Design of nanobody targeting SARS-CoV-2 spike glycoprotein using CDR-grafting assisted by molecular simulation and machine learning

The design of proteins capable to effectively bind to specific protein targets is crucial for developing therapies, diagnostics, and vaccine candidates for viral infections. Here, we introduce a complementarity-determining regions (CDRs)-grafting approach for designing nanobodies (Nbs) that target specific epitopes, with the aid of computer simulation and machine learning. As a proof-of-concept, we designed, evaluated, and characterized a high-affinity Nb against the spike protein of SARS-CoV-2, the causative agent of the COVID-19 pandemic. The designed Nb, referred to as Nb Ab.2, was synthesized and displayed high-affinity for both the purified receptor-binding domain protein and to the virus-like particle, demonstrating affinities of 9 nM and 60 nM, respectively, as measured with microscale thermophoresis. Circular dichroism showed the designed protein's structural integrity and its proper folding, whereas molecular dynamics simulations provided insights into the internal dynamics of Nb Ab.2. This study shows that our computational pipeline can be used to efficiently design high affinity Nbs with diagnostic and prophylactic potential, which can be tailored to tackle different viral targets.