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

<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

Double knockdown of α1,6-fucosyltransferase () and GDP-mannose 4,6-dehydratase () in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC-1

<p><b>Copyright information:</b></p><p>Taken from "Double knockdown of α1,6-fucosyltransferase () and GDP-mannose 4,6-dehydratase () in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC"</p><p>http://www.biomedcentral.com/1472-6750/7/84</p><p>BMC Biotechnology 2007;7():84-84.</p><p>Published online 30 Nov 2007</p><p>PMCID:PMC2216013.</p><p></p> line 32-05-12 (filled squares) was cultured as a control. Viable cell density (A, solid lines), antibody concentration in the culture supernatant (A, dotted lines), and cell viability (B) were analyzed in the fed-batch culture. The oligosaccharide structures of the final products from 32-05-12 (C), FG1 (D), and FG16 (E) were analyzed using MALDI-TOF MS. The relative composition of each peak is shown as the relative amount to the total amount of oligosaccharide detected

Double knockdown of α1,6-fucosyltransferase () and GDP-mannose 4,6-dehydratase () in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC-0

<p><b>Copyright information:</b></p><p>Taken from "Double knockdown of α1,6-fucosyltransferase () and GDP-mannose 4,6-dehydratase () in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC"</p><p>http://www.biomedcentral.com/1472-6750/7/84</p><p>BMC Biotechnology 2007;7():84-84.</p><p>Published online 30 Nov 2007</p><p>PMCID:PMC2216013.</p><p></p>n or hygromycin resistance gene and a short hairpin siRNA expression cassette controlled by the human tRNApromoter (B or C). The siRNA tandem expression plasmid consisted of a hygromycin resistance gene and two short hairpin siRNA expression cassettes targeting and (D). The transcribed shRNAs and tRNA-shRNA fusion product were processed into siRNAs by Dicer

Double knockdown of α1,6-fucosyltransferase () and GDP-mannose 4,6-dehydratase () in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC-2

<p><b>Copyright information:</b></p><p>Taken from "Double knockdown of α1,6-fucosyltransferase () and GDP-mannose 4,6-dehydratase () in antibody-producing cells: a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC"</p><p>http://www.biomedcentral.com/1472-6750/7/84</p><p>BMC Biotechnology 2007;7():84-84.</p><p>Published online 30 Nov 2007</p><p>PMCID:PMC2216013.</p><p></p>A). The antigen-binding activity of the antibody was measured by ELISA (B). Antibody purified from the serum-free fed-batch cultures of cells transformed by the and tandem siRNA expression vector, FG1 (open circles), FG16 (open squares), and the parental cell line 32-05-12 (filled circles) are shown. Cytotoxicity (%) and absorbance are indicated as the mean values ± SD of triplicates