Imai-Nishiya Harue, Mori Katsuhiro, Inoue Miho, Wakitani Masako, Iida Shigeru, Shitara Kenya, Satoh Mitsuo
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co,, Ltd,, 3-6-6 Asahi-machi, Machida-shi, Tokyo 194-8533, Japan.
BMC Biotechnol. 2007 Nov 30;7:84. doi: 10.1186/1472-6750-7-84.
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. alpha1,6-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.
抗体依赖的细胞毒性作用（ADCC）会因附着于Fc的寡糖核心岩藻糖缺失而大大增强，且与体内人类抗癌活性的临床疗效密切相关。不幸的是，所有已获许可的治疗性抗体以及几乎所有目前正在研发的治疗性抗体都高度岩藻糖基化，无法优化ADCC，这导致癌症患者接受抗体治疗时需要大剂量给药，成本极高。在本研究中，我们探索了将已建立的抗体产生细胞转化为完全缺乏核心岩藻糖基化的抗体产生细胞的可能性，以促进下一代治疗性抗体的快速开发。
首先，使用针对参与寡糖岩藻糖修饰的三个关键基因，即α1,6-岩藻糖基转移酶（FUT8）、GDP-甘露糖4,6-脱水酶（GMD）和GDP-岩藻糖转运体（GFT）的小干扰RNA（siRNA）进行功能丧失分析，结果显示，即使使用了最有效的siRNA（靶mRNA抑制率>90%），对每个靶点进行单基因敲低也不足以使抗体产生细胞中的产物完全去岩藻糖基化。有趣的是，出乎我们意料的是，观察到FUT8和GMD siRNA对岩藻糖基化减少具有协同作用，但与GFT siRNA联合使用时则未观察到这种协同作用。其次，我们成功开发了一种有效的短发夹siRNA串联表达载体，该载体促进了FUT8和GMD的双重敲低，并且在两个月内将抗体产生的中国仓鼠卵巢（CHO）细胞转化为完全无岩藻糖基化的抗体产生细胞，且转化频率很高。最后，使用转化后的细胞在无血清补料分批培养中证实了能够稳定生产具有增强ADCC的完全无岩藻糖基化抗体。
我们的结果表明，FUT8和GMD在细胞内寡糖岩藻糖基化过程中协同发挥作用。我们还证明，在抗体产生细胞中对FUT8和GMD进行双重敲低可作为一种新策略，用于生产完全缺乏核心岩藻糖基化且具有增强ADCC的下一代治疗性抗体。这种方法为下一代治疗性抗体的开发提供了巨大的成本和时间节省优势。
