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Induced T-to-natural killer feeder cell as well as preparation method and application thereof

A feeder cell, NK cell technology, applied in the biological field, to achieve the effect of strong tumor killing ability and stable killing function

Pending Publication Date: 2021-04-02
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, reprogrammed NK cells are different from NK cells. They have dual functions of T cells and NK cells. At present, there is no effective method for in vitro expansion and activation of reprogrammed NK cells.

Method used

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  • Induced T-to-natural killer feeder cell as well as preparation method and application thereof
  • Induced T-to-natural killer feeder cell as well as preparation method and application thereof
  • Induced T-to-natural killer feeder cell as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Construction of reprogrammed NK cells

[0047] (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;

[0048] 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.

[0049] (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;

[0050] 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...

Embodiment 2

[0052] Example 2 Construction of reprogrammed NK feeder cells

[0053] (1) Artificially synthesized T2A-linked nucleic acid molecules containing genes encoding IL-12 and CD8α transmembrane regions, CD19, CD64 and CD86, and added EcoRI and BamHI restriction sites and their protective bases at both ends;

[0054] Use restriction endonucleases EcoRI and BamHI to perform double digestion on nucleic acid molecules, incubate in a water bath at 37°C for 30 minutes, and use 1.5% agarose gel electrophoresis to recover the digested products containing sticky ends;

[0055] The digested product was ligated into the linearized pLVX-EF1-MCS plasmid (containing cohesive ends) digested with EcoRI and BamHI, and the ligation system was shown in Table 1 to obtain a lentiviral vector.

[0056] Table 1

[0057] components Dosage (μL) pLVX-EF1-MCS plasmid 2(50ng) IL-12-CD19-CD64-CD86 nucleic acid molecule 10(150ng) T4 DNA Ligation Buffer 2 T4 DNA Ligase (NEB)...

Embodiment 3

[0061] Example 3 In vitro expansion of reprogrammed NK cells

[0062] In this example, the reprogrammed NK 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, add an equal proportion of reprogrammed NK feeder cells to the culture medium on day 0 and day 7, and add IL-2 at a concentration of 50 U / mL, at 37°C, 5% CO 2 Co-cultivation in the incubator under 500Gy irradiation conditions.

[0063] Such as figure 1 As shown, after the reprogrammed NK cells were co-cultured with IL-12-CD19-CD64-CD86 reprogrammed NK feeder cells, the number of cells increased significantly. After 2 weeks of culture, the number of reprogrammed NK cells increased by about 120 times, which was significantly higher than that of the control Group.

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Abstract

The invention provides an induced T-to-natural killer feeder cell as well as a preparation method and application thereof. The induced T-to-natural killer feeder cell expresses membrane proteins IL-12, CD19, CD64 and CD86. The induced T-to-natural killer feeder cell is constructed by adopting combination of the IL-12, the CD19, the CD64 and the CD86 for induced T-to-natural killer cell culture, and the obtained induced T-to-natural killer cell is high in quantity increasing speed, remarkable in tumor killing capacity and good in functional stability, and has important application prospects inthe field of immunotherapy.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a reprogrammed NK feeder cell and its preparation method and application, in particular to a reprogrammed NK feeder cell, its preparation method and its application in culturing reprogrammed NK cells. Background technique [0002] Reprogrammed NK cells (induced T-to-natural killer cells, ITNK cells) are transgenic cells with dual functions of T cells and NK cells obtained by gene editing T cells using CRISPR / Cas9 technology. Compared with T cells or NK cells, reprogrammed NK cells do not depend on histocompatibility complex (MHC), have significantly improved tumor killing function, and show great potential in cancer therapy. [0003] At present, a major obstacle restricting the application of reprogrammed NK cells in immunotherapy is that it is difficult to obtain sufficient and fully functional reprogrammed NK cells. The large-scale expansion of reprogrammed NK cells in vitro is a key...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867C12N13/00
CPCC12N5/0694C12N5/0646C12N5/0636C07K14/70503C07K14/70535C07K14/70532C12N15/86C12N13/00C07K14/82C12N2510/00C07K2319/03C12N2740/15043C12N2800/107
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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