Discovery of Nanosota-2, -3, and -4 as super potent and broad-spectrum therapeutic nanobody candidates against COVID-19.

Nanobodies are single-domain antibodies derived from camelid animals. Here, we discovered three anti-SARS-CoV-2 nanobodies, namely, Nanosota-2, -3, and -4, from an alpaca immunized with SARS-CoV spike protein. We further characterized the antiviral activities of these Fc-tag-fused nanobodies. Notably, Nanosota-2 inhibits the prototypic SARS-CoV-2 strain in vitro (with an IC 50 of 2 pM) and in mice (at a dosage of 4 mg/kg or administered 18 hours post-challenge). These potency metrics are the best among known SARS-CoV-2 entry inhibitors. Moreover, Nanosota-3 effectively inhibits the omicron variant, both in vitro and in mice, regardless of the administration route (intraperitoneal or intranasal). Furthermore, Nanosota-3 has been biochemically engineered to inhibit both early and currently circulating subvariants of omicron. Additionally, Nanosota-4 uniquely inhibits both SARS-CoV-1 and SARS-CoV-2. Cryo-EM data revealed that the three nanobodies bind to functionally critical and non-overlapping regions in the spike protein. Given their cost-effectiveness, ease of adaptation to new viral strains, and potential use as inhalers, the Nanosota series are powerful therapeutic tools against coronavirus pandemics. IMPORTANCE The COVID-19 pandemic exposed limitations of conventional antibodies as therapeutics, including high cost, limited potency, ineffectiveness against new viral variants, and primary reliance on injection-only delivery. Nanobodies are single-domain antibodies with therapeutic potentials. We discovered three anti-SARS-CoV-2 nanobodies, named Nanosota-2, -3, and -4, from an immunized alpaca. Nanosota-2 is super potent against prototypic SARS-CoV-2, Nanosota-3 is highly potent against the omicron variant, and Nanosota-4 is effective against both SARS-CoV-1 and SARS-CoV-2. In addition to their super potency and combined broad antiviral spectrum, these nanobodies are cost-effective, can be easily adapted to new viral variants through phage display, and can potentially be administered as inhalers. The Nanosota series are powerful therapeutic candidates to combat circulating SARS-CoV-2 and prepare for possible future coronavirus pandemics.