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Method for preparing NK cells with stable and high expression of chimeric receptors by non-viral method

A chimeric receptor, non-viral technology, applied in the field of medical bioengineering, can solve the problems of low efficiency of chimeric receptor modification, regulatory obstacles, high production costs, etc., and achieve the effect of complex production process, high cost, and increased positive rate

Pending Publication Date: 2022-01-11
杭州朔溪生物医药有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] In order to overcome the problem of low efficiency of chimeric receptor modification of primary NK cells by current technical means, as well as the problems of regulatory obstacles and high production costs caused by the use of retroviruses, the present invention provides a non-viral preparation of chimeric receptors Receptor-modified NK cell method, using the transposon system to introduce stable and highly expressed chimeric receptors into NK cells to achieve efficient genetic modification of NK cells

Method used

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  • Method for preparing NK cells with stable and high expression of chimeric receptors by non-viral method
  • Method for preparing NK cells with stable and high expression of chimeric receptors by non-viral method
  • Method for preparing NK cells with stable and high expression of chimeric receptors by non-viral method

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Experimental program
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Effect test

Embodiment 1

[0079] Embodiment 1 vector construction

[0080] (1) Construction of vectors for preparing engineered K562-NK1 cells

[0081] DNA fragment 1: the nucleotide sequence of CMV promoter, the coding nucleotide sequence of puromycin, and the coding nucleotide sequence of EGFP were synthesized by an outsourced service company;

[0082] DNA fragment 2: CMV promoter nucleotide sequence, bmIL-15 coding nucleotide sequence, P2A coding nucleotide sequence, mbIL-21 coding nucleotide sequence were synthesized by an outsourced service company;

[0083] DNA fragment 1 and DNA fragment 2 were cloned into pFastBac1 plasmid (purchased from Thermofisher) and named as plasmid 1.

[0084] (2) Construction of CAR vector

[0085] In order to construct the NKG2D CAR vector, the extracellular antigen-binding domain (ED) of NKG2D was fused with the IgG4 hinge region, CD28 transmembrane region, 4-1BB and CD3ζ signaling domain to generate the second-generation NKG2D CAR vector; Cytometry was used to de...

Embodiment 2

[0097] Example 2 Preparation of engineered K562-NK1 cells

[0098] Resuspend wild-type K562 cells in 10 mL of Opti-MEM, centrifuge at 300×g for 10 min, resuspend the cell pellet in 100 μL of P3 buffer, add 10 μg of plasmid 1, mix well and transfer to Lonza electric shock cup;

[0099] Place the electric shock cup in the Lonza 4D-NucleofectorTM X Unit (in the single electric shock cup module) for electroporation. After the electroporation, slowly transfer the K562 cell suspension in a cuvette to a well of a 6-well plate, and add K562 medium (IMDM+10%FBS);

[0100] On day 5 after electroporation, puromycin selection was performed at a concentration of 200 μg / mL for 1 month, and the medium was changed every 2 days;

[0101] One month later, use the cell sorter BD FACSAria (BD Biosciences) to sort single cells into 96-well plates, and perform flow cytometry analysis on the amplified single cell clones to detect the expression of fusion genes and detect antibodies APC-bound Strep...

Embodiment 3

[0103] Example 3 Preparation of CAR-NK using piggyBac transposon system

[0104] The preparation flow chart is as image 3 Shown:

[0105] On day 0, the 5×10 6 PBMCs and 5×10 6 K562-NK1 (100Gy γ-ray irradiation) was resuspended in 10mL NK cell medium, inoculated in a T25 cell culture flask, and hrIL-2 was added to make the final concentration 100IU / mL;

[0106] On the second day, take out the suspended cells for counting, centrifuge at 300×g for 10min; resuspend the cell pellet in 10mL Opti-MEM, and centrifuge at 300×g for 10min; resuspend the cell pellet in 100μL P3 buffer, add 5μg plasmid 4 and 10μg Plasmid 3, mix well and transfer to the electric Lonza electric shock cup; place the electric shock cup in the Lonza 4D-NucleofectorTM X Unit (in the single electric shock cup module) for electroporation, after the electroporation, slowly transfer the NK cell suspension in a cuvette To a new T25 cell culture flask, add 10 mL of NK cell culture medium containing hrIL-2 at a fi...

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Abstract

The invention provides a method for preparing a natural killer (NK) cell with stable and high expression of chimeric receptors by non-viral method and application thereof, and the method comprises the following steps: (1) transferring a encoding gene of the chimeric receptor into the natural killer cell by using a transposon system to obtain an initial chimeric receptor modified natural killer cell; and (2) amplifying the initial chimeric receptor modified natural killer cells by using artificial antigen presenting cells. According to the invention, the stable and high-expression chimeric receptor is introduced into the NK cell by applying a transposon system, compared with the prior art, the method disclosed by the invention is simple and convenient to operate, rapid, efficient, accurate, good in repeatability, high in cost benefit and relatively good in safety, and the prepared NK cell modified by chimeric receptor has relatively strong tumor killing capability, shows a good anti-tumor effect in vitro, and has a wide application prospect in various fields of biomedicine, especially in the field of cell therapy of diseases.

Description

technical field [0001] The invention belongs to the technical field of medical bioengineering, and relates to a method for preparing stable and high-expression chimeric receptor-modified NK cells using a non-viral method and an application thereof. Background technique [0002] Natural killer (NK) cells are an important part of the innate immune system, and the proportion of NK cells in peripheral blood lymphocytes is about 10%, which is the third largest lymphocyte population after B cells and T cells. NK cells have a variety of biological functions and are the body's first line of defense against infection and tumors. NK cell function is regulated by a sophisticated and complex network of activating and inhibitory receptors. Most circulating NK cells are in a quiescent phase that can be activated by cytokines and infiltrate into pathogen-infected or malignant-cell-containing tissues. When the receptors of NK cells are combined with the corresponding ligands, NK cells can...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/90C12N15/62C12N5/10A61K39/00A61P35/00
CPCC12N15/85C12N15/907C07K16/2851C07K14/70521C07K14/70578C07K14/7051C12N5/0646A61P35/00C12N2800/107C12N2800/90C07K2319/03C07K2319/33C07K2319/74C12N2510/00A61K39/464429A61K2239/50A61K39/464417A61K2239/59A61K39/4631A61K2239/48A61K39/4613C12N5/10C12N15/62C12N15/90C07K14/705C07K19/00
Inventor 肖琳裘新红彭群武张亮朱苏闽徐芳芳
Owner 杭州朔溪生物医药有限公司
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