An ultra-high affinity synthetic nanobody blocks SARS-CoV-2 infection by locking Spike into an inactive conformation

Schoof, M.; Faust, B.; Saunders, R. A.; Sangwan, S.; Rezelj, V. V.; Hoppe, N.; Boone, M.; Billesboelle, C.; Zimanyi, M.; Deshpande, I.; Liang, J.; Anand, A. A.; Dobzinski, N.; Zha, B. S.; Barsi-Rhyne, B.; Bleyy, V.; Barile-Hill, A. W.; Gupta, S.; Simoneau, C. R.; Leon, K.; White, K. M.; Nock, S.; Liu, Y.; Krogan, N. J.; Ralston, C. Y.; Swaney, D. L.; Garcia-Sastre, A.; Ott, M.; Vignuzzi, M.; Quantitative Biosciences Institute (QBI) Coronavirus Research Group Structural Biology Consortium,; Walter, P.; Manglik, A.

doi:10.1101/2020.08.08.238469

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bioRxiv · 10.1101/2020.08.08.238469

An ultra-high affinity synthetic nanobody blocks SARS-CoV-2 infection by locking Spike into an inactive conformation

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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.

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Schoof, M., Faust, B., Saunders, R. A., Sangwan, S., Rezelj, V. V., Hoppe, N., Boone, M., Billesboelle, C., Zimanyi, M., Deshpande, I., Liang, J., Anand, A. A., Dobzinski, N., Zha, B. S., Barsi-Rhyne, B., Bleyy, V., Barile-Hill, A. W., Gupta, S., Simoneau, C. R., Leon, K., White, K. M., Nock, S., Liu, Y., Krogan, N. J., Ralston, C. Y., Swaney, D. L., Garcia-Sastre, A., Ott, M., Vignuzzi, M., Quantitative Biosciences Institute (QBI) Coronavirus Research Group Structural Biology Consortium,, Walter, P., Manglik, A.. 2020-08-10. An ultra-high affinity synthetic nanobody blocks SARS-CoV-2 infection by locking Spike into an inactive conformation. https://doi.org/10.1101/2020.08.08.238469

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