MOE/NHC Key Laboratory of Medical Molecular Virology, Shanghai Institute of Infectious Disease and Biosecurity, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai, 200032, China.
Department of Ophthalmology and Vision Science, Eye, Ear, Nose and Throat Hospital, Fudan University, Shanghai, 200031, China.
Small Methods. 2022 Feb;6(2):e2100966. doi: 10.1002/smtd.202100966. Epub 2021 Nov 28.
With increasing interest in applying recombinant monoclonal antibodies (mAbs) in human medicine, engineered mAb fragments with reduced size and improved stability are in demand to overcome current limitations in clinical use. Herein, a novel Fab-like antibody fragment generated via an in silico-based engineering approach where the CH1 and CL domains of Fab are replaced by the IgG1 CH3 domains is described. This construct, designated as FabCH3, maintains the natural N-terminus and C-terminus of IgG antibody, can be expressed at a high level in bacterial cells and, importantly, exhibits much higher stability and affinity than the parental Fab when tested in a mesothelin-specific Fab m912, as well as a vascular endothelial growth factor A (VEGFA)-specific Fab Ranibizumab (in vivo). The high-resolution crystal structures of m912 FabCH3 and m912 Fab are determined, and the comparative analysis reveals more rigid structures in both constant domains and complementarity-determining regions of FabCH3, explaining its enhanced stability and affinity. Overall, the stabilized FabCH3 described in this report provides a versatile platform for engineering Fab-like antibody fragments with higher stability and antigen-binding affinity that can be used as a distinct class of antibody therapeutics.
随着人们对将重组单克隆抗体 (mAb) 应用于人类医学的兴趣日益浓厚，需要具有更小尺寸和更高稳定性的工程化 mAb 片段来克服当前临床应用中的限制。在此，描述了一种通过基于计算的工程方法产生的新型 Fab 样抗体片段，其中 Fab 的 CH1 和 CL 结构域被 IgG1 的 CH3 结构域取代。该构建体被命名为 FabCH3，保留了 IgG 抗体的天然 N 端和 C 端，能够在细菌细胞中高水平表达，并且在测试时表现出比亲本 Fab 更高的稳定性和亲和力，特别是在针对间皮素的 Fab m912 以及针对血管内皮生长因子 A (VEGFA) 的 Fab Ranibizumab（体内）。确定了 m912 FabCH3 和 m912 Fab 的高分辨率晶体结构，比较分析表明 FabCH3 的恒定结构域和互补决定区的结构更加刚性，解释了其增强的稳定性和亲和力。总体而言，本报告中描述的稳定化 FabCH3 为工程化具有更高稳定性和抗原结合亲和力的 Fab 样抗体片段提供了一个多功能平台，可作为一类独特的抗体治疗药物。
