title: Establishment of a GDP-mannose 4,6-dehydratase (GMD) knockout host cell line: a new strategy for generating completely non-fucosylated recombinant therapeutics.
abstract: Currently, removal of core fucose from the Fc oligosaccharides of therapeutic antibodies is widely recognized as being of great importance for the effector function of antibody-dependent cellular cytotoxicity, and alpha-1,6-fucosyltransferase (FUT8) knockout cells have been generated as an ideal host cell line for manufacturing such therapeutics. Here, we attempted to identify genes other than FUT8 that could be targeted for the manufacture of non-fucosylated therapeutics. Loss-of-function analyses using siRNAs against three key genes involved in oligosaccharide fucosylation in Chinese hamster ovary (CHO) cells revealed that there was a positive correlation between the Fc oligosaccharide fucosylation and the mRNA expression through the origin in the cases of both GDP-fucose 4,6-dehydratase (GMD) and FUT8, but not for the GDP-fucose transporter, suggesting that there is no functional redundancy in GMD and FUT8. GMD knockout CHO/DG44 cells were successfully established, and were confirmed to be devoid of intracellular GDP-fucose and to produce completely non-fucosylated antibodies. GMD knockout cells recovered their fucosylation capability through the salvage pathway upon addition of l-fucose into the culture medium, and exhibited equable morphology, growth kinetics and recombinant protein productivity, demonstrating that loss of oligosaccharide fucosylation has no impact on these cellular phenotypes. Our results demonstrate that GMD knockout is a new strategy applicable to the manufacture of non-fucosylated therapeutic antibodies, and completely O-fucose-negative therapeutics as well.
authors.affiliation: Tokyo Research Laboratories, Kyowa Hakko Kogyo Co. Ltd., 3-6-6 Asahi-machi, Machida-shi, Tokyo 194-8533, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Kanda
authors.forename: Harue
authors.initials: H
authors.lastname: Imai-Nishiya
authors.forename: Reiko
authors.initials: R
authors.lastname: Kuni-Kamochi
authors.forename: Katsuhiro
authors.initials: K
authors.lastname: Mori
authors.forename: Miho
authors.initials: M
authors.lastname: Inoue
authors.forename: Kazuko
authors.initials: K
authors.lastname: Kitajima-Miyama
authors.forename: Akira
authors.initials: A
authors.lastname: Okazaki
authors.forename: Shigeru
authors.initials: S
authors.lastname: Iida
authors.forename: Kenya
authors.initials: K
authors.lastname: Shitara
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Satoh
mesh_terms: D000818:Animals; D018951:Antigens, CD20 / Q000276:immunology; D001709:Biotechnology / Q000379:methods*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D016895:Culture Media, Serum-Free; D018076:DNA, Complementary; D005643:Fucose / Q000378:metabolism*; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D006836:Hydro-Lyases / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D007074:Immunoglobulin G / Q000276:immunology; D051379:Mice; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D037121:Plant Lectins / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000494:pharmacology*
chemical_list: D018951:Antigens, CD20; D016895:Culture Media, Serum-Free; D018076:DNA, Complementary; D007074:Immunoglobulin G; D009004:Monosaccharide Transport Proteins; D009844:Oligosaccharides; D037121:Plant Lectins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C033619:lentil lectin; D005643:Fucose; D005647:Fucosyltransferases; C020559:Glycoprotein 6-alpha-L-fucosyltransferase; D006836:Hydro-Lyases; C044971:GDPmannose 4,6-dehydratase