Directed evolution of a stem-helix-targeting antibody enables MERS-CoV cross-neutralization through enhanced binding affinity.

Broadly neutralizing antibodies (bnAbs) targeting conserved regions of the betacoronavirus spike are important for pan-betacoronavirus protection and pandemic preparedness. Here, we report on the isolation of a human monoclonal antibody, CC65.1, from a SARS-CoV-2 convalescent donor that targets the conserved S2 stem helix region. CC65.1 neutralizes various sarbecoviruses, including SARS-CoV-2, and binds the MERS-CoV spike but lacks MERS-CoV-neutralizing activity due to insufficient binding affinity. We utilized directed evolution to enhance the binding affinity of CC65.1 for the MERS-CoV S2 stem helix, yielding engineered antibody variants with newly acquired MERS-CoV-neutralizing activity. High-resolution structural analysis reveals key paratope mutations that optimize binding and stabilize epitope engagement. Our findings demonstrate the potential of rational antibody engineering to expand bnAb breadth across divergent betacoronaviruses. This work supports the development of engineered bnAbs and S2-targeted vaccines for broad betacoronavirus countermeasures and highlights strategies to achieve cross-lineage immunity for future pandemic threats.