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A kind of anti-tumor NK cell and its preparation method

A technology of NK cells and genetic modification, applied in the field of new high-efficiency anti-tumor NK cells and its development, can solve the problems of high toxicity and side effects, application limitations, and difficult control of cytokine expression levels

Active Publication Date: 2021-07-02
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the concentration of cytokines used in the whole body to act on NK cells is not high, and the toxic and side effects are large; transgenic technology makes certain cells, such as tumor cells, highly express these cytokines, but the disadvantage is that the expression level of cytokines is difficult to control, and the direct effect The concentration in NK cells is not high
More importantly, the mode of cytokine transgene modification is controlled by the restriction of major histocompatibility complex gene MHC molecules, which limits its application

Method used

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  • A kind of anti-tumor NK cell and its preparation method
  • A kind of anti-tumor NK cell and its preparation method
  • A kind of anti-tumor NK cell and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1: IL2, IL12 lentiviral vector plasmid construction

[0061] 1.1 Design gRNA primers (see Table 1)

[0062] Principle of gene editing technology: the present invention uses a transcription-activating CRISPR-CAS9 system to increase the transcriptional activity of IL-2 and IL-12. In the present invention, IL-12A and IL-12B refer to the two subunits of IL-12. The principle is as follows Figure 11 shown, outlined below in Section 3.1.

[0063] According to the aforementioned principle, the gene manipulation of IL-2, IL-12A, and IL-12B was carried out. The method and process were as follows: the IL-2 target genes were respectively obtained from the database of the National Center for Information Technology (NCBI) in the United States (GenBank number: NM_000586) , IL-12A target gene (GenBank number: NM_000882), IL-12B target gene (GenBank number: NM_002187) transcription start site upstream 5KB DNA fragment sequence design and synthesis of sgRNA, according to http: / ...

Embodiment 2

[0074] Example 2: NK92-IL2-IL12 cell line construction

[0075] Method and process:

[0076] Culture NK92 cells in a T25 culture flask (α-MEM medium, 12.5% ​​FBS, 12.5% ​​horse serum, 1% penicillin and streptomycin, 100U / ml IL2), and take the NK92 cells in good growth state to infect, and the cell concentration is about 65-80%. Set the volume to 2ml in the T25 culture bottle, take centrifuged recombinant virus solution 3ml (about 10^6 of virus titer) and 50 μl of concentrated virus solution (about 10^7 of virus titer) prepared in Example 1 after centrifugation and change after 12h hours solution, replaced with 5ml of complete medium, and observed the cell infection efficiency under a fluorescent microscope after 48h and 72h. In order to further improve the transfection efficiency, 5ul polybrene was added at a ratio of 1:1000 to improve the infection efficiency. At the same time, repeated infection could be repeated at intervals of two days; After adding 5 μl of puromycin (co...

Embodiment 3

[0079] Embodiment 3: ELISA and qPCR expression and identification

[0080] Method and process:

[0081] Take the logarithmic growth phase lentivirus transfection group (infected with the recombinant lentivirus described in Example 1) and the wild type group NK92 cells (α-MEM medium, 12.5% ​​FBS, 12.5% ​​horse serum, 1% Streptomyces penicillin prime, 100U / ml IL2 (1000pg / ml)), count 10 7 Add one to a T25 culture flask, dilute to 5ml, place at 37°C, 5% CO 2 After culturing in the incubator for 48 hours, the culture medium was taken by centrifugation, and the expression levels of IL2 and IL12 proteins were detected by referring to the ELISA kit protocol. Each group was divided into blank medium group, factor modification group, and wild-type NK92 group with 3 pairs of wells for medium detection. ;See Figure 5 . The cells in the factor modification group and wild-type NK92 group were extracted by Triol method, reverse-transcribed into cDNA by Takara kit, and the expression lev...

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Abstract

The present invention uses CRISPR-CAS9 gene editing technology to make NK92 constantly and highly express cytokines IL2 and IL12. Experiments show that the constantly highly expressed IL2 and IL12 act on NK92 cells themselves and other adjacent cells through an autocrine pathway, significantly improving the proliferation ability of NK92 cells and the killing activity against K562 tumor cells. Thus, the present invention has successfully developed a new type of high-efficiency NK cell capable of sustained proliferation and significantly enhanced anti-tumor activity.

Description

technical field [0001] The invention belongs to the field of medical immunotherapy, and relates to a novel high-efficiency anti-tumor NK cell with sustainable proliferation and its development. Background technique [0002] At present, tumor immunotherapy including CAR-T, NK cells and immune checkpoint inhibitors has become the most promising tumor treatment method. Among them, NK cells have a strong natural anti-tumor ability, and can recognize and attack tumor cells without prior sensitization of tumor cell antigens (targets), avoiding the obstacle that solid tumor cells lack ideal targets. However, the small number and low activity of activated NK cells in tumor tissues are the main obstacles to the efficacy of NK cell immunotherapy for solid tumors. [0003] The use of in vitro expansion to obtain high-quantity and high-purity NK cells has become a hot spot in the study of NK cell adoptive immunotherapy in recent years. With the improvement of the expansion method, the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/867C12N15/26C12N15/24C12N15/113A61K35/17A61P35/00
CPCA61K35/17A61P35/00C07K14/5434C07K14/55C12N5/0646C12N15/113C12N15/86C12N2310/10C12N2510/02C12N2740/15043
Inventor 黄常新王聪洁李永强杨丽丽张嗣玉苏萌高岚岚葛钻敏
Owner HANGZHOU NORMAL UNIVERSITY