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Method for knocking out FUT8 gene

A gene and gene silencing technology, applied in genetic engineering, plant genetic improvement, chemical instruments and methods, etc., can solve problems such as mediating ADCC activity, and achieve the effect of low time cost and easy operation.

Pending Publication Date: 2022-05-10
SHENGHE CHINA BIOPHARMACEUTICAL CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Trastuzumab cannot effectively mediate ADCC activity in patients with CD16a 158F / F type, which is also a reason why trastuzumab is ineffective in some patients

Method used

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  • Method for knocking out FUT8 gene
  • Method for knocking out FUT8 gene
  • Method for knocking out FUT8 gene

Examples

Experimental program
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Effect test

Embodiment

[0045] This embodiment relates to a method for knocking out the FUT8 gene to improve the ADCC activity of an antibody, and the specific steps are as follows:

[0046] 1. Construction of anti-HER2 antibody expression vector

[0047] The target gene expression vector is the CHO matching vector pCHO1.0, and the DHFR amplification system is used. The target gene carrier sequence is synthesized by codon optimization and cloned into the expression vector. In the cloned plasmid, the promoters of the light and heavy chains of the antibody are CMV / EF1 and EF2 / CMV hybrid promoters, respectively, and the terminators are simian vacuolus virus PloyA (SV40 PA) and cytomegalovirus PloyA (CMVPA), respectively. Contains a puromycin resistance gene (PuroR) and a kanamycin resistance gene (KanR) as selectable markers. The restriction restriction map verification and sequence verification of the expression vector were carried out, and the results showed that the restriction restriction map was ...

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Abstract

The invention discloses a method for knocking out an FUT8 gene to improve the ADCC activity of an antibody. And coding the FUT8 gene of the CHO cell by using a CRISPR / Cas9 technology to obtain the CHO cell silenced by the FUT8 gene. Through detection of physicochemical properties and biological activity of the engineered cell expression antibody, a glycoform detection result shows that fucose of the engineered cell expression antibody is completely eliminated, and by taking trastuzumab as a contrast, the affinity of the engineered cell expression complete fucose-free antibody and Fc gamma Rs antigen is improved by 20 times, and the ADCC effect is improved by 19 times.

Description

technical field [0001] The invention relates to the biological field, and in particular provides a method for knocking out the FUT8 gene to improve the ADCC activity of an antibody. Background technique [0002] ADCC (antibody dependent cellular cytotoxicity, antibody-dependent cell-mediated cytotoxicity) refers to a series of immune responses triggered by NK (natural killer) cells. NK cells bind to the constant region (Fc) of antibody molecules through FcγRIII receptors, Cellular degradation and apoptosis are then induced on the target cell surface by perforin and granzymes. Studies have shown that when fucose is removed from the sugar chain structure of the CH2 domain of the Fc fragment of IgG antibodies, the binding force between FcγRIIIa of NK cells and the Fc fragment of IgG antibodies can be enhanced, thereby enhancing the ADCC effect of antibodies. [0003] Existing studies have also shown that Fc receptors on the surface of immune cells have genetic polymorphisms, a...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10C12N15/54C07K16/32A61K39/395A61P35/00
CPCC12N15/85C12N5/0682C12N9/1051C12Y204/01068C07K16/32A61P35/00C07K2317/14C07K2317/92C07K2317/732C12N2510/02A61K2039/505
Inventor 姜晓玲周冲吴崇兵高超马昀肖哲媛
Owner SHENGHE CHINA BIOPHARMACEUTICAL CO LTD
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