A high-affinity RBD-targeting nanobody improves fusion partner's potency against SARS-CoV-2.

A key step to the SARS-CoV-2 infection is the attachment of its Spike receptor-binding domain (S RBD) to the host receptor ACE2. Considerable research has been devoted to the development of neutralizing antibodies, including llama-derived single-chain nanobodies, to target the receptor-binding motif (RBM) and to block ACE2-RBD binding. Simple and effective strategies to increase potency are desirable for such studies when antibodies are only modestly effective. Here, we identify and characterize a high-affinity synthetic nanobody (sybody, SR31) as a fusion partner to improve the potency of RBM-antibodies. Crystallographic studies reveal that SR31 binds to RBD at a conserved and 'greasy' site distal to RBM. Although SR31 distorts RBD at the interface, it does not perturb the RBM conformation, hence displaying no neutralizing activities itself. However, fusing SR31 to two modestly neutralizing sybodies dramatically increases their affinity for RBD and neutralization activity against SARS-CoV-2 pseudovirus. Our work presents a tool protein and an efficient strategy to improve nanobody potency. Author summary: SARS-CoV-2 relies on the receptor-binding domain (RBD) of its envelope Spike protein to recognize and infect host cells. RBD is therefore an immunological hotspot to generate antibodies for therapeutic and detection purposes. Binding affinity is one of the key characteristics of such antibodies. Here, we report a single-chain antibody (nanobody, ~14 kDa) that binds RBD with nanomolar affinity. The nanobody, dubbed SR31, binds RBD at an epitope distal to the receptor-binding motif (RBM) which is the target of most neutralizing antibodies. SR31 can therefore bind RBD in addition to RMB-binders, and increase their affinity and potency by avidity effects when used as a fusion partner. Compared to other in vitro affinity maturation techniques such as library screening and structure-based design, the fusion strategy offers advantages in speed and simplicity. In addition, SR31, together with RBD-targeting nanobodies recognizing a wide spectrum of epitopes, provides a useful toolkit to probe epitopes of uncharacterized antibodies by competitive binding. [ABSTRACT FROM AUTHOR]