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

被引：19

作者：

Beuger, Vincent ^{[1
]}

Kuenkele, Klaus-Peter ^{[1
]}

Koll, Hans ^{[1
]}

Gaertner, Achim ^{[1
]}

Baehner, Monika ^{[1
]}

Burtscher, Helmut ^{[1
]}

Klein, Christian ^{[1
]}

机构：

[1] Roche Diagnost GmbH, D-82372 Penzberg, Germany

来源：

BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY | 2009年 / 53卷

关键词：

antibody; Chinese-hamster ovary cells (CHO cells); ESI-MS; electrospray-ionization MS; fucosyltransferase 8 (Fut8); gene silencing; immune effector function; short-hairpin RNA (shRNA); FC-GAMMA-RIIIA; NONFUCOSYLATED THERAPEUTIC ANTIBODIES; DEPENDENT CELLULAR CYTOTOXICITY; ANTI-CD20; MONOCLONAL-ANTIBODY; MANNOSE 4,6-DEHYDRATASE GMD; HUMAN IGG1; MAMMALIAN-CELLS; GLYCOFORMS; RECEPTORS; BINDING;

D O I：

10.1042/BA20080220

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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-IR (insulin-like-growth-factor-I receptor)] antibodies (up to 88%) that exhibited considerably enhanced ADCC compared with anti-IGF-IR 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.

引用

页码：31 / 37

页数：7

共 28 条

[1] Anti-CD20 monoclonal antibody with enhanced affinity for CD16 activates NK cells at lower concentrations and more effectively than rituximab [J].