A potent bispecific nanobody protects hACE2 mice against SARS-CoV-2 infection via intranasal administration

The dramatically expanding COVID-19 needs multiple effective countermeasures. Neutralizing antibodies are a potential therapeutic strategy for treating COVID-19. A number of neutralizing nanobodies (Nbs) were reported for their in vitro activities. However, in vivo protection of these nanobodies was not reported in animal models. In the current report, we characterized several RBD-specific Nbs isolated from a screen of an Nb library derived from an alpaca immunized with SARS-CoV-2 spike glycoprotein (S); among them, three Nbs exhibited picomolar potency against SARS-CoV-2 live virus, pseudotyped viruses, and 15 circulating SARS-CoV-2 variants. To improve the efficacy, various configurations of Nbs were engineered. Nb 15 -Nb H -Nb 15 , a novel trimer constituted of three Nbs, was constructed to be bispecific for human serum albumin (HSA) and RBD of SARS-CoV-2. Nb 15 -Nb H -Nb 15 exhibited sub-ng/ml neutralization potency against the wild-type and currently circulating variants of SARS-CoV-2 with a long half-life in vivo . In addition, we showed that intranasal administration of Nb 15 -Nb H -Nb 15 provided 100% protection for both prophylactic and therapeutic purposes against SARS-CoV-2 infection in transgenic hACE2 mice. Nb 15 -Nb H -Nb 15 is a potential candidate for both prevention and treatment of SARS-CoV-2 through respiratory administration. One sentence summary Nb 15 -Nb H -Nb 15 , with a novel heterotrimeric bispecific configuration, exhibited potent and broad neutralization potency against SARS-CoV-2 in vitro and provided in vivo protection against SARS-CoV-2 infection in hACE2 transgenic mice via intranasal delivery. Graphical abstract: Highlights We described a novel heterotrimeric configuration of Nb-Nb H -Nb (Nb 15 -Nb H -Nb 15 ) that exhibited improved viral inhibition and stability. Nb 15 -Nb H -Nb 15 provides ultrahigh neutralization potency against SARS-CoV-2 wild type and 18 mutant variants, including the current circulating variants of D614G and N501Y predominantly in the UK and South Africa. It is the first to demonstrate the Nbs efficacy in preventing and treating SARS-CoV-2 infection in hACE2 transgenic mice via intranasal delivery.