An ultra-potent synthetic nanobody neutralizes SARS-CoV-2 by locking Spike into an inactive conformation

Abstract

Without an effective prophylactic solution, infections from SARS-CoV-2 continue to rise worldwide with devastating health and economic costs. SARS-CoV-2 gains entry into host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). Disruption of this interaction confers potent neutralization of viral entry, providing an avenue for vaccine design and for therapeutic antibodies. Here, we develop single-domain antibodies (nanobodies) that potently disrupt the interaction between the SARS-CoV-2 Spike and ACE2. By screening a yeast surface-displayed library of synthetic nanobody sequences, we identified a panel of nanobodies that bind to multiple epitopes on Spike and block ACE2 interaction via two distinct mechanisms. Cryogenic electron microscopy (cryo-EM) revealed that one exceptionally stable nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for SARS-CoV-2 Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains stability and function after aerosolization, lyophilization, and heat treatment. These properties may enable aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia, promising to yield a widely deployable, patient-friendly prophylactic and/or early infection therapeutic agent to stem the worst pandemic in a century.

Ultrapotent human antibodies protect against SARS-CoV-2 challenge via multiple mechanisms

“…Two recent reports describe the identification of a mAb and of a nanobody targeting quaternary epitopes, spanning two neighboring RBDs, which are present in the closed S trimer. Nb6 was identified from a naïve nanobody library, affinity matured and trimerized to achieve an IC50 of 160pM, however without the ability to exert effector functions (46). C144 was isolated from a COVID-19 convalescent serum sample, uses VH3-53 and VL2-14 genes, harbors a 25residue long CDRH3 and potently neutralizes SARS-CoV-2 (47).…”

A potent synthetic nanobody targets RBD and protects mice from SARS-CoV-2 infection

“…Of relevance to SARS-CoV-2, nanobodies can survive nebulization and an inhaler nanobody drug (ALX-0171) has gone into clinical trials for the treatment of the Respiratory Syncytial Virus 31 . Recent weeks have witnessed the generation of nanobodies that neutralize SARS-CoV-2 from several independent groups [14][15][16][17][18] . However, the in vivo efficacy of such nanobodies remains to be investigated.…”

A potent synthetic nanobody targets RBD and protects mice from SARS-CoV-2 infection

“…Increasing valency is a common technique to enhance potency for nanobodies 18,31 . To this end, we engineered three types of divalent sybodies, including the biparatopic fusion of two different sybodies, the Fc-fusion and direct fusion of the same sybody.…”

“…Of relevance to SARS-CoV-2, nanobodies can survive nebulization and an inhaler nanobody drug (ALX-0171) has gone into clinical trials for the treatment of the Respiratory Syncytial Virus 31 . Recent weeks have witnessed the generation of nanobodies that neutralize SARS-CoV-2 from several independent groups 14–18 . However, the in vivo efficacy of such nanobodies remains to be investigated.…”

“… ABSTRACT SARS-CoV-2, the causative agent of COVID-19 1 , recognizes host cells by attaching its receptor-binding domain (RBD) to the host receptor ACE2 2–7 . Neutralizing antibodies that block RBD-ACE2 interaction have been a major focus for therapeutic development 8–18 . Llama-derived single-domain antibodies (nanobodies, ∼15 kDa) offer advantages including ease of production and possibility for direct delivery to the lungs by nebulization 19 , which are attractive features for bio-drugs against the global respiratory disease.…”