TY - JOUR T1 - An ultra-high affinity synthetic nanobody blocks SARS-CoV-2 infection by locking Spike into an inactive conformation JF - bioRxiv DO - 10.1101/2020.08.08.238469 SP - 2020.08.08.238469 AU - Michael Schoof AU - Bryan Faust AU - Reuben A. Saunders AU - Smriti Sangwan AU - Veronica Rezelj AU - Nick Hoppe AU - Morgane Boone AU - Christian Bache Billesbølle AU - Marcell Zimanyi AU - Ishan Deshpande AU - Jiahao Liang AU - Aditya A. Anand AU - Niv Dobzinski AU - Beth Shoshana Zha AU - Benjamin Barsi-Rhyne AU - Vladislav Belyy AU - Andrew W. Barile-Hill AU - Sayan Gupta AU - Camille R. Simoneau AU - Kristoffer Leon AU - Kris M. White AU - Silke Nock AU - Yuwei Liu AU - Nevan J. Krogan AU - Corie Y. Ralston AU - Danielle L. Swaney AU - Adolfo García-Sastre AU - Melanie Ott AU - Marco Vignuzzi AU - QCRG Structural Biology Consortium AU - Peter Walter AU - Aashish Manglik Y1 - 2020/01/01 UR - http://biorxiv.org/content/early/2020/08/10/2020.08.08.238469.abstract N2 - 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 StatementM.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. ER -