Engineering a β-Sheet Enables Bispecific Binding in Single VHH Domains

Bispecific protein binding empowers unique functionality in multiple therapeutic contexts. Bispecificity is typically accomplished using multidomain assemblies of antibodies or other ligand proteins. However, their molecular complexity and large size hinder development and limit utility. Herein we address this technological gap by engineering a VHH nanobody to contain a second binding paratope─in the β-sheet─independent from the native complementarity-determining region (CDR) paratope to achieve bispecific binding within a single domain. We used this platform to engineer multiple dual binders to two target combinations: EGFR/PD-L1 and HER2/TfR. One variant, v6, exhibits affinities of 1.7 nM for PD-L1 and 3.6 nM for EGFR. Grafting the PD-L1-binding v6 β-sheet paratope into two HER2-binding VHHs and the trastuzumab scFv and IgG all enabled single-digit nM dual binding, thereby establishing the modularity of these novel binding sites. We also demonstrated the modularity of full VHHs by reformatting them into cell surface receptors that retained dual binding. Combined, these results outline a novel method for engineering broad bivalency into single immunoglobulin scaffold domains and highlight the utility of β-sheet paratopes in high-affinity binding.