Résumé
Adenovirus-derived nanoparticles (ADDomer) comprise 60 copies of adenovirus penton base protein (PBP). ADDomer is thermostable, rendering the storage, transport, and deployment of ADDomer-based therapeutics independent of a cold chain. To expand the scope of ADDomers for new applications, we engineered ADDobodies, representing PBP crown domain, genetically separated from PBP multimerization domain. We inserted heterologous sequences into hyper-variable loops, resulting in monomeric, thermostable ADDobodies expressed at high yields in Escherichia coli. The X-ray structure of an ADDobody prototype validated our design. ADDobodies can be used in ribosome display experiments to select a specific binder against a target, with an enrichment factor of ∼104-fold per round. ADDobodies can be re-converted into ADDomers by genetically reconnecting the selected ADDobody with the PBP multimerization domain from a different species, giving rise to a multivalent nanoparticle, called Chimera, confirmed by a 2.2 Å electron cryo-microscopy structure. Chimera comprises 60 binding sites, resulting in ultra-high, picomolar avidity to the target.
[Display omitted]
•Design of ADDobody with high expression yields, thermostability, and loop variability•Crystal structures of ADDobody validate design•104-fold enrichment of binders over non-binders by ADDobody ribosome display selections•ADDobody conversion into nanoparticles with 60 binding sites and ultra-high avidity
Based on an adenoviral protein, Buzas et al. engineer a novel protein scaffold, the ADDobody, validated by high-resolution structure analyses. ADDobody can be functionalized to interact with a chosen antigen. Fusing ADDobody to a multimerization domain results in a megadalton-sized protein nanoparticle binding the antigen extremely tightly due to avidity.