Discovery of a junctional epitope antibody that stabilizes IL-6 and gp80 protein:protein interaction and modulates its downstream signaling

Ralph Adams,Michael Wright,Jörg Kinne,Robert Griffin,Richard D Taylor,David Mcmillan,Omar Qureshi,Stephen Rapecki,Alastair D G Lawson,Prashant Mori,Chris Meier,Rebecca J Burnley,Simon Lumb,Chiara R Valenzano,C Kuyler Doyle ,Laura Griffin ,Ulrich Wernery,María Del Carmen Avendaño López ,Terry Baker ,Anna Ettorre

doi:10.1038/srep37716

Ralph Adams, Michael Wright + Show 18 more

Open Access

https://doi.org/10.1038/srep37716

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Abstract

Protein:protein interactions are fundamental in living organism homeostasis. Here we introduce VHH6, a junctional epitope antibody capable of specifically recognizing a neo-epitope when two proteins interact, albeit transiently, to form a complex. Orthogonal biophysical techniques have been used to prove the “junctional epitope” nature of VHH6, a camelid single domain antibody recognizing the IL-6–gp80 complex but not the individual components alone. X-ray crystallography, HDX-MS and SPR analysis confirmed that the CDR regions of VHH6 interact simultaneously with IL-6 and gp80, locking the two proteins together. At the cellular level, VHH6 was able to alter the response of endothelial cells to exogenous IL-6, promoting a sustained STAT3 phosphorylation signal, an accumulation of IL-6 in vesicles and an overall pro-inflammatory phenotype supported further by transcriptomic analysis. Junctional epitope antibodies, like VHH6, not only offer new opportunities in screening and structure-aided drug discovery, but could also be exploited as therapeutics to modulate complex protein:protein interactions.

Highlights

The concept of the interactome and the understanding of the role played in disease by binary protein:protein interactions (PPIs) have opened alternative possibilities for therapeutic intervention[1]
We investigated whether increased and sustained activation of STAT3 due to VHH6 was ascribable to an altered internalization of IL-6–gp[80] using IL-6 labelled with NT647
In this study we introduce VHH6, a prototype locking antibody which recognizes an epitope newly formed at the junction where IL-6 and gp[80] interact to form a transient complex[7] before recruiting the signaling receptor gp[130]

Summary

Introduction

The concept of the interactome and the understanding of the role played in disease by binary protein:protein interactions (PPIs) have opened alternative possibilities for therapeutic intervention[1]. Orthosteric and in particular allosteric modulation of PPIs is considered an emerging frontier in drug discovery[2,3,4], to date few PPIs have been found “druggable” using small molecules and peptides[5] Due to their high specificity and affinity for a particular epitope, antibodies are a natural choice to explore the fine modulation of relevant biological axes by interfering with specific PPIs. In particular, an antibody able to stabilize a transient complex at the junction formed when the two proteins interact would be an invaluable tool for a deeper molecular understanding of PPIs, and to aid the screening or rational design of new biologicals and small molecules with improved targeting capabilities[6]. IL-6 can signal in cis or trans, depending on whether gp[80] is cell membrane-expressed or in a soluble form generated by shedding[9]. Both forms of gp[80] are active and bind IL-610. VHH6 recognizes an epitope that spans the junction between IL-6 and gp[80], locking them together in a stable functional unit

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