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

CN106701823APending Publication Date: 2017-05-24SHANGHAI JIAO TONG UNIV +2
5 Cites 45 Cited by

Patent Information

Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Publication Date
2017-05-24

Smart Images

  • Figure 1
    Figure 1
  • Figure 2
    Figure 2
  • Figure 3
    Figure 3
Patent Text Reader

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.
Need to check novelty before this filing date? Find Prior Art

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...

Examples

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...