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

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.

Cytokine “fine tuning” of enthesis tissue homeostasis as a pointer to spondyloarthritis pathogenesis with a focus on relevant TNF and IL-17 targeted therapies

“…TNF exerts its affects through two different receptors: TNFRI/p55 and TNFRII/p75 [ 53 ]. TNFRI is expressed ubiquitously and constitutively, whilst TNFRII is only expressed on immune cells.…”

Size-Based Characterization of Adalimumab and TNF-α Interactions Using Flow Induced Dispersion Analysis: Assessment of Avidity-Stabilized Multiple Bound Species

“…The fact that no interaction is observed at low adalimumab concentration indicates a fast dissociation of the primary complexes (TA, T 2 A and TA 2 ) relative to the mobilization time inside the capillary, whereas the avidity stabilized (TA) 2 displays significantly slower dissociation kinetics as expected from SPR 10 and hydrodynamic friction measurements 25 . According to Fig.…”

“…found the structure of TNF-α as trimeric at 100 nM (pH 7.4) using hydrodynamic friction measurements 25 . The determined hydrodynamic radius of adalimumab was consistent with previous TDA measurements (5.6 -5.9 nm) 26 .…”

“…In particular, surfacebased methodologies lack the possibility of easily controlling the concentrations of both binding partners, which complicates quantitative assessment of avidity effects. For instance, the TNF-αadalimumab interaction was recently characterized using hydrodynamic friction measurements 25 , which confirmed strong avidity effects and the presence of higher order complexes. The existing studies of the TNF-α -adalimumab system have been primarily conducted in neat buffer solutions, hence, little is known on TNF-α -adalimumab interactions under native (in-solution) and biorelevant conditions (e.g., in human plasma).…”

Unexpected similarity between HIV-1 reverse transcriptase and tumor necrosis factor revealed by binding site image processing

“…The MST signal is based on ligand-dependent temperature-induced changes (thermophoresis, temperature-related fluorescence intensity) of the fluorescence emission of the labelled protein target. The 17.3 kDa homotrimeric TNF-α that spontaneously assemble in solution [28,29] was therefore labelled by a REDfluorescent probe for MST experiments in presence of increasing concentrations of the three HIV-1 RT inhibitors (Fig 3). MST traces in presence of efavirenz and delavirdine showed distinct states (from bound to unbound), indicating a direct interaction of these two inhibitors with TNF-α (Fig 3A, B).…”