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Protein applied to culture of NK cells, culture medium component combination and preparation method

A culture medium formula and NK cell technology, applied in the field of NK cell culture, can solve the problems of clinical trial limitations, high lethality, and affecting the use effect, etc., to avoid contamination of exogenous cell lines, high adaptability, improve efficiency and stability sexual effect

Active Publication Date: 2019-04-16
NOVOPROTEIN SCI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] NK cells have high application prospects due to their broad spectrum and high lethality, but the proportion of primary NK cells in PBMC (peripheral bloodmoNonuclear cells, peripheral blood mononuclear cells) is low, about 5-10%, and in vitro Expansion culture has always been a problem
At present, the most efficient method is the inactivated K562 trophoblast cells. After 2-3 weeks of culture, the NK positive rate can reach more than 90%, but the inactivated trophoblast cells are difficult to remove, and it is difficult to achieve therapeutic levels.
In the past two years, some recombinant cytokine culture programs have appeared on the market, most of which can achieve a stable positive rate of about 40-50%, and there will be a large number of T cells remaining, which will affect the subsequent use effect and may cause GVHD The risk of high-efficiency and stable NK non-trophoblast culture has always been a difficult problem for the program with a positive rate of more than 80%, or the amplification efficiency is average, or the individual differences are serious, making most of the treatment programs relying on NK are in the early stage of research , causing significant limitations to clinical trials

Method used

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  • Protein applied to culture of NK cells, culture medium component combination and preparation method
  • Protein applied to culture of NK cells, culture medium component combination and preparation method
  • Protein applied to culture of NK cells, culture medium component combination and preparation method

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1: Construction and expression purification of recombinant MICA-Ig eukaryotic expression vector

[0037] 1. Design of recombinant MICA-Ig eukaryotic expression construction scheme

[0038] In the present invention, human MICA (MHC class I polypeptide-related sequence A, histocompatibility complex I polypeptide-related sequence A) protein on the surface of tumor cells and the Fc segment of human immunoglobulin IgG1 are fused into a recombinant MICA-Ig protein , in order to secrete and express the recombinant MICA-Ig protein in mammalian cells, the 1-23 amino acids of the human MICA signal peptide were used to help its secretion and expression, and the 1-308 amino acids of the human MICA protein were combined with the human immunoglobulin The 99th-330th amino acids of protein IgG1 are linked by a flexible linker peptide IEGR.

[0039] The following sequences are all 5' to 3' unless otherwise specified.

[0040] Specifically, the nucleotide sequence of the human ...

Embodiment 2

[0056] Embodiment 2: Expression and purification of recombinant MICA-Ig

[0057] 1. Expression of recombinant MICA-Ig

[0058]1.1. The passage density of HEK 293F cells (purchased from Thermo Fisher Scientific) 1 day before transfection was 0.5-0.6×10 6 / ml;

[0059] 1.2. Count the cell density on the day of transfection, when the density is 1~1.4×10 6 / ml, when the activity is >90%, it can be used for plasmid transfection;

[0060] 1.3. Preparation of transfection complex:

[0061] Two centrifuge tubes / bottles need to be prepared, take 20ml as an example, place them separately, and take the recombinant plasmid prepared in Example 1:

[0062] Add 600μl PBS and 20μg recombinant plasmid to tube ①, mix well;

[0063] Add 600μl PBS, 20ul FreeStyle to tube ② TM MAX Transfection Reagent (purchased from Thermo Fisher Scientific company), mixing;

[0064] 1.4. Add the diluted transfection reagent to the diluted recombinant plasmid, mix well, and prepare a transfection complex; ...

Embodiment 5

[0099] Embodiment 5 NK cell in vitro expansion culture

[0100] Take blood from 2 healthy volunteers, use Ficoll (purchased from GE) to separate PBMCs, and after cell counting, adjust the cell density to 2X10 with X vivo 15 (purchased from Lonza) medium 6 / ml, the cells were added to the pre-used 4-1BBL working concentration of 1-10ug / ml (product number: CS18, purchased from Shanghai Jinan Technology Co., Ltd.), NovoNectin working concentration of 5-50ug / ml (product number: CH38, purchased from Shanghai Nearshore Technology Co., Ltd.) and MICA-Ig (using the methods of the above-mentioned examples 1 to 4, self-made) working concentration of 2-20ug / ml in the orifice plate, add culture factor IL-18 working concentration of 10-500ng / ml ( Product number: CD72, purchased from Shanghai Jinan Technology Co., Ltd.), IL15RA&IL15 fusion protein (self-made) working concentration 10-500ng / ml, anti-HER2 working concentration 0.1-10ug / ml (product number: GMP-A062, purchased from Shanghai Jin...

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Abstract

The invention provides protein applied to culture of NK cells, a culture medium component and a preparation method, and further relates to application of the protein. The protein and the culture medium component combination are used for culture of the NK cells to avoid exogenous cell line contamination of trophoblast cells and improve the efficiency and stability of NK culture, and the cultured NKcells are high in purity and adaptability. On one hand, the protein is applied to culture of the NK cells, and is MICA-Ig, and the MICA-Ig is a recombined MICA-Ig protein which is formed by fusing human MICA protein and the Fc segment of human immune globulin IgG1 of the surface of a tumor cell.

Description

technical field [0001] The invention belongs to the field of molecular and cell biology, and particularly relates to NK cell culture. Background technique [0002] NK (natural killer, natural killer) cells are a kind of toxic lymphocytes that are crucial in the innate immune system, can respond quickly to virus infection, and have an effect on tumor cells at the same time. Unlike other killer immune cells, NK cells are unique in that they can quickly recognize and respond to stressed cells without antibodies or MHC (majorhistocompatibility complex, major histocompatibility complex). In recent years, CAR-T (Chimeric antigen receptors-T cell, chimeric antigen receptor-T cells) has become a research hotspot, which can effectively treat blood cancers, but high-risk side effects and limited role in solid tumors have become CAR-T T development constraints. Several recent studies have shown that CAR-NK (Chimeric antigen receptors-T cell, chimeric antigen receptor-NK cells) has lo...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K1/22C12N15/62C12N15/85C12N5/0783
CPCC12N5/0646C12N15/85C07K14/47C07K2319/30C12N2501/50C12N2501/599C12N2501/2318C12N2501/2315C12N2501/165C12N2501/2302C12N2501/998
Inventor 李德彬崔利兰王笃强周哲
Owner NOVOPROTEIN SCI INC
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