Bai Zixuan, Wang Jiewen, Li Jiaqi, Yuan Haibin, Wang Ping, Zhang Miao, Feng Yuanhang, Cao Xiangtong, Cao Xiangan, Kang Guangbo, de Marco Ario, Huang He
Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, China.
Frontiers Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin 300072, China.
Comput Struct Biotechnol J. 2022 Dec 16;21:601-613. doi: 10.1016/j.csbj.2022.12.021. eCollection 2023.
Random mutagenesis is the natural opportunity for proteins to evolve and biotechnologically it has been exploited to create diversity and identify variants with improved characteristics in the mutant pools. Rational mutagenesis based on biophysical assumptions and supported by computational power has been proposed as a faster and more predictable strategy to reach the same aim. In this work we confirm that substantial improvements in terms of both affinity and stability of nanobodies can be obtained by using combinations of algorithms, even for binders with already high affinity and elevated thermal stability. Furthermore, approaches allowed the development of an optimized bispecific construct able to bind simultaneously the two clinically relevant antigens TNF-α and IL-23 and, by means of its enhanced avidity, to inhibit effectively the apoptosis of TNF-α-sensitive L929 cells. The results revealed that salt bridges, hydrogen bonds, aromatic-aromatic and cation-pi interactions had a critical role in increasing affinity. We provided a platform for the construction of high-affinity bispecific constructs based on nanobodies that can have relevant applications for the control of all those biological mechanisms in which more than a single antigen must be targeted to increase the treatment effectiveness and avoid resistance mechanisms.
随机诱变是蛋白质进化的自然契机，在生物技术领域，人们利用它来创造多样性，并在突变库中鉴定具有改进特性的变体。基于生物物理假设并借助计算能力的理性诱变已被提出，作为实现同一目标的更快且更具可预测性的策略。在这项工作中，我们证实，即使对于已经具有高亲和力和高热稳定性的结合物，通过使用算法组合，也可以在纳米抗体的亲和力和稳定性方面取得实质性改进。此外，这些方法使得能够开发出一种优化的双特异性构建体，它能够同时结合两种临床相关抗原TNF-α和IL-23，并通过增强的亲和力有效抑制TNF-α敏感的L929细胞的凋亡。结果表明，盐桥、氢键、芳香-芳香相互作用和阳离子-π相互作用在提高亲和力方面起着关键作用。我们提供了一个基于纳米抗体构建高亲和力双特异性构建体的平台，该平台可在控制所有那些必须靶向多种抗原以提高治疗效果并避免耐药机制的生物机制方面具有相关应用。
