Method for proliferating NK cells in vitro by virtue of magnetic beads coupled with multiple stimulating proteins and application of NK cells

Abstract

The invention discloses a method for proliferating NK cells in vitro by virtue of magnetic beads coupled with multiple stimulating proteins. The method comprises the following steps: culturing NK cells by adopting an NK cell culture medium, adding magnetic beads coupled with IL-15, 4-1BBligand and IL-21 (of which the molar ratio is (1-3):(1-3):(1-3)), culturing for 12-15 days, and harvesting and washing the NK cells, wherein the NK cell culture medium is prepared by adopting a method comprising the step of adding 10% of human AB serum or autologous serum, 500IU / mL of IL-2 and 5ng / ml of IL-15 into an X-VIVO 20 basic culture medium. The method disclosed by the invention has the advantages that the magnetic beads are coupled with three specificity stimulating factors, the aim of stimulating the NK cells by multiple factors is achieved, and usage of a trophoblastic cell line is also avoided, so that GMP production requirement and amplification efficiency requirement are considered at the same time, and the harvested NK cells can be used for NK allotransplantation.

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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