ABSTRACT
SARS-CoV-2, the Covid-19 causative virus, adheres to human cells through binding of its envelope Spike protein to the receptor ACE2. The Spike receptor-binding domain (S-RBD) mediates this key event and thus is a primary target for therapeutic neutralizing antibodies to mask the ACE2-interacting interface. Here, we generated 99 synthetic nanobodies (sybodies) using ribosome and phage display. The best sybody MR3 binds the RBD with KD of 1.0 nM and neutralizes SARS-CoV-2 pseudovirus with IC50 of 0.40 μg mL-1. Crystal structures of two sybody-RBD complexes reveal a common neutralizing mechanism through which the RBD-ACE2 interaction is competitively inhibited by sybodies. The structures allowed the rational design of a mutant with higher affinity and improved neutralization efficiency by ∼24-folds, lowering the IC50 from 12.32 to 0.50 μg mL-1. Further, the structures explain the selectivity of sybodies between SARS-CoV strains. Our work presents an alternative approach to generate neutralizers against newly emerged viruses.
One sentence summary Structural and biochemical studies revealed the molecular basis for the neutralization mechanism of in vitro-selected and rationally designed nanobody neutralizers for SARS-CoV-2 pseudovirus.
Competing Interest Statement
The authors have declared no competing interest.