Optimization of nanobody caplacizumab <i>via</i> computational design

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder characterized by the formation of microvascular thrombosis caused by ultra-large von Willebrand factor (VWF) multimers. Caplacizumab, the first FDA-approved nanobody for TTP treatment, binds to the A1 domain of VWF, thereby inhibiting its interaction with platelet glycoprotein Ib (GPIb). In this study, molecular dynamics (MD) simulations were employed to dynamically analyze the regulatory mechanism of caplacizumab on the interaction between VWF A1 and GPIb. Subsequently, alanine scanning was conducted to identify critical hotspot residues at the caplacizumab-VWF A1 binding interface and evaluate their contributions to binding free energy. Single-point saturation mutagenesis was performed on coldspot and warmspot residues of caplacizumab, and the binding free energy changes of the resulting mutants with VWF A1 were calculated. Following the identification of mutants with significantly enhanced binding affinity, combinatorial mutations were designed, resulting in an optimized caplacizumab variant with four mutations. Computational results demonstrated that this four-point mutant substantially improved the binding free energy with VWF A1. This study provides valuable theoretical insights into the allosteric regulation mechanisms of nanobody therapeutics and establishes a foundation for designing next-generation caplacizumab variants. The findings open new avenues for the development of more effective and precise antithrombotic therapies.