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

Wu, Xilin; Cheng, Lin; Fu, Ming; Huang, Bilian; Zhu, Linjing; Xu, Shijie; Shi, Haixia; Zhang, Doudou; Yuan, Huanyun; Nawaz, Waqas; Yang, Ping; Hu, Qinxue; Liu, Yalan; Wu, Zhiwei

The dramatically expanding COVID-19 needs multiple effective countermeasures. Neutralizing nanobodies (Nbs) are a potential therapeutic strategy for treating COVID-19. Here, we characterize several RBD-specific Nbs isolated from an Nb library derived from an alpaca immunized with 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 the efficacy, various configurations of Nbs are engineered. Nb15-NbH-Nb15, a trimer constituted of three Nbs, is constructed to be bispecific for human serum albumin (HSA) and RBD of SARS-CoV-2. Nb15-NbH-Nb15 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 Nb15-NbH-Nb15 provides effective protection for both prophylactic and therapeutic purposes against SARS-CoV-2 infection in transgenic hACE2 mice. Nb15-NbH-Nb15 is a potential candidate for both prevention and treatment of SARS-CoV-2 through respiratory administration.