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

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 Statement B.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. D.T. is employed by ExeVir Bio and is 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.