An Effective Way to Introduce Disulfide Bonds into Nanobodies: Using an Anticarbaryl Nanobody Structure as a Model

The unique characteristics of nanobodies make them ideal candidates for diagnostic and therapeutic applications. Introducing additional disulfide bonds is one of the most effective ways to enhance a nanobody's stability. However, it always negatively affects the nanobody's affinity and expression in host cells. In this study, an anticarbaryl nanobody (NbCBR) was used as a model to investigate the strategy of additional disulfide bond introduction. The crystal structure of the NbCBR-carbaryl complex was determined, based on which molecular dynamics simulations and virtual screening were carried out. Seven disulfide bond-introduced mutants were then constructed. Compared with the wild-type nanobody, all the mutants retained good sensitivity. The results demonstrate that introducing disulfide bonds between two β-sheets or anchoring the flexible CDR3 to a β-sheet can significantly enhance the nanobody's stability under thermal, organic solvent, and pH stress conditions. This study provides a theoretical basis for rational engineering of nanobodies to improve their stability, potentially expanding their further applications.