Modifying Binding Affinity of Nanobody Caplacizumab to treat Acquired Thrombotic Thrombocytopenic Purpura

Acquired thrombotic thrombocytopenic purpura(aTTP) is a disorder caused by a lack of the ADAMTS13 enzyme, which acts as a blood-clot regulator. Caplacizumab is a nanobody treatment for this disease and interacts with the A1 domain of von Willebrand factor (vWF) to prevent pathological clot formations. We utilized computational biology tools to predict how mutations will affect the binding affinity and stability of Caplacizumab for interaction with vWF. With these tools, mutations with positive ΔΔG values indicated beneficial outcomes. Certain mutations only had a positive effect on affinity or stability. Single-mutation affinity predictions identified that residues G67, S64, E66, etc, had a positive effect on affinity, highlighting it as a mutational hotspot. However, D63K, D63R, D63I, and E66M had the most stable and positive effects on the nanobody. Residues with a positive effect are located in complementarity-determining region 2 (CDR2), making it a key engineering hotspot. Beneficial mutations were then modeled using Alpha Fold. Overall, results showed a clear trade-off between binding affinity and stability, with few residues maintaining both values with positive ΔΔG.