The trimer to monomer transition of Tumor Necrosis Factor-Alpha is a dynamic process that is significantly altered by therapeutic antibodies

Herwin Daub,Ulrich Rant,Aymelt Itzen,Fjolla Ismajli,Bastian Groitl,Lukas Traxler

doi:10.1038/s41598-020-66123-5

Abstract

The cytokine tumor necrosis factor-alpha (TNF-α) readily forms homotrimers at sub-nM concentrations to promote inflammation. For the treatment of inflammatory diseases with upregulated levels of TNF-α, a number of therapeutic antibodies are currently used as scavengers to reduce the active TNF-α concentration in patients. Despite their clinical success, the mode-of-action of different antibody formats with regard to a stabilization of the trimeric state is not entirely understood. Here, we use a biosensor with dynamic nanolevers to analyze the monomeric and trimeric states of TNF-α together with the binding kinetics of therapeutic biologics. The intrinsic trimer-to-monomer decay rate k = 1.7 × 10−3 s−1 could be measured directly using a microfluidic system, and antibody binding affinities were analyzed in the pM range. Trimer stabilization effects are quantified for Adalimumab, Infliximab, Etanercept, Certolizumab, Golimumab for bivalent and monovalent binding formats. Clear differences in trimer stabilization are observed, which may provide a deeper insight into the mode-of-action of TNF-α scavengers.

Highlights

Adalimumab Fab, which binds in the interface between two monomeric subunits, whereas each Infliximab Fab binds to a single monomeric subunit within a TNF-α trimer[20,21]
As the monomerization of TNF-α when complexed with its scavengers reflects an increase in free TNF-α, we investigated the stability of TNF-α-trimer-TNF-scavenger complexes with Adalimumab, Infliximab, Golimumab, Certolizumab, and Etanercept as well as Fab fragments derived from Adalimumab, Infliximab and Golimumab
The complex nature of TNF-α being expressed as a trimeric membrane-bound, proteinogenic cytokine that undergoes a conversion to a soluble trimer has been subject of several studies since the early 1990s5–7,12,25

Summary

Introduction

Adalimumab Fab, which binds in the interface between two monomeric subunits, whereas each Infliximab Fab binds to a single monomeric subunit within a TNF-α trimer[20,21]. Previous studies focus on monomer exchange rates in the equilibrium state, employing Förster Resonance Energy Transfer (FRET) as well as analytical size exclusion chromatographic assays[23]. In contrast to these studies, we determined a universal monomerization rate by continuous measurements in real time using a novel Electro Switchable Biosurface technique (ESB)[24]. Some do not (fully) suppress monomerization of bound TNF-α, leading to release of TNF-α monomers These monomeric subunits of TNF-α have not shown to form clinically relevant amounts of trimeric TNF-α. Concentration-dependent re-trimerization has been shown in vitro[7]

Methods

Results

Conclusion