Drug development of an affinity enhanced, broadly neutralizing heavy chain-only antibody that restricts SARS-CoV-2 in rodents

Schepens, Bert; van Schie, Loes; Nerinckx, Wim; Roose, Kenny; Van Breedam, Wander; Fijalkowska, Daria; Devos, Simon; Weyts, Wannes; De Cae, Sieglinde; Vanmarcke, Sandrine; Lonigro, Chiara; Eeckhaut, Hannah; Van Herpe, Dries; Borloo, Jimmy; Oliveira, Ana Filipa; Catani, Joao Paulo; Creytens, Sarah; De Vlieger, Dorien; Michielsen, Gitte; Zavala Marchan, Jackeline Cecilia; Moschonas, George D.; Rossey, Iebe; Sedeyn, Koen; Van Hecke, Annelies; Zhang, Xin; Langendries, Lana; Jacobs, Sofie; ter Horst, Sebastiaan; Seldeslachts, Laura; Liesenborghs, Laurens; Boudewijns, Robbert; Thibaut, Hendrik Jan; Dallmeier, Kai; Velde, Greetje Vande; Weynand, Birgit; Beer, Julius; Schnepf, Daniel; Ohnemus, Annette; Remory, Isabel; Foo, Caroline S.; Abdelnabi, Rana; Maes, Piet; Kaptein, Suzanne J. F.; Rocha-Pereira, Joana; Jochmans, Dirk; Delang, Leen; Peelman, Frank; Staeheli, Peter; Schwemmle, Martin; Devoogdt, Nick; Tersago, Dominique; Germani, Massimiliano; Heads, James; Henry, Alistair; Popplewell, Andrew; Ellis, Mark; Brady, Kevin; Turner, Alison; Dombrecht, Bruno; Stortelers, Catelijne; Neyts, Johan; Callewaert, Nico; Saelens, Xavier

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.