Arsenal of Nanobodies for Broad-Spectrum Countermeasures against Current and Future SARS-CoV-2 Variants of Concerns

Abstract

Nanobodies offer several potential advantages over mAbs for the control of SARS-CoV-2. Their ability to access cryptic epitopes conserved across SARS-CoV-2 variants of concern (VoCs) and feasibility to engineer modular, multimeric designs, make these antibody fragments ideal candidates for developing broad-spectrum therapeutics against current and continually emerging SARS-CoV-2 VoCs. Here we describe a diverse collection of 37 anti-SARS-CoV-2 spike glycoprotein nanobodies extensively characterized as both monovalent and IgG Fc-fused bivalent modalities. The panel of nanobodies were shown to have high intrinsic affinity; high thermal, thermodynamic and aerosolization stability; broad subunit/domain specificity and cross-reactivity across many VoCs; wide-ranging epitopic and mechanistic diversity; high and broad in vitro neutralization potencies; and high neutralization efficacies in hamster models of SARS-CoV-2 infection, reducing viral burden by up to six orders of magnitude to below detectable levels. In vivo protection was demonstrated with anti-RBD and previously unreported anti-NTD and anti-S2 nanobodies. This collection of nanobodies provides a therapeutic toolbox from which various cocktails or multi-paratopic formats could be built to tackle current and future SARS-CoV-2 variants and SARS-related viruses. Furthermore, the high aerosol-ability of nanobodies provides the option for effective needle-free delivery through inhalation.

Structural and Computational Studies of the SARS-CoV-2 Spike Protein Binding Mechanisms with Nanobodies: From Structure and Dynamics to Avidity-Driven Nanobody Engineering

“…A panel of 37 anti-SARS-CoV-2 spike nanobodies was characterized as both monovalent and IgG Fc-fused bivalent modalities [ 102 ]. The analysis of binding characteristics showed that both monovalent and bivalent nanobodies have high affinity and high cross-reactivity against spike variants.…”

“…A total of 12 out of 20 tested bivalent nanobodies exhibited high efficacy in neutralizing the Delta variant. Of these nanobodies, nine were capable of neutralizing all tested variants [ 102 ] For the first time, this study demonstrated high epitope diversity, revealing that several NTD- and S2-specific nanobodies were potent and efficacious neutralizers in vitro and in vivo as well as remaining potent against the Alpha, Beta, Gamma, Delta, and Kappa variants [ 102 ]. Using computer-aided design, multi-specific VHH antibodies fused to human IgG1 Fc domains were constructed based on the epitope predictions for leading VHHs [ 103 ].…”

Ultrapotent SARS coronavirus-neutralizing single-domain antibodies that bind a conserved membrane proximal epitope of the spike

“…In general, however, S2-specific monoclonal antibodies exhibit poor virus neutralizing activity. Single-domain antibodies (VHHs, also known as nanobodies) directed against the SARS-CoV-2 S2 subunit have also been reported, which, even as Fc-fusions, have low SARS-CoV-2-neutralizing activity (11, 12).…”