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Method for in-vitro large-scale induction of NK cell expansion

A large-scale technology of NK cells, applied in the biological field, can solve the problems of NK cell expansion, artificial antigen-presenting cells complex and cumbersome, etc., and achieve obvious tumor cell killing effect, stable and efficient cytotoxicity, and good cell proliferation.

Active Publication Date: 2019-10-15
侯宗柳
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the expansion methods at home and abroad are different and cumbersome. Therefore, the acquisition of high-quality NK cells is still the key to restricting their clinical application. It is a huge task to generate a large number of NK cells that meet GMP (Good Manufacturing Practice) standards for the treatment of patients. challenge
[0004] CN 108251376 A discloses an artificial antigen-presenting cell and its construction method and its application in the expansion of chimeric antigen receptor T cells. However, research has found that the constructed artificial antigen-presenting cells cannot expand NK cells , and the artificial antigen-presenting cells are relatively complex and costly, and at the same time, the C expansion of D19-CAR T cells is slow
[0005] CN 108300693 A discloses a method for in vitro expansion of natural killer cells. The NK cells amplified by this method have poor antitumor activity, and only when NK cells:tumor target cells=10:1 can they produce good antitumor effects , not conducive to clinical application
At the same time, the study found that the expanded NK cells had poor tumoricidal activity under hypoxic conditions.

Method used

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  • Method for in-vitro large-scale induction of NK cell expansion
  • Method for in-vitro large-scale induction of NK cell expansion
  • Method for in-vitro large-scale induction of NK cell expansion

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Embodiment 1

[0054] Example 1 Construction method of artificial antigen-presenting cells CD86CD64mIL-15CD19-K562.

[0055] In the present invention, unless otherwise stated, the percentage symbols represent volume percentages.

[0056] 1. Obtain the target sequence fragment by PCR method

[0057] 1) Obtain CD86, CD64 and mIL-15 genes by using the method of total gene synthesis.

[0058] 2) Primer design, the upstream and downstream primers of the target gene are respectively added with the homologous sequences on both sides of NotI and BamHI on the LV6 vector, which are used for subcloning of the vector. The primer sequences of CD86, CD64 and mIL-15 genes are as follows:

[0059] Table 1 CD64 gene primer sequence

[0060]

[0061]

[0062] Table 2 CD86 gene primer sequence

[0063]

[0064]

[0065]

[0066] Table 3 m-IL15 gene primer sequence

[0067]

[0068]

[0069] 3) The above oligos were synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd.

[00...

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Abstract

The invention relates to a method for in-vitro large-scale induction of NK cell expansion, and belongs to the technical field of biology. According to the invention, the constructed novel artificial antigen presenting cell CD86 CD64 mIL-15-aAPC is taken as an expanded feeder cell, NK cells are directly expanded from peripheral blood lymphocytes, and the NK cells expanded by the method has the advantages of good proliferation, high purity, strong cytotoxicity and obvious tumor cell killing effect. The invention solves the problems of limited number of cell expansion, low expression level of surface antigen CD3- / CD56+ and low cell killing activity faced by traditional culture methods, and can meet the clinical requirement on NK cells.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for inducing NK cell expansion on a large scale in vitro. Background technique [0002] Natural killer cells (natural killer cells, NK) are mainly distributed in peripheral blood, accounting for 5-10% of PBMC. NK cells are important immune cells in the body, which are related to anti-tumor, anti-viral infection and immune regulation. In recent years, with the development of immunology and molecular biology technology, cell biological therapy has become the fourth mode of tumor treatment. At present, various studies have shown that NK cells have the ability to recognize and lyse transformed tumor cells without any antigen stimulation, and play an important role in tumor immunity. Although they can quickly mount a defense and attack tumor cells directly, NK cells are only a small part of the immune system, making up only 10 percent of white blood cells. NK cells are an impo...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C12N5/10C12N15/867
CPCC12N5/0646C12N15/86C12N2500/90C12N2501/2312C12N2501/2315C12N2501/599C12N2502/1114C12N2740/15043
Inventor 侯宗柳王文举黄文文李琳孟明耀王晓丹赵旖旖唐维伟解燕华何珊
Owner 侯宗柳
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