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

Abstract

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 have 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.

Integrative overview of antibodies against SARS-CoV-2 and their possible applications in COVID-19 prophylaxis and treatment

“…The cryo-EM structure suggested Sb23 as a Class 2 indicating that interacts with the S protein, wherein two RBDs are in the “up” conformation [ 250 ]. SR31, another Sb isolated from a synthetic library, interacts with the RBD, distorting it, and does not neutralize SARS-CoV-2 pseudovirus [ 251 ]. Since SR31 displays high affinity, its fusion with other neutralizing Sbs, such as SR31-MR17 or SR31-MR6, increases the neutralization activity against SARS-CoV-2 pseudovirus (IC 50 : 52.8 µg/mL or 2.7 µg/mL, respectively) [ 251 ].…”

Discovery of nanobodies against SARS-CoV-2 and an uncommon neutralizing mechanism

“…In this study, we report two high-affinity RBD binders isolated from immunized alpaca and their structural and biological characterization. Most monovalent RBDtargeting nanobodies bind S or RBD with KD in the nanomolar ranges [9][10][11][12][13][14][15][16][17][18]21 .…”

“…Being small, nanobodies are ultra-stable, relatively easy to produce (in microbial) with low costs and high yields, and amenable to protein engineering such as fusion in various forms. Such fusion can result in improved potency -binding affinity and neutralizing activity can increase by hundreds to thousands of fold [9][10][11] . In addition, nanobodies that recognize non-competing epitopes can be conveniently fused to make biparatopic nanobodies that are potentially more tolerant to escape mutant strains 9,10,12 .…”

“…Such fusion can result in improved potency -binding affinity and neutralizing activity can increase by hundreds to thousands of fold [9][10][11] . In addition, nanobodies that recognize non-competing epitopes can be conveniently fused to make biparatopic nanobodies that are potentially more tolerant to escape mutant strains 9,10,12 . The heat stability of nanobodies opens the possibility of using them as inhaling drugs for respiratory diseases 8 (and indeed potentially for SARS-CoV-2 as demonstrated in hamsters 13 ) and offers convenience in storage and transport.…”

Perspective on therapeutic and diagnostic potential of camel nanobodies for coronavirus disease-19 (COVID-19)

“…The last one (Sb23) can also bind the RBD in both "up" and "down" conformation (Custodio et al 2020). Also, Yao et al (2020) identified a new synthetic Nb. Selected sybody (named SR31) displayed poor neutralization activity against SARS-CoV-2 pseudo-virus.…”