Therapeutic TNF Inhibitors can Differentially Stabilize Trimeric TNF by Inhibiting Monomer Exchange.
Tumor necrosis factor (TNF) is a homotrimeric cytokine that is a key mediator of inflammation. It is unstable at physiological concentrations and slowly converts into an inactive form. Here, we investigated the mechanism of this process by using a Förster resonance energy transfer (FRET) assay that allowed monitoring of monomeric subunit exchange in time. We observed continuous exchange of monomeric subunits even at concentrations of TNF high enough to maintain its bioactivity. The kinetics of this process closely corresponds with the appearance of monomeric subunits and disappearance of trimeric TNF in time at ng/ml concentrations as monitored by high-performance size-exclusion chromatography (HP-SEC). Furthermore, of the five therapeutic TNF inhibitors that are currently used in the clinic, three (adalimumab, infliximab, etanercept) were found to completely inhibit the monomer exchange reaction and stabilize TNF trimers, whereas golimumab and certolizumab could not prevent monomer exchange, but did slow down the exchange process. These differences were not correlated with the affinities of the TNF inhibitors, measured with both surface plasmon resonance (SPR) and in fluid phase using fluorescence-assisted HP-SEC. The stabilizing effect of these TNF inhibitors might result in prolonged residual TNF bioactivity under conditions of incomplete blocking, as observed in vitro for adalimumab.
Sanquin Research, Department of Immunopathology and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, The Netherlands.
Karin A
KA
van Schie
Sanquin Research, Department of Immunopathology and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, The Netherlands.
Pleuni
P
Ooijevaar-de Heer
Sanquin Research, Department of Immunopathology and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, The Netherlands.
Lisanne
L
Dijk
Sanquin Research, Department of Immunopathology and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, The Netherlands.
Simone
S
Kruithof
Sanquin Research, Department of Immunopathology and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, The Netherlands.|Jan van Breemen Research Institute | Reade, Amsterdam, The Netherlands.
Gertjan
G
Wolbink
Sanquin Research, Department of Immunopathology and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, The Netherlands.
Theo
T
Rispens
D000068879:Adalimumab / Q000378:metabolism* / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000378:metabolism / Q000494:pharmacology; D002850:Chromatography, Gel; D000068800:Etanercept / Q000378:metabolism* / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D000069285:Infliximab / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D055503:Protein Multimerization; D055550:Protein Stability; D020349:Surface Plasmon Resonance; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D014409:Tumor Necrosis Factor-alpha; C529000:golimumab; D000069285:Infliximab; D000068879:Adalimumab; D000068800:Etanercept