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Method for efficiently amplifying NK cells in vitro

A cell and nuclear cell technology, applied in the field of cell expansion, can solve the problems of low killing ability and lack of efficient amplification, and achieve the effect of high killing ability

Active Publication Date: 2020-05-19
山东省齐鲁细胞治疗工程技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In view of the current lack of methods for efficiently expanding NK cells and low killing ability, the present invention provides a method for expanding NK cells using genetically modified K562 trophoblast cells, which can efficiently obtain NK cells with high purity and strong killing ability

Method used

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  • Method for efficiently amplifying NK cells in vitro
  • Method for efficiently amplifying NK cells in vitro
  • Method for efficiently amplifying NK cells in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Construction of genetically modified K562 trophoblast cells

[0036] (1) Construction of lentiviral vector: Synthesize the target gene according to the sequence of CD8asp-IL21-CD8TM-T2A-CD8asp-IL27-CD8TM-IRES-CD70, the sequence is shown in SEQ ID NO: 1, and SpeI enzyme is added at the 5' end cutting site (actagt), and a NotI restriction site (gcggccgc) is added to the 3' end. After double-enzyme digestion, it was transferred into the same double-enzyme-digested lentiviral backbone plasmid (pHR-mCheery) to obtain a lentiviral backbone plasmid containing IL-21, IL-27 and CD70 genes. The signal peptide (MALPVTALLLPLALLLLHAARP) gene of CD8 is added to the 5' end of IL-21 and IL-27 sequence, and the transmembrane region gene of CD8 is added to the 3' end respectively, so that these two proteins can be anchored on the cell membrane; CD70 itself transmembrane Membrane proteins, which can be anchored to the cell membrane;

[0037] (2) The backbone plasmid and packag...

Embodiment 2

[0042] Example 2 The cultivation and identification of NK cells

[0043](1) Collect 30-50 mL of fresh blood from healthy volunteers on day 0, separate peripheral blood mononuclear cells (PBMC) with lymphocyte separation medium, wash 2 times, and take 4×10 7 Use culture medium (551-H3, TAKARA) to adjust the cell density to 1.5×10 6 / mL;

[0044] (2) Add 1×10 at a ratio of 1:4 to the number of PBMCs 7 / mL K562 trophoblast cells and IL-2 (Quangang) at a final concentration of 200U / mL; 37°C, 5%CO 2 , 100% humidity conditions for cell culture, observe the cell state and replenish fluid in time; on the 7th day, add 4×10 7 / mL K562 trophoblast cells are used for the continuous activation of NK cells, and bagged; cultured until the 15th day, and collected NK1 cells;

[0045] According to the method of Zhang H. (Activating signals dominate inhibitory signals in CD137L / IL-15 activated natural killer cells. J Immunother 2011 (34): 187–195), the classic K562 engineered cells and PBMCs...

Embodiment 3

[0047] Example 3 In vitro killing test of NK cells on tumor cell lines

[0048] HepG2 cells (liver cancer cell line) were seeded in xCELLigence RTCA S16 cell proliferation plate (ACEA), 1×10 per well 5 Place the plate in a cell culture incubator (37°C 5% CO 2 ) on the RTCA Station in ), cultured for 24 hours, respectively inoculated NK cells cultured for 14 days according to E:T=1:1, 3:1, 6:1, and 10:1, and the NK cells cultured by K562 trophoblast cells obtained in Example 1 The NK cells cultured from NK1 and classical K562 engineered cells were used as NK2 as a control, with 3 replicate wells in each group. Put the plate back on the RTCA Station, incubate for 24 hours, pipette 100 μL supernatant from each well and store at -80°C for later use. The result is as Figure 5 As shown, NK1 cells can specifically kill HepG2 cells, and the average killing rates are 21.5%, 47.5%, 68.5%, and 88.5% when the effect-to-target ratio is 1:1, 3:1, 6:1, and 10:1. The killing rate increas...

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Abstract

The invention provides a method for amplifying NK cells by utilizing gene modified K562 trophoblast cells. IL-21, IL-27 and CD70 genes are transfected into K562 cells through a lentivirus reprint system, so that K562 engineering cells capable of stably expressing IL-21, IL-27 and CD70 protein can be obtained; the K562 engineering cells are mixed with PBMC to perform cultivation after being inactivated, so that the NK cells can be amplified in a serum-free medium under the presence of IL-2, and the amplification multiples can be about 800-1,100; the effective purity of the NK cells can reach 91%, and the secretion volume of IFN-gamma can be five times that of the NK cells cultivated by common methods; and the killing rate of the NK cells on hepatoma carcinoma cell HepG2 can reach 88.5% under the condition that effector-target ratio is 10: 1, which is about 20% higher than that of the NK cells cultivated under normal conditions, and therefore, the NK cells cultivated by the method have stronger killing abilities.

Description

technical field [0001] The invention belongs to the field of cell expansion, and in particular relates to a method for efficiently expanding NK cells in vitro. Background technique [0002] Natural killer cells (NK cells) are a special group of lymphocytes different from T and B cells. The killing effect of NK cells on target cells is an immediate effect, without prior sensitization, and the killing effect will be exerted within 4 hours after mixing with target cells. [0003] NK cells can not only directly kill target cells without the restriction of major histocompatibility complex (MHC), but also interfere with virus replication and further activate phagocytes by secreting cytokines such as IFN-γ and TNF-α, and can also activate phagocytes through antibodies. Antibody-dependent cell-mediated cytotoxicity (ADCC) kills tumor cells coated with specific IgG. [0004] Therefore, NK cells play a very important role in anti-infection and anti-tumor therapy. However, NK cells,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867
CPCC07K14/54C07K14/70575C12N5/0646C12N15/86C12N2740/15043C12N2510/02C12N2501/2302Y02A50/30
Inventor 梁晨谭毅
Owner 山东省齐鲁细胞治疗工程技术有限公司
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