Double knockdown of alpha1,6-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.
Double knockdown of alpha1,6-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.
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DOI：
10.1186/1472-6750-7-84
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发表时间：
2007-11-30
影响因子：
3.5
通讯作者：
Satoh M
中科院分区：
文献类型：
--
作者：
Imai-Nishiya H;Mori K;Inoue M;Wakitani M;Iida S;Shitara K;Satoh M
抗体依赖性细胞毒性（ADCC）由于缺乏与Fc连接的寡糖的核心岩藻糖而大大增强，并且与人体体内抗癌活性的临床疗效密切相关。不幸的是，所有许可的治疗性抗体和几乎所有目前开发的治疗性抗体都是严重岩藻糖基化的，并且不能优化ADCC，这导致对癌症患者施用抗体疗法需要大剂量，成本非常高。在这项研究中，我们探索了将已经建立的抗体产生细胞转化为产生完全缺乏核心岩藻糖基化的抗体的细胞的可能性，以促进下一代治疗性抗体的快速开发。首先，使用小干扰RNA（siRNA）针对参与寡糖岩藻糖修饰的三个关键基因，即α 1，6-岩藻糖基转移酶（FUT 8）、GDP-甘露糖4，6-转移酶（GMD）和GDP-岩藻糖转运蛋白（GFT）的功能丧失分析揭示，每个靶点的单基因敲低不足以使抗体产生细胞中的产物完全去岩藻糖基化，即使使用了最有效的siRNA（靶mRNA抑制>90%）。有趣的是，超出我们的预期，观察到FUT 8和GMD siRNA对岩藻糖基化减少的协同作用，但当这些与GFT siRNA组合使用时没有。其次，我们成功地开发了一种有效的短发夹siRNA串联表达载体，该载体促进FUT 8和GMD的双敲低，并且我们在两个月内将产生抗体的中国仓鼠卵巢（CHO）细胞转化为完全非岩藻糖基化的抗体产生者，并且具有高转化频率。最后，在无血清补料分批培养中使用转化的细胞证实了具有增强的ADCC的完全非岩藻糖基化抗体的稳定生产。我们的研究结果表明，FUT 8和GMD协同细胞内寡糖岩藻糖基化的过程中。我们还证明，在抗体产生细胞中FUT 8和GMD的双敲低可以作为用于产生完全缺乏核心岩藻糖基化并且具有增强的ADCC的下一代治疗性抗体的新策略。这种方法为开发下一代治疗性抗体提供了巨大的成本和时间节省优势。
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 in vivo. 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. 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 (FUT8), GDP-mannose 4,6-dehydratase (GMD), and GDP-fucose transporter (GFT), 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 FUT8 and GMD siRNAs on the reduction in fucosylation were observed, but not when these were used in combination with GFT siRNA. Secondly, we successfully developed an effective short hairpin siRNA tandem expression vector that facilitated the double knockdown of FUT8 and GMD, 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. Our results suggest that FUT8 and GMD collaborate synergistically in the process of intracellular oligosaccharide fucosylation. We also demonstrated that double knockdown of FUT8 and GMD 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.
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影响因子：
4.8
作者：
Hellbusch, Christina C.;Sperandio, Markus;Korner, Christian
通讯作者：
Korner, Christian
影响因子：
3.8
作者：
Ferrara, C;Brünker, P;Umaña, P
通讯作者：
Umaña, P
影响因子：
11.5
作者：
Iida, S;Misaka, H;Satoh, M
通讯作者：
Satoh, M
影响因子：
11.5
作者：
Niwa, R;Sakurada, M;Shitara, K
通讯作者：
Shitara, K
影响因子：
4.1
作者：
Kanda, Yutaka;Imai-Nishiya, Harue;Satoh, Mitsuo
通讯作者：
Satoh, Mitsuo