Affinity maturation and rational design have a raised importance in the application of nanobody (VHH), and its unique structure guaranteed these processes quickly done in vitro. An anti-CD47 nanobody, Nb02, was screened via a synthetic phage display library with 278 nM of KD value. In this study, a new strategy based on homology modeling and Rational Mutation Hotspots Design Protocol (RMHDP) was presented for building a fast and efficient platform for nanobody affinity maturation. A three-dimensional analytical structural model of Nb02 was constructed and then docked with the antigen, the CD47 extracellular domain (CD47ext). Mutants with high binding affinity are predicted by the scoring of nanobody-antigen complexes based on molecular dynamics trajectories and simulation. Ultimately, an improved mutant with an 87.4-fold affinity (3.2 nM) and 7.36 °C higher thermal stability was obtained. These findings might contribute to computational affinity maturation of nanobodies via homology modeling using the recent advancements in computational power. The add-in of aromatic residues which formed aromatic-aromatic interaction plays a pivotal role in affinity and thermostability improvement. In a word, the methods used in this study might provide a reference for rapid and efficient in vitro affinity maturation of nanobodies.
Keywords: homology modeling; in vitro affinity maturation; nanobody; rational design.