Human Health Therapeutics Research Centre, National Research Council Canada, 6100 Royalmount Avenue, Montreal, Quebec, H4P 2R2, Canada.
Institute of Parasitology, McGill University, 21111 Lakeshore Road, Sainte-Anne-de-Bellevue, Quebec, H9X 3V9, Canada.
Sci Rep. 2018 Feb 2;8(1):2260. doi: 10.1038/s41598-018-20599-4.
Assisted Design of Antibody and Protein Therapeutics (ADAPT) is an affinity maturation platform interleaving predictions and testing that was previously validated on monoclonal antibodies (mAbs). This study expands the applicability of ADAPT to single-domain antibodies (sdAbs), a promising class of recombinant antibody-based biologics. As a test case, we used the camelid sdAb A26.8, a VH that binds Clostridium difficile toxin A (TcdA) relatively weakly but displays a reasonable level of TcdA neutralization. ADAPT-guided A26.8 affinity maturation resulted in an improvement of one order of magnitude by point mutations only, reaching an equilibrium dissociation constant (K) of 2 nM, with the best binding mutants having similar or improved stabilities relative to the parent sdAb. This affinity improvement generated a 6-fold enhancement of efficacy at the cellular level; the A26.8 double-mutant T56R,T103R neutralizes TcdA cytotoxicity with an IC of 12 nM. The designed mutants with increased affinities are predicted to establish novel electrostatic interactions with the antigen. Almost full additivity of mutation effects is observed, except for positively charged residues introduced at adjacent positions. Furthermore, analysis of false-positive predictions points to general directions for improving the ADAPT platform. ADAPT guided the efficacy enhancement of an anti-toxin sdAb, an alternative therapeutic modality for C. difficile.
抗体和蛋白治疗物的辅助设计（ADAPT）是一种亲和力成熟平台，它交错进行预测和测试，此前已在单克隆抗体（mAb）上得到验证。本研究将 ADAPT 的适用性扩展到单域抗体（sdAb），这是一类有前途的重组抗体生物药物。作为一个测试案例，我们使用了骆驼科 sdAb A26.8，它的 VH 与艰难梭菌毒素 A（TcdA）的结合相对较弱，但显示出一定水平的 TcdA 中和能力。ADAPT 指导的 A26.8 亲和力成熟仅通过点突变就提高了一个数量级，达到 2 nM 的平衡解离常数（K），最佳结合突变体相对于亲本 sdAb 具有相似或改善的稳定性。这种亲和力的提高使细胞水平的疗效提高了 6 倍；A26.8 双突变体 T56R，T103R 以 IC 为 12 nM 的浓度中和 TcdA 的细胞毒性。预测具有更高亲和力的设计突变体与抗原建立新的静电相互作用。除了在相邻位置引入带正电荷的残基外，观察到突变效应几乎完全相加。此外，对假阳性预测的分析指出了改进 ADAPT 平台的一般方向。ADAPT 指导了抗毒素 sdAb 的疗效增强，这是艰难梭菌的另一种治疗方式。
