Peptidisc-AssistedHydrophobic Clustering Toward theProduction of Multimeric and Multispecific Nanobody Proteins

Multimerization is a powerful engineering strategy for enhancing protein structural stability, diversity and functional performance. Typical methods for clustering proteins include tandem linking, fusion to self-assembly domains and cross-linking. Here we present a novel approach that leverages the Peptidisc membrane mimetic to stabilize hydrophobic-driven protein clusters. We apply the method to nanobodies (Nbs), effective substitutes to traditional antibodies due to their production efficiency, cost-effectiveness and lower immunogenicity, and we demonstrate the formation of multimeric assemblies termed “polybodies” (Pbs). Starting with Nbs directed against the green fluorescent protein (GFP), we produce Pbs that display an increased affinity for GFP due to the avidity effect. The benefit of this increased avidity in affinity-based assays is demonstrated with Pbs directed against the human serum albumin. Using the same autoassembly principle, we produce bispecific and auto-fluorescent Pbs, validating our method as a versatile engineering strategy to generate multispecific and multifunctional protein entities. Peptidisc-assisted hydrophobic clustering thus expand the protein engineering toolbox to broaden the scope of protein multimerization in life sciences.