Update and reuse: Structure-guided nanobody evolution against SARS-CoV-2 escape.

SARS-CoV-2 continues to accumulate spike mutations that erode the efficacy of antibody therapeutics. The Q493E mutation in the spike RBD, present in recent Omicron subvariants, enables escape from many antibodies and nanobodies, including our Nanosota-9A nanobody, which neutralizes Omicron JN.1 (Q493) but not KP.3 (E493). To address this, we applied a structure-guided in vitro evolution strategy to engineer Nanosota-9A, generating Nanosota-9B, which binds the KP.3 RBD with high affinity but shows reduced binding to JN.1 RBD. To regain breadth, we engineered a bispecific nanobody combining Nanosota-9A and -9B, which effectively neutralizes both JN.1 and KP.3 in infection assays. Our results provide proof of concept for an "update and reuse" strategy: applying structure-guided engineering to update and reuse validated nanobodies to overcome variant escape. This strategy offers a practical path to maintain therapeutic coverage as the virus evolves, supporting more efficient use of research resources and faster responses to emerging variants.