Development of an Escape-resistant SARS CoV-2 Neutralizing Synthetic Nanobody

An emerging COVID-19 pandemic resulted in a global crisis, but also accelerated vaccine development and antibody discovery. In this work, we identified a number of nanomolar-range affinity VHH binders to SARS-CoV-2 variants of concern (VoC) receptor binding domains (RBD), by screening synthetic humanized antibody library with more than 1011 diversity. In order to explore the most robust and fast method for affinity improvement, we performed affinity maturation by CDR1 and CDR2 shuffling and avidity engineering by multivalent trimeric VHH fusion protein construction. As a result, H7-Fc and G12×3-Fc binders were developed with the affinities in nM and pM range respectively. Importantly, their affinities are weakly influenced by SARS-CoV-2 VoC mutations. The plaque reduction neutralization test (PRNT) resulted in IC50 = 100 ng\ml and 9.6 ng\ml for H7-Fc and G12×3-Fc antibodies respectively for emerging Omicron variant. Therefore, these VHH could expand the present landscape of SARS-CoV-2 neutralization binders with the therapeutic potential for present and future SARS-CoV-2 variants. ### Competing Interest Statement DD and MS have a pending patent application for the RBD-targeted antibodies from this study.