Short-hairpin-RNA-mediated silencing of fucosyltransferase 8 in Chinese-hamster ovary cells for the production of antibodies with enhanced antibody immune effector function

Biotechnol Appl Biochem. 2009 May;53(Pt 1):31-7. doi: 10.1042/BA20080220.

Abstract

Antibody-producing Chinese-hamster ovary cells (CHO-DG44) were converted into cells producing antibodies with strongly enhanced ADCC (antibody-dependent cellular cytotoxicity) by knocking down FuT8 (alpha-1,6-fucosyltransferase or fucosyltransferase 8) via constitutive expression of shRNA (short-hairpin RNA) against FuT8. After the introduction of a FuT8 shRNA expression plasmid under the control of a U6 promoter, CHO-DG44 clones with less than 5% residual FuT8 mRNA expression were isolated by selection for neomycin resistance, followed by low affinity nerve growth factor receptor enrichment and selection for LCA [Lens culinaris (culinary lentil) agglutinin] resistance. The CHO-DG44 clones identified produced highly afucosylated anti-[IGF-1R (insulin-like-growth-factor-1 receptor)] antibodies (up to 88%) that exhibited considerably enhanced ADCC compared with anti-IGF-1R wild-type antibodies produced by parental CHO cells. At the same time, antibody productivity was not significantly decreased. Analysis of stability showed that the clones obtained may be suitable for up-scaling, since low residual levels of FuT8 mRNA and production of afucosylated antibodies were maintained for at least 4 weeks.

MeSH terms

  • Animals
  • Antibodies / immunology*
  • Antibody Formation
  • Antibody-Dependent Cell Cytotoxicity*
  • CHO Cells
  • Cloning, Molecular
  • Cricetinae
  • Cricetulus
  • Fucosyltransferases / genetics*
  • Fucosyltransferases / metabolism
  • Gene Expression
  • RNA Interference*
  • RNA, Small Interfering*
  • Receptor, IGF Type 1 / immunology*
  • Receptor, IGF Type 1 / metabolism
  • Recombinant Proteins / immunology
  • Recombinant Proteins / metabolism

Substances

  • Antibodies
  • RNA, Small Interfering
  • Recombinant Proteins
  • Fucosyltransferases
  • Glycoprotein 6-alpha-L-fucosyltransferase
  • Receptor, IGF Type 1