Establishment of a GDP-mannose 4,6-dehydratase (GMD) knockout host cell line: A new strategy for generating completely non-fucosylated recombinant therapeutics
Establishment of a GDP-mannose 4,6-dehydratase (GMD) knockout host cell line: A new strategy for generating completely non-fucosylated recombinant therapeutics
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DOI：
10.1016/j.jbiotec.2007.04.025
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发表时间：
2007-06-30
影响因子：
4.1
通讯作者：
Satoh, Mitsuo
中科院分区：
文献类型：
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作者：
Kanda, Yutaka;Imai-Nishiya, Harue;Satoh, Mitsuo
目前，从治疗性抗体的Fc寡糖中去除核心岩藻糖被广泛认为对于抗体依赖性细胞毒性的效应子功能非常重要，并且已经产生了α-1，6-岩藻糖基转移酶（FUT 8）敲除细胞作为用于制造此类治疗剂的理想宿主细胞系。在这里，我们试图鉴定除FUT 8以外的基因，其可以被靶向用于制造非岩藻糖基化治疗剂。使用siRNA针对中国仓鼠卵巢（CHO）细胞中涉及寡糖岩藻糖基化的三个关键基因进行的功能丧失分析显示，在GDP-岩藻糖4，6-脱氢酶（GMD）和FUT 8的情况下，Fc寡糖岩藻糖基化与通过起源的mRNA表达之间存在正相关性，但对于GDP-岩藻糖转运蛋白，这表明在GMD和FUT 8中不存在功能冗余。成功建立了GMD敲除CHO/DG 44细胞，并证实其缺乏细胞内GDP-岩藻糖，并产生完全非岩藻糖基化的抗体。在培养基中加入L-岩藻糖后，GMD敲除细胞通过补救途径恢复了其岩藻糖基化能力，并表现出稳定的形态学、生长动力学和重组蛋白生产率，表明寡糖、岩藻糖基化的损失对这些细胞表型没有影响。我们的结果表明，GMD敲除是一种新的策略，适用于生产非岩藻糖基化的治疗性抗体，以及完全O-岩藻糖阴性的治疗剂。(c)2007 Elsevier B. V.保留所有权利。
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. (c) 2007 Elsevier B.V. All rights reserved.