An ultra-potent synthetic nanobody neutralizes SARS-CoV-2 by locking Spike into an inactive conformation
Reuben A. Saunders,
Christian B. Billesbølle,
Caleigh M Azumaya,
Huong T Kratochvil,
Henry C. Nguyen,
Cynthia M Chio,
Gregory E Merz,
Michael C Thompson,
Aditya A. Anand,
Camille R. Simoneau,
Kris M. White,
Un Seng Chio,
Amber M. Smith,
Alexandrea N. Rizo,
Axel F. Brilot,
Andrew W. Barile-Hill,
Nevan J. Krogan,
Corie Y. Ralston,
Danielle L. Swaney,
QCRG Structural Biology Consortium,
Posted 10 Aug 2020
bioRxiv DOI: 10.1101/2020.08.08.238469
Posted 10 Aug 2020
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. ### Competing Interest Statement M.Schoof, B.Faust, R.Saunders, N.Hoppe, P.Walter, and A.Manglik are inventors on a provisional patent describing anti-Spike nanobodies described in this manuscript.
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