Method and application of in vitro expansion of NK cells coupled with magnetic beads and various stimulatory proteins

Abstract

The invention discloses a method for in vitro expansion of NK cells with magnetic beads coupled to various stimulatory proteins: NK cells are cultured with NK cell medium, and magnetic beads coupled with IL‑15, 4‑1BBligand and IL‑21 are added ( The molar ratio of the three is 1~3:1~3:1~3), cultivated for 12~15 days, harvested and washed NK cells; the NK cell culture medium was prepared by the following method: in X-VIVO 20 Add 10% human AB serum or autologous serum, 500IU / mL IL-2, and 5ng / ml IL-15 to the culture medium. The method of the present invention uses magnetic beads to couple three specific stimulatory factors, which not only achieves the purpose of stimulating NK cells with multiple factors, but also avoids the use of trophoblast cell lines, thereby taking into account the requirements of GMP production and amplification efficiency. Harvested NK cells can be used for allogeneic NK transplantation.

Description

technical field [0001] The invention relates to a method and application of in vitro expansion of NK cells coupled with magnetic beads to various stimulating proteins, and belongs to the field of novel tumor immunotherapy Background technique [0002] NK cells are a group of large granular lymphocytes different from T and B lymphocytes. They are part of the innate immune system. They can identify and dissolve tumor cells and virus-infected cells, as well as produce regulatory cytokines. They are the body's anti-infection and anti-tumor cells important immune cells that grow. Generally, CD3-CD56+ lymphocytes in the human body are identified as NK cells, and according to the difference in the expression density of CD56 molecules on the cells, NK cells are divided into two subgroups, CD56dim and CD56bright. CD56dim accounts for more than 90% of NK cells, mainly for cytotoxicity, and has stronger killing activity; CD56bright can produce a large number of cytokines, mainly for i...

AI Technical Summary

Problems solved by technology

[0009] 1. Add soluble growth factors IL2 and IL15, the disadvantage is that the amplification factor is limited;

[0010] 2. Adding growth factors or monoclonal antibodies to coat the bottom of the culture flask, the disadvantage is that the amplification factor is limited and the operation is inconvenient;

[0011] 3. Using feeder cells (Feeder Cell) K562-IL15-4-1BBligand, etc., after gamma radiation, the disadvantage is that it does not meet the GMP standard

[0012] 4. Using 100Gy-irradiated EBV transformed LCL trophoblast cells, use the first 100Gy gamma radiation, and expand 800 times after 3 weeks of culture. The disadvantage is that it does not meet the GMP standard

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