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Engineered cell for activating NK-like cell, and preparation method and application of engineered cell

An engineering and cellular technology, applied in the field of genetic engineering, to achieve the effects of improving proliferation, enhancing tumor killing ability, and significant tumor cytotoxicity

Pending Publication Date: 2021-04-09
广东昭泰细胞生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, NK-like cells are different from NK cells, which have dual functions of T cells and NK cells. At present, there is no effective method for expanding and activating NK-like cells in vitro.

Method used

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  • Engineered cell for activating NK-like cell, and preparation method and application of engineered cell
  • Engineered cell for activating NK-like cell, and preparation method and application of engineered cell
  • Engineered cell for activating NK-like cell, and preparation method and application of engineered cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Construction of Example 1 NK-like cells

[0048] (1) Design sgRNA (SEQ ID NO:6:gaccatgaactgctcacttg) according to the Bcl11b gene, and add restriction enzyme sites EcoRI and SalI at the 5' end and 3' end of the sequence after artificial synthesis;

[0049] The synthesized sequence fragment was digested with EcoRI and SalI, then connected to the PX458-gBCL11b vector containing the T7 promoter, and the recombinant plasmid was successfully constructed by sequencing.

[0050] (2) will 1×10 7 T cells were resuspended in 3 mL of T cell medium, seeded in one well of a 6-well plate, and activated by adding a combination of 100 ng / mL anti-human CD3 antibody and 100 ng / mL anti-human CD28 antibody;

[0051] After 3 days, remove the suspended cells for counting, centrifuge at 300×g for 10 min, resuspend the cell pellet in 10 mL of Opti-MEM, centrifuge at 300×g for 10 min, resuspend the cell pellet in 100 μL of Opti-MEM, add CRISPR at a concentration of 40 μM / Cas9 plasmid, after ...

Embodiment 2

[0053] Example 2 Construction of engineered cells

[0054] (1) Artificially synthesize T2A-linked nucleic acid molecules containing MICA-encoding genes, IL-12 and CD28 transmembrane regions, CD64 and CD86-encoding genes, and add Pme1 and Spe1 restriction sites and their protective bases at both ends ;

[0055] The nucleic acid molecules were double-digested with restriction endonucleases Pme1 and Spe1, incubated in a water bath at 37°C for 30 minutes, and recovered by 1.5% agarose gel electrophoresis to obtain the digested products containing sticky ends;

[0056] The digested product was ligated into the linearized pWPXLd-eGFP plasmid (containing cohesive ends) digested by Pme1 and Spe1, and the ligation reaction was carried out with the participation of T4 DNA polymerase (Invitrogent) to obtain a lentiviral vector.

[0057] (2) Use 293T cells to prepare recombinant lentivirus. When 293T cells are spread on the bottom of a 100mm culture dish to reach 80-90%, carry out lentiv...

Embodiment 3

[0065] Example 3 In vitro expansion of NK-like cells

[0066] In this embodiment, NK-like cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, and the cells were counted after 24 hours of culture, and the cell concentration was adjusted to 2×10 6 cells / mL, an equal proportion of engineered cells were added to the culture medium on day 0, followed by a culture medium containing 2×10 6 Each / mL engineered cells, 10% fetal bovine serum and 50 U / mL IL-2 in RPMI-1640 medium continued to culture for 14 days at 37°C, 5% CO 2 Co-cultivation in the incubator under 500Gy irradiation conditions.

[0067] Such as figure 1 As shown, after NK-like cells were co-cultured with MICA-IL-12-CD64-CD86 engineered cells, the number of cells increased significantly. After 2 weeks of culture, the number of NK-like cells increased by about 100 times, which was significantly higher than that of the control group.

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Abstract

The invention provides an engineered cell for activating a NK-like cell, and a preparation method and application of the engineered cell. The engineered cell is a mammalian cell for expressing membrane proteins MICA, IL-21, CD64 and CD86. The engineered cell disclosed by the invention expresses and activates the membrane protein of the NK-like cell; the amplification capacity of the NK-like cell and the toxicity of tumour cells are improved; and a strategy for standardizing in-vitro amplification of the NK-like cell is provided.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to an engineered cell for activating NK-like cells, a preparation method and application thereof. Background technique [0002] NK-like cells are cells obtained by reprogramming T cells using CRISPR / Cas9 technology. NK-like cells have dual functions of T cells and NK cells, and do not depend on the major histocompatibility complex (MHC). Compared with T cells or NK cells have significantly enhanced tumor killing function and have shown great potential in cancer therapy. [0003] A major obstacle to the application of NK-like cells in immunotherapy is the difficulty of producing large numbers of fully functional NK-like cells and the lack of standard methods for expanding NK-like cells in vitro. At present, the commonly used culture methods of NK cells include blood cell separation method, cytokine or chemical drug stimulation method and feeder cell method. Very suitable ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867C12N7/01C12N13/00A61K35/17A61P35/00C12R1/93
CPCC12N5/0694C12N15/86C12N7/00C12N5/0646C12N5/0636C12N13/00C07K14/70539C07K14/54C07K14/70535C07K14/70532A61K35/17A61P35/00C12N2501/51C12N2501/599C12N2501/2321C12N2501/50C07K2319/03C12N2740/15043C12N2510/00C12N2800/107C12N2740/15021C12N2502/99Y02A50/30
Inventor 汤朝阳秦乐吴迪冯世忠冯嘉昆杨乐旋其他发明人请求不公开姓名
Owner 广东昭泰细胞生物科技有限公司
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