Liu Yaohui, Gu Manping, Wu Yaxing, Wang Wei, Wang Ruikun, Du Mingjuan, Ma Peixiang, Zhou Xingdong, Wang Yuan, Cao Youjia, Zhang Hongkai
State Key Laboratory of Medicinal Chemical Biology, 94 Weijin Road, Tianjin, China.
College of Life Sciences, Nankai University, 94 Weijin Road, Tianjin, China.
Protein Eng Des Sel. 2018 Nov 1;31(11):427-436. doi: 10.1093/protein/gzz004.
Single-chain variable fragment (scFv) is the most common format for phage display antibody library. The isolated scFvs need to be reformatted to full-length IgGs for further characterization. High throughput reformatting of scFv to IgG without disrupting VH-VL pairing is of great demanding for exhaustive screening of all antibodies in IgG format. Herein, we developed a strategy based on the overlap extension PCR in emulsion to reformat scFv to IgG while maintain the accuracy and complexity of variable region pairing. Using CD40 as an example target, we reformatted phage display derived CD40 binding scFv library to IgG mammalian display library and isolated high affinity CD40 binding IgGs. This robust and reliable antibody reformatting approach could be integrated into any phage display based antibody drug discovery.
单链可变片段（scFv）是噬菌体展示抗体库最常见的形式。分离得到的scFv需要重新构建为全长IgG以进行进一步表征。在不破坏VH-VL配对的情况下将scFv高通量重新构建为IgG，对于全面筛选所有IgG形式的抗体具有很高的要求。在此，我们开发了一种基于乳液中重叠延伸PCR的策略，将scFv重新构建为IgG，同时保持可变区配对的准确性和复杂性。以CD40作为示例靶点，我们将噬菌体展示衍生的CD40结合scFv库重新构建为IgG哺乳动物展示库，并分离出高亲和力的CD40结合IgG。这种强大且可靠的抗体重新构建方法可整合到任何基于噬菌体展示的抗体药物发现中。
