Short-hairpin-RNA-mediated silencing of fucosyltransferase 8 in Chinese-hamster ovary cells for the production of antibodies with enhanced antibody immune effector function.
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.
Beuger
Vincent
V
Roche Diagnostics GmbH, Nonnenwald 2, 82372 Penzberg, Federal Republic of Germany.
Künkele
Klaus-Peter
KP
Koll
Hans
H
Gärtner
Achim
A
Bähner
Monika
M
Burtscher
Helmut
H
Klein
Christian
C
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000917:Antibody Formation; D000920:Antibody-Dependent Cell Cytotoxicity; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D005647:Fucosyltransferases / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D034622:RNA Interference; D034741:RNA, Small Interfering; D017526:Receptor, IGF Type 1 / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism
D000906:Antibodies; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D005647:Fucosyltransferases; C020559:Glycoprotein 6-alpha-L-fucosyltransferase; D017526:Receptor, IGF Type 1