Abstract

Summary The dramatically expanding coronavirus disease 2019 (COVID-19) needs multiple effective countermeasures. Neutralizing nanobodies (Nbs) are a potential therapeutic strategy for treating COVID-19. Here, we characterize several receptor binding domain (RBD)-specific Nbs isolated from an Nb library derived from an alpaca immunized with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (S); among them, three Nbs exhibit picomolar potency against SARS-CoV-2 live virus, pseudotyped viruses, and circulating SARS-CoV-2 variants. To improve their efficacy, various configurations of Nbs are engineered. Nb 15 -Nb H -Nb 15 , a trimer constituted of three Nbs, is constructed to be bispecific for human serum albumin (HSA) and RBD of SARS-CoV-2. Nb 15 -Nb H -Nb 15 exhibits single-digit ng/ml neutralization potency against the wild-type and Delta variants of SARS-CoV-2 with a long half-life in vivo . In addition, we show that intranasal administration of Nb 15 -Nb H -Nb 15 provides effective 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 the prevention and treatment of SARS-CoV-2 through respiratory administration.