Engineering Chinese hamster ovary cells to maximize effector function of produced antibodies using FUT8 siRNA
Engineering Chinese hamster ovary cells to maximize effector function of produced antibodies using FUT8 siRNA
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DOI：
10.1002/bit.20326
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发表时间：
2004-12-30
影响因子：
3.8
通讯作者：
Satoh, M
中科院分区：
文献类型：
--
作者：
Mori, K;Kuni-Karnochi, R;Satoh, M
我们探索了将已建立的产生抗体的细胞转化为产生高抗体依赖性细胞毒性（ADCC）抗体的细胞的可能性。转化是通过小干扰RNA （siRNA）对alpha1,6 focusyltransferase （FUT8）的组成性表达实现的。我们发现了两种有效的sirna，当引入中国仓鼠卵巢(CHO)/DG44细胞时，它们将FUT8 mRNA的表达降低了20%。在引入FUT8 siRNA表达质粒后，选择具有LCA抗性的Lens culinaris凝集素（LCA）抗性的克隆，产生高度去聚焦化（约60%）的抗体，其ADCC比亲本细胞产生的抗体（约10%去聚焦化）高出100倍以上。此外，所选择的克隆保持稳定，即使在无血清间歇培养中也能产生去聚焦抗体。我们的研究结果表明，组成型FUT8 siRNA的表达可以控制CHO细胞产生的重组抗体的寡糖结构，从而产生具有显著增强ADCC的抗体。(C) 2004 Wiley期刊有限公司
We explored the possibility of converting established antibody-producing cells to cells producing high anti body-dependent cellular cytotoxicity (ADCC) antibodies. The conversion was made by constitutive expression of small interfering RNA (siRNA) against alpha1,6 fucosyltransferase (FUT8). We found two effective siRNAs, which reduce FUT8 mRNA expression to 20% when introduced into Chinese hamster ovary (CHO)/DG44 cells. Selection for Lens culinaris agglutinin (LCA)-resistant clones after introduction of the FUT8 siRNA expression plasmids yields clones producing highly defucosylated (approximate to 60%) antibody with over 100-fold higher ADCC compared to antibody produced by the parental cells (approximate to 10% defucosylated). Moreover, the selected clones remain stable, producing defucosylated antibody even in serum-free fed-batch culture. Our results demonstrate that constitutive FUT8 siRNA expression can control the oligosaccharide structure of recombinant antibody produced by CHO cells to yield antibodies with dramatically enhanced ADCC. (C) 2004 Wiley Periodicals, Inc.