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

Abstract

SummaryAdenovirus-derived dodecamer (ADDomer) nanoparticles 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 ADDomer nanoparticles for new applications, we engineered ADDobodies. ADDobodies represent the crown domain of the PBP, genetically separated from its multimerization domain. We inserted heterologous sequences into hyper-variable loops in the crown domain. The resulting ADDobodies were expressed at high yields inEscherichia coli,are monomeric and maintain thermostability. We solved the X-ray structure of an ADDobody prototype validating our design. We demonstrated that ADDobodies can be used to select a specific binder against a target inin vitroselection experiments using ribosome display, with an enrichment factor of ∼104-fold in one selection round. We show that ADDobodies can be converted back 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 structure determined by cryogenic electron microscopy (cryo-EM). Chimera comprises 60 binding sites, resulting in ultra-high, picomolar avidity to the target.

ADDovenom: Thermostable Protein-Based ADDomer Nanoparticles as New Therapeutics for Snakebite Envenoming

“…The bipartite architecture of the protomer suggested a possibility to separate the crown from the jelly-roll fold. Thus, we designed ADDobody, comprising only the crown domain and produced the prototype in Escherichia coli with excellent yields as a monomeric protein ( Figure 1 d) [ 23 ]. Next, the VL and the RGD loops of the crown domain were randomised in length and sequence, mimicking the CDR loops of antibodies ( Figure 1 d).…”

“…Selected specific binders are biophysically characterised and tested for toxin neutralisation using enzymatic or cell-based assays. In a next step, the selected toxin-neutralising ADDobody can be rejoined with the jelly-roll fold multimerisation domain to produce the ADDomer-based superbinders [ 23 ] ( Figure 1 ). The resulting antitoxin nanoparticle displays 60 binding domains against the toxin target, ensuring high-avidity binding and highly efficient neutralisation of the snake toxin targets.…”

ADDovenom: Thermostable Protein-Based ADDomer Nanoparticles as New Therapeutics for Snakebite Envenoming

“…The bipartite architecture of the protomer suggested a possibility to separate the crown from the jelly-roll fold. Thus, we designed ADDobody, comprising only the crown domain and produced the prototype in Escherichia coli with excellent yields as a monomeric protein (Figure 1d) [23]. Next, the VL and the RGD loops of the crown domain were randomised in length and sequence, mimicking the CDR loops of antibodies (Figure 1d).…”