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Establishment and application of CHO cell line for producing fucose-free monoclonal antibody

A monoclonal antibody, fucus-free technology, applied in the field of bioengineering, can solve the problems of time-consuming, high cost, cumbersome operation, etc., and achieve the effect of simple operation, low time cost and effective editing

Pending Publication Date: 2017-05-24
SHANGHAI JIAO TONG UNIV +2
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  • Abstract
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  • Claims
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Problems solved by technology

[0011] The purpose of the present invention is to overcome the disadvantages of cumbersome operation, long time consumption and high cost in the above-mentioned prior art, and provide a CHO cell line establishment and its application for producing fucose-free monoclonal antibody

Method used

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  • Establishment and application of CHO cell line for producing fucose-free monoclonal antibody
  • Establishment and application of CHO cell line for producing fucose-free monoclonal antibody
  • Establishment and application of CHO cell line for producing fucose-free monoclonal antibody

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Embodiment

[0053] This example relates to the establishment and application of a CHO cell line producing a fucose-free monoclonal antibody; its construction process and analysis process are as follows figure 1 , 2 As shown, the specific steps are as follows:

[0054] 1: Construction of FUT8 knockout vector PX330-sgRNA plasmid

[0055] From the protein sequence (NCBI: XP_003501783.1, SEQ ID NO.12) corresponding to the FUT8 gene (its mRNA sequence (CHO-WT) is shown in SEQ ID NO.1), it can be seen that the amino acid position corresponding to exon9 has The function is the binding site of GDP-fucose, which can transfer GDP-fucose to the N-acetylglucosamine core site of the expressed protein. If the gene sequence is mutated, the function of α-1,6 fucosyltransferase can be lost.

[0056] The pX330 plasmid contains human codon-optimized streptococcal hspCas9 endonuclease coding sequence and U6 RNA polymerase promoter sequence. The schematic diagram of the partial structure of the plasmid is...

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Abstract

The invention discloses establishment and application of a CHO cell line for producing a fucose-free monoclonal antibody. A CRISPR / Cas9 technology is utilized to encode an FUT8 gene of a CHO cell to obtain the FUT8 gene silenced CHO cell line for stabilizing FUT8 gene deletion (hereinafter referred to as FUT8- / -cell line). By adopting physicochemical property and bioactivity detection of an FUT8- / -cell expressed antibody, a glycoform detection result indicates that fucose of the FUT8- / -cell expressed monoclonal antibody is completely eliminated, a fucose-containing monoclonal antibody expressed by a wild CHO cell serves as a contrast, the affinity with a Fcgamma RIIIa antigen of the fucose-free monoclonal antibody is improved by 7 times, and the ADCC effect is improved by 25 times.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to the establishment and application of a CHO cell line for producing fucose-free monoclonal antibodies. Background technique [0002] The initial use of antibodies in disease treatment can be traced back a century ago, but its rapid development was mainly in the past 30 years. In 1975, Koehler and Milstein founded the in vitro hybridoma technology, obtained mouse-derived monoclonal antibodies, and started a new era of polyclonal antibodies moving towards monoclonal antibodies (Wang Zhiming, Gao Jian and Li Geng, Therapeutic monoclonal antibody drugs Current situation and development trend. China Biotechnology Journal, 2013(06): p.117-124). Monoclonal antibodies have the advantages of high specificity, high titer, high purity, strong reproducibility, low cost, and mass production. However, the application of mouse-derived monoclonal antibodies to humans has strong...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/113C12N5/10C07K16/44
CPCC07K16/44C07K2317/51C07K2317/515C12N15/113C12N15/85C12N2310/10C12N2800/107
Inventor 朱建伟宗会芳张宝红江华谢跃庆韩雷丁凯孙涛
Owner SHANGHAI JIAO TONG UNIV
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