Redirecting a Broad-Spectrum Nanobody Against the Receptor-Binding Domain of SARS-CoV-2 to Target Omicron Variants

The urgent need for an effective COVID-19 therapy has propelled the exploration of innovative strategies to combat the fast-mutating SARS-CoV-2 virus. This study attempted to develop nanobodies (Nbs) against the SARS-CoV-2 Omicron variants by redirecting the 1.29 neutralizing Nb, a receptor-binding domain (RBD)-specific Nb that can protect against various SARS-CoV-2 variants other than Omicron, to target SARS-CoV-2 Omicron subvariant BA.5, the variant used for the development of the bivalent vaccine. Error-prone libraries of the 1.29 Nb were constructed. Following two rounds of selection using the functional ligand-binding identification by Tat-based recognition of associating proteins (FLI-TRAP) technique, we rapidly identified two Nbs, namely, C11 and K9, that could target the RBD of the Omicron subvariant BA.5, XBB.1.5, and XBB.1.16 subvariants. Molecular docking provided insights into how these Nbs interact with the RBD of the BA.5 and JN.1 variants. The application of directed evolution via utilization of error-prone PCR and the synthetic E. coli applied in the FLI-TRAP selection method may be a powerful tool for facilitating simple, fast and economical selection to redirect existing antibodies and to generate antibody fragments to target proteins susceptible to autonomous mutation, not only for viral infection but also other diseases, such as cancer.