng / ml, 1-80 ng / ml, 2-80 ng / ml, 1-70 ng / ml, 1-60 ng / ml, 1-50 ng / ml, 1-40 ng / ml, 1-30 ng / ml, 1-20 ng / ml, 1-18 ng / ml, 1-16 ng / ml, 1-15 ng / ml, 1-14 ng / ml, 1-13 ng / ml, or 1-12 ng / ml. l, 1-11ng / ml, 1-10ng / ml, 1-8ng / ml, 1-6ng / ml, 1-5ng / ml, 3-100ng / ml, 3-80ng / ml, 3-70ng / ml, 3-60ng / ml, 3-50ng / ml, 3-40ng / ml, 3-30ng / ml, 3-20ng / ml, 3-18ng / ml, 3-16ng / ml, 3-15ng / ml, 3-14ng / ml, 3-13ng / ml, 3-12ng / ml, 3- 11ng / ml, 3-10ng / ml, 3-8ng / ml, 3-6ng / ml, 3-5ng / ml, 5-100ng / ml, 5-80ng / ml, 5-70ng / ml, 5-60ng / ml, 5-50ng / ml, 5-40ng / ml, 5-30ng / ml, 5-20ng / ml, 5-18ng / ml, 5-16ng / ml, 5-15ng / ml, 5-14ng / ml, 5-13ng / ml, 5-12ng / ml, 5-11ng / ml, 5-10ng / ml, 5-8ng / ml, 5-6ng / ml, 8-100ng / ml, 8-80ng / ml, 8-70ng / ml, 8-60ng / ml, 8-50ng / ml, 8-40ng / ml, 8-3 0ng / ml, 8-20ng / ml, 8-18ng / ml, 8-16ng / ml, 8-15ng / ml, 8-14ng / ml, 8-13ng / ml, 8-12ng / ml, 8-11ng / ml or 8-10ng / ml; Preferably, the IL15 content in the culture medium is 1-100 ng / ml, 2-80 ng / ml, 5-50 ng / ml, or 5-20 ng / ml.

8. The culture medium according to any one of claims 1-7, comprising a basal culture medium for cell culture; The basal culture medium is a general basal culture medium used for cell culture; The general basal culture medium is either serum-free or serum-containing; Preferably, the serum-free culture medium is selected from any one or more of the following: OpTmizer™ CTS™, Immunocult™ XF, CellGro™, TexMacs™, Stemline™, Xvivo15™, PrimeXV, ImmunoCult™-XF medium, SCGM medium, X-VIVO series serum-free immune cell medium, X-VIV010 serum-free immune aOHQiS medium, X-VIV015 serum-free immune cell medium, X-VIVO 20 serum-free cell medium, OptiVitro® T cell serum-free medium, lymphocyte HIPP-T009 serum-free medium, lymphocyte serum-free medium KBM581, SuperCulture® L500 lymphocyte serum-free medium, as well as StemXVivo, CTS NK-Xpander medium, NK MACS Medium / NK cell medium, OptiVitro® NK cell expansion kit, DKW37-NKP series medium, and NK MACS® series medium. Preferably, the culture medium may also be 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 NK cells, comprising culturing peripheral blood mononuclear cells (PBMCs), umbilical cord blood mononuclear cells (UCBs), NK cells, or any combination thereof using the culture medium described in any one of claims 1-8; Preferably, the culture time is 7-30 days; More preferably, the culture time is 14-28 days.

10. The method according to claim 9, wherein the NK cells are natural NK cells and / or genetically modified NK cells; Preferably, the genetically modified NK cells contain a gene fragment encoding a chimeric antigen receptor CAR; Preferably, the gene-modified NK cells are CAR-NK cells.

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