Zhong Xiaotian, Schenk Jennifer, Sakorafas Paul, Chamberland John, Tam Amy, Thomas L Michael, Yan Grace, D' Antona Aaron M, Lin Laura, Nocula-Lugowska Malgorzata, Zhang Yan, Sousa Eric, Cohen Justin, Gu Ling, Abel Molica, Donahue Jacob, Lim Sean, Meade Caryl, Zhou Jing, Riegel Logan, Birch Alex, Fennell Brian J, Franklin Edward, Gomes Jose M, Tzvetkova Boriana, Scarcelli John J
BioMedicine Design, Medicinal Sciences, Pfizer Worldwide R&D, 610 Main Street, Cambridge, MA 02139, USA.
Analytical R&D, Biotherapeutics Pharmaceutical Sciences, Medicinal Sciences, Pfizer Worldwide R&D, 1 Burtt Road, Andover, MA 01810, USA.
J Biotechnol. 2022 Dec 10;360:79-91. doi: 10.1016/j.jbiotec.2022.10.016. Epub 2022 Oct 27.
This study has employed mammalian transient expression systems to generate afucosylated antibodies and antibody Fc mutants for rapid candidate screening in discovery and early development. While chemical treatment with the fucose analogue 2-fluoro-peracetyl-fucose during transient expression only partially produced antibodies with afucosylated N-glycans, the genetic inactivation of the FUT8 gene in ExpiCHO-S™ by CRISPR/Cas9 enabled the transient production of fully afucosylated antibodies. Human IgG and murine IgG generated by the ExpiCHOfut8KO cell line possessed a 8-to-11-fold enhanced FcγRIIIa binding activity in comparison with those produced by ExpiCHO-S™. The Fc mutant S239D/S298A/I332E produced by ExpiCHO-S™ had an approximate 2-fold higher FcγRIIIa affinity than that of the afucosylated wildtype molecule, although it displayed significantly lower thermal-stability. When the Fc mutant was produced in the ExpiCHOfut8KO cell line, the resulting afucosylated Fc mutant antibody had an additional approximate 6-fold increase in FcγRIIIa binding affinity. This synergistic effect between afucosylation and the Fc mutations was further verified by a natural killer (NK) cell activation assay. Together, these results have not only established an efficient large-scale transient CHO system for rapid production of afucosylated antibodies, but also confirmed a cooperative impact between afucosylation and Fc mutations on FcγRIIIa binding and NK cell activation.
本研究采用哺乳动物瞬时表达系统来生成去岩藻糖基化抗体和抗体Fc突变体，用于在发现和早期开发阶段进行快速候选筛选。虽然在瞬时表达过程中用岩藻糖类似物2-氟-全乙酰岩藻糖进行化学处理仅部分产生具有去岩藻糖基化N-聚糖的抗体，但通过CRISPR/Cas9对ExpiCHO-S™中的FUT8基因进行基因失活能够瞬时产生完全去岩藻糖基化的抗体。与ExpiCHO-S™产生的抗体相比，ExpiCHOfut8KO细胞系产生的人IgG和鼠IgG具有增强8至11倍的FcγRIIIa结合活性。ExpiCHO-S™产生的Fc突变体S239D/S298A/I332E的FcγRIIIa亲和力比去岩藻糖基化野生型分子高约2倍，尽管其热稳定性显著较低。当在ExpiCHOfut8KO细胞系中产生Fc突变体时，所得的去岩藻糖基化Fc突变体抗体的FcγRIIIa结合亲和力又增加了约6倍。自然杀伤（NK）细胞激活试验进一步验证了去岩藻糖基化与Fc突变之间的这种协同效应。总之，这些结果不仅建立了一种高效的大规模瞬时CHO系统，用于快速生产去岩藻糖基化抗体，而且证实了去岩藻糖基化与Fc突变在FcγRIIIa结合和NK细胞激活方面的协同作用。
