Double knockdown of α1,6-fucosyltransferase (<it>FUT8</it>) and GDP-mannose 4,6-dehydratase (<it>GMD</it>) in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC

• Main Authors: Shitara Kenya, Iida Shigeru, Wakitani Masako, Inoue Miho, Mori Katsuhiro, Imai-Nishiya Harue, Satoh Mitsuo
• Format: Article
• Language: English
• Published: BMC 2007-11-01
• Series: BMC Biotechnology
• Online Access: http://www.biomedcentral.com/1472-6750/7/84

<p>Abstract</p> <p>Background</p> <p>Antibody-dependent cellular cytotoxicity (ADCC) is greatly enhanced by the absence of the core fucose of oligosaccharides attached to the Fc, and is closely related to the clinical efficacy of anticancer activity in humans <it>in vivo</it>. Unfortunately, all licensed therapeutic antibodies and almost all currently-developed therapeutic antibodies are heavily fucosylated and fail to optimize ADCC, which leads to a large dose requirement at a very high cost for the administration of antibody therapy to cancer patients. In this study, we explored the possibility of converting already-established antibody-producing cells to cells that produce antibodies fully lacking core fucosylation in order to facilitate the rapid development of next-generation therapeutic antibodies.</p> <p>Results</p> <p>Firstly, loss-of-function analyses using small interfering RNAs (siRNAs) against the three key genes involved in oligosaccharide fucose modification, i.e. α1,6-fucosyltransferase (<it>FUT8</it>), GDP-mannose 4,6-dehydratase (<it>GMD</it>), and GDP-fucose transporter (<it>GFT</it>), revealed that single-gene knockdown of each target was insufficient to completely defucosylate the products in antibody-producing cells, even though the most effective siRNA (>90% depression of the target mRNA) was employed. Interestingly, beyond our expectations, synergistic effects of <it>FUT8 </it>and <it>GMD </it>siRNAs on the reduction in fucosylation were observed, but not when these were used in combination with <it>GFT </it>siRNA. Secondly, we successfully developed an effective short hairpin siRNA tandem expression vector that facilitated the double knockdown of <it>FUT8 </it>and <it>GMD</it>, and we converted antibody-producing Chinese hamster ovary (CHO) cells to fully non-fucosylated antibody producers within two months, and with high converting frequency. Finally, the stable manufacture of fully non-fucosylated antibodies with enhanced ADCC was confirmed using the converted cells in serum-free fed-batch culture.</p> <p>Conclusion</p> <p>Our results suggest that FUT8 and GMD collaborate synergistically in the process of intracellular oligosaccharide fucosylation. We also demonstrated that double knockdown of <it>FUT8 </it>and <it>GMD </it>in antibody-producing cells could serve as a new strategy for producing next-generation therapeutic antibodies fully lacking core fucosylation and with enhanced ADCC. This approach offers tremendous cost- and time-sparing advantages for the development of next-generation therapeutic antibodies.</p>