Development of a bispecific nanobody conjugate broadly neutralizes diverse SARS-CoV-2 variants and structural basis for its broad neutralization

The continuous emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with increased transmissibility and profound immune-escape capacity makes it an urgent need to develop broad-spectrum therapeutics. Nanobodies have recently attracted extensive attentions due to their excellent biochemical and binding properties. Here, we report two high-affinity nanobodies (Nb-015 and Nb-021) that target non-overlapping epitopes in SARS-CoV-2 S-RBD. Both nanobodies could efficiently neutralize diverse viruses of SARS-CoV-2. The neutralizing mechanisms for the two nanobodies are further delineated by high-resolution nanobody/S-RBD complex structures. In addition, an Fc-based tetravalent nanobody format is constructed by combining Nb-015 and Nb-021. The resultant nanobody conjugate, designated as Nb-X2-Fc, exhibits significantly enhanced breadth and potency against all-tested SARS-CoV-2 variants, including Omicron sub-lineages. These data demonstrate that Nb-X2-Fc could serve as an effective drug candidate for the treatment of SARS-CoV-2 infection, deserving further in-vivo evaluations in the future. The pandemic of SARS-CoV-2 has led to a severe public health crisis. In view of the wide clinical-applications of antibody drugs, it has always been a research focus to identify new antibodies and/or nanobodies with anti-viral neutralizing activity. In this study, we identified two neutralizing nanobodies (Nb-015 and Nb-021) that target non-overlapping epitopes in SARS-CoV-2 S-RBD with high affinity. Both nanobodies could broadly neutralize diverse SARS-CoV-2 variants. Furthermore, we revealed two distinct neutralization mechanisms of Nb-015 and Nb-021 by high-resolution nanobody/S-RBD complex structures. Eventually, we engineered a bispecific nanobody conjugate which exhibits excellent inhibitory activity against SARS-CoV-2, including the original strain and the VOI and VOC variants. We believe that the nanobody conjugate identified in this study could serve as an effective anti-viral drug reserve for potential treatment of COVID-19.