PT - JOURNAL ARTICLE AU - Justin D. Walter AU - Cedric A.J. Hutter AU - Alisa A. Garaeva AU - Melanie Scherer AU - Iwan Zimmermann AU - Marianne Wyss AU - Jan Rheinberger AU - Yelena Ruedin AU - Jennifer C. Earp AU - Pascal Egloff AU - Michèle Sorgenfrei AU - Lea M. Hürlimann AU - Imre Gonda AU - Gianmarco Meier AU - Sille Remm AU - Sujani Thavarasah AU - Gert Zimmer AU - Dirk J. Slotboom AU - Cristina Paulino AU - Philippe Plattet AU - Markus A. Seeger TI - Highly potent bispecific sybodies neutralize SARS-CoV-2 AID - 10.1101/2020.11.10.376822 DP - 2020 Jan 01 TA - bioRxiv PG - 2020.11.10.376822 4099 - http://biorxiv.org/content/early/2020/11/10/2020.11.10.376822.short 4100 - http://biorxiv.org/content/early/2020/11/10/2020.11.10.376822.full AB - The COVID-19 pandemic has resulted in a global crisis. Here, we report the generation of synthetic nanobodies, known as sybodies, against the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. We identified a sybody pair (Sb#15 and Sb#68) that can bind simultaneously to the RBD, and block ACE2 binding, thereby neutralizing pseudotyped and live SARS-CoV-2 viruses. Cryo-EM analyses of the spike protein in complex with both sybodies revealed symmetrical and asymmetrical conformational states. In the symmetric complex each of the three RBDs were bound by both sybodies, and adopted the up conformation. The asymmetric conformation, with three Sb#15 and two Sb#68 bound, contained one down RBD, one up-out RBD and one up RBD. Bispecific fusions of the sybodies increased the neutralization potency 100-fold, as compared to the single binders. Our work demonstrates that linking two binders that recognize spatially-discrete binding sites result in highly potent SARS-CoV-2 inhibitors for potential therapeutic applications.Competing Interest StatementIwan Zimmermann, Pascal Egloff and Markus A. Seeger are founders and shareholders of Linkster Therapeutics AG.