Abstract
The remarkable clinical success of Fc-fusion proteins has driven intense investigation for even more potent replacements. Using quality-by-design (QbD) approaches, we generated hexameric-Fc (hexa-Fc), a ~20 nm oligomeric Fc-based scaffold that we here show binds low-affinity inhibitory receptors (FcRL5, FcγRIIb, and DC-SIGN) with high avidity and specificity, whilst eliminating significant clinical limitations of monomeric Fc-fusions for vaccine and/or cancer therapies, in particular their poor ability to activate complement. Mass spectroscopy of hexa-Fc reveals high-mannose, low-sialic acid content, suggesting that interactions with these receptors are influenced by the mannose-containing Fc. Molecular dynamics (MD) simulations provides insight into the mechanisms of hexa-Fc interaction with these receptors and reveals an unexpected orientation of high-mannose glycans on the human Fc that provides greater accessibility to potential binding partners. Finally, we show that this biosynthetic nanoparticle can be engineered to enhance interactions with the human neonatal Fc receptor (FcRn) without loss of the oligomeric structure, a crucial modification for these molecules in therapy and/or vaccine strategies where a long plasma half-life is critical.
Highlights
The remarkable clinical success of Fc-fusion proteins has driven intense investigation for even more potent replacements
FccRIIb, FcRL5, and dendritic cell (DC)-SIGN may limit immune cell activation against chronic pathogens or self-reactive antigen, and approaches that have the potential to target these receptors with high affinity/avidity may prove beneficial in therapies, including intravenous immunoglobulin (IVIG), aimed at controlling pro-inflammatory disease[1,4]
To test if hexa-Fc could bind to other, non-classical Fc-receptors that are believed to be involved in controlling disease[9], we investigated the interaction of hexa-Fc with soluble recombinant human dendritic cell-specific intercellular adhesion molecule-3grabbing non-integrin (DC-SIGN) tetramers by multichannel surface plasmon resonance analysis (MCSPR)[26,27]
Summary
The remarkable clinical success of Fc-fusion proteins has driven intense investigation for even more potent replacements. Using quality-by-design (QbD) approaches, we generated hexameric-Fc (hexa-Fc), a ,20 nm oligomeric Fc-based scaffold that we here show binds low-affinity inhibitory receptors (FcRL5, FccRIIb, and DC-SIGN) with high avidity and specificity, whilst eliminating significant clinical limitations of monomeric Fc-fusions for vaccine and/or cancer therapies, in particular their poor ability to activate complement. F c-fusion proteins are a well-established class of therapeutics[1], presently exhibiting the greatest growth rate of all biologics in the United States[2] Notwithstanding this success though, there is great interest in identifying novel approaches to improve their efficacy and safety while expanding their range of potential clinical applications to other areas such as vaccines[3] and replacements for intravenous immunoglobulin (IVIG) therapy[1,4]. Multimerization would be expected to significantly enhance their interaction with the www.nature.com/scientificreports salvage neonatal Fc-receptor (FcRn), a crucial association that significantly prolongs the plasma half-life and likewise therapeutic and/ or vaccine activity of any Fc-containing protein[1,20]
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