Engineering the ADDobody protein scaffold for generation of high-avidity ADDomer super-binders

Dora Buzas; Huan Sun; Christine Toelzer; Sathish K N Yadav; Ufuk Borucu; Gunjan Gautam; Kapil Gupta; Joshua C Bufton; Julien Capin; Richard B Sessions; Frederic Garzoni; Imre Berger; Christiane Schaffitzel

doi:10.1016/j.str.2023.12.010

Engineering the ADDobody protein scaffold for generation of high-avidity ADDomer super-binders

Structure. 2024 Mar 7;32(3):342-351.e6. doi: 10.1016/j.str.2023.12.010. Epub 2024 Jan 9.

Authors

Affiliations

¹ School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK; Max Planck Bristol Centre for Minimal Biology, Cantock's Close, Bristol BS8 1TS, UK.
² School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK.
³ School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK; Imophoron Ltd, Science Creates Old Market, Midland Road, Bristol BS2 0JZ, UK.
⁴ Imophoron Ltd, Science Creates Old Market, Midland Road, Bristol BS2 0JZ, UK.
⁵ School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK; Max Planck Bristol Centre for Minimal Biology, Cantock's Close, Bristol BS8 1TS, UK; School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 1TS, UK. Electronic address: imre.berger@bristol.ac.uk.
⁶ School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK. Electronic address: cb14941@bristol.ac.uk.

PMID: 38198950
DOI: 10.1016/j.str.2023.12.010

Abstract

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.

Keywords: ADDobody; ADDomer nanoparticle super-binder; Protein engineering; alternative scaffold; ribosome display selection.

MeSH terms

Binding Sites
Protein Engineering*