TY - JOUR T1 - Drug development of an affinity enhanced, broadly neutralizing heavy chain-only antibody that restricts SARS-CoV-2 in hamsters JF - bioRxiv DO - 10.1101/2021.03.08.433449 SP - 2021.03.08.433449 AU - Bert Schepens AU - Loes van Schie AU - Wim Nerinckx AU - Kenny Roose AU - Wander Van Breedam AU - Daria Fijalkowska AU - Simon Devos AU - Wannes Weyts AU - Sieglinde De Cae AU - Sandrine Vanmarcke AU - Chiara Lonigro AU - Hannah Eeckhaut AU - Dries Van Herpe AU - Jimmy Borloo AU - Ana Filipa Oliveira AU - Joao Paulo Catani AU - Sarah Creytens AU - Dorien De Vlieger AU - Gitte Michielsen AU - Jackeline Cecilia Zavala Marchan AU - Georgios D. Moschonas AU - Iebe Rossey AU - Koen Sedeyn AU - Annelies Van Hecke AU - Xin Zhang AU - Lana Langendries AU - Sofie Jacobs AU - Sebastiaan ter Horst AU - Laura Seldeslachts AU - Laurens Liesenborghs AU - Robbert Boudewijns AU - Hendrik Jan Thibaut AU - Kai Dallmeier AU - Greetje Vande Velde AU - Birgit Weynand AU - Julius Beer AU - Daniel Schnepf AU - Annette Ohnemus AU - Caroline S. Foo AU - Rana Abdelnabi AU - Piet Maes AU - Suzanne J. F. Kaptein AU - Joana Rocha-Pereira AU - Dirk Jochmans AU - Leen Delang AU - Frank Peelman AU - Peter Staeheli AU - Martin Schwemmle AU - Dominique Tersago AU - Massimiliano Germani AU - James Heads AU - Alistair Henry AU - Andrew Popplewell AU - Mark Ellis AU - Kevin Brady AU - Alison Turner AU - Bruno Dombrecht AU - Catelijne Stortelers AU - Johan Neyts AU - Nico Callewaert AU - Xavier Saelens Y1 - 2021/01/01 UR - http://biorxiv.org/content/early/2021/03/08/2021.03.08.433449.abstract N2 - We have identified camelid single-domain antibodies (VHHs) that cross-neutralize SARS-CoV-1 and -2, such as VHH72, which binds to a unique highly conserved epitope in the viral receptor-binding domain (RBD) that is difficult to access for human antibodies. Here, we establish a protein engineering path for how a stable, long-acting drug candidate can be generated out of such a VHH building block. When fused to human IgG1-Fc, the prototype VHH72 molecule prophylactically protects hamsters from SARS-CoV-2. In addition, we demonstrate that both systemic and intranasal application protects hACE-2-transgenic mice from SARS-CoV-2 induced lethal disease progression. To boost potency of the lead, we used structure-guided molecular modeling combined with rapid yeast-based Fc-fusion prototyping, resulting in the affinity-matured VHH72_S56A-Fc, with subnanomolar SARS-CoV-1 and -2 neutralizing potency. Upon humanization, VHH72_S56A was fused to a human IgG1 Fc with optimized manufacturing homogeneity and silenced effector functions for enhanced safety, and its stability as well as lack of off-target binding was extensively characterized. Therapeutic systemic administration of a low dose of VHH72_S56A-Fc antibodies strongly restricted replication of both original and D614G mutant variants of SARS-CoV-2 virus in hamsters, and minimized the development of lung damage. This work led to the selection of XVR011 for clinical development, a highly stable anti-COVID-19 biologic with excellent manufacturability. Additionally, we show that XVR011 is unaffected in its neutralizing capacity of currently rapidly spreading SARS-CoV-2 variants, and demonstrate its unique, wide scope of binding across the Sarbecovirus clades.Competing Interest StatementB.S., L.v.S., W.N., K.R., W.V.B., D.F., H.E., D.D.V., S.J.F.K., J.R.-P., L.D., B.D., C.S., J.N., N.C., and X.S. are named as inventors on patent applications related to the use of single domain antibody constructs to prevent and treat COVID-19. N.C. and X.S. are scientific founders of ExeVir Bio and are in receipt of ExeVir Bio share options. A.H, A.P., K.B., and A.T are in receipt of UCB shares and share options. ER -