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 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.

Computationally Designed ACE2 Decoy Receptor Binds SARS-CoV-2 Spike (S) Protein with Tight Nanomolar Affinity

“… 59 The computationally designed FFWF mutant has ninefold higher affinity than the wild-type for RBD, with the increased binding mainly attributed to the slower off-rate. The K D of 1.8 nM for the FFWF mutant is in line with SARS CoV-2 monoclonal antibodies 56 − 59 and other therapeutics such as nanobodies 60 − 63 that potently neutralize SARS CoV-2. Although FFWF did not bind as tightly as v2 or v2.4 designed mutants, these designs were constructed after multiple rounds of experimental mutagenesis.…”

A synthetic nanobody targeting RBD protects hamsters from SARS-CoV-2 infection

“…They include monoclonal antibodies CB6 35 ( c ), CV30 36 ( d ), REGN10987 12 ( e ), REGN10933 12 ( f ), and the two sybodies SR4 ( g ) and MR17 ( h ) characterized in this study. i The MR3-RBD binding is compatible with SR31, which targets a non-RBM surface 37 . In c – h , the epitopes of the antibodies are highlighted in cyan except for SR4 and MR17, which are shown in Fig.…”

“…3a ). Third, we conducted cross-competition assays using ACE2 and structurally characterized antibodies, including four monoclonal antibodies (mAbs; CB6 35 , CV30 36 , REGN10987, and REGN10933 12 ), and three sybodies (SR4, MR17, and SR31 37 ). MR3 competed with ACE2 and all the RBM antibodies (Fig.…”

Anti-SARS-CoV-1 and −2 nanobody engineering towards avidity-inspired therapeutics

“…The authors showed in a subsequent report that their homotrimeric Nb21 (PiN-21) require low-dosage (0.2–0.6 mg/kg) to protected infected Syrian hamster [94] . Many other recent studies have demonstrated and confirmed the potential of linker-based bi-paratopic or multi-paratopic nanobodies for enhanced blocking and neutralization of SARS-CoV-2 variants [63] , [95] , [96] , [97] .…”