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Therapeutic TNF Inhibitors can Differentially Stabilize Trimeric TNF by Inhibiting Monomer Exchange

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.


Stability of trimeric TNF.(A) TNF-488 (DOL 0.6; 3 ng/mL) was incubated at 37 °C and samples were analyzed by HP-SEC. Column was calibrated using a sample of IgG and Fab fragments (resp. 150 and 50 kDa; grey line, detected at 215 nm). Monomeric TNF was only partially recovered. (B) Disappearance of trimeric TNF-488 (3 ng/mL) in time. (C) TNF-488 (3 ng/mL) was incubated alone or in the presence of 27 or 297 ng/mL of unlabeled TNF for 72 hrs and recovery of trimeric TNF-488 was evaluated as in (A). (D) TNF or TNF-488 (3 ng/mL) was incubated at 37 °C for various times and analyzed using an ELISA that only measures trimeric TNF. (A–C) representative of n = 2; D average of triplicate experiment, error bars indicate SEM. F = fluorescence.
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f3: Stability of trimeric TNF.(A) TNF-488 (DOL 0.6; 3 ng/mL) was incubated at 37 °C and samples were analyzed by HP-SEC. Column was calibrated using a sample of IgG and Fab fragments (resp. 150 and 50 kDa; grey line, detected at 215 nm). Monomeric TNF was only partially recovered. (B) Disappearance of trimeric TNF-488 (3 ng/mL) in time. (C) TNF-488 (3 ng/mL) was incubated alone or in the presence of 27 or 297 ng/mL of unlabeled TNF for 72 hrs and recovery of trimeric TNF-488 was evaluated as in (A). (D) TNF or TNF-488 (3 ng/mL) was incubated at 37 °C for various times and analyzed using an ELISA that only measures trimeric TNF. (A–C) representative of n = 2; D average of triplicate experiment, error bars indicate SEM. F = fluorescence.

Mentions: Next, we investigated the dissociation of TNF trimers. TNF-488 was incubated at 3 ng/mL for various times at 37 °C and analyzed using high-performance size-exclusion chromatography (HP-SEC), monitoring the elution of TNF by in-line fluorescence detection. Disappearance of trimeric TNF in time was observed, accompanied by the appearance of monomeric TNF (Fig. 3A). However, recovery of total TNF was not complete, indicating that monomeric TNF may have precipitated, or otherwise, remained on the column. The time-course of dissociation roughly corresponded to that observed for the monomer exchange (Fig. 3B). In the presence of increasing amounts of (unlabeled) TNF, the dissociation was diminished (Fig. 3C). This is consistent with a dynamic process of monomer exchange, with faster re-association at higher TNF concentrations preventing progressive loss of trimeric and monomeric TNF by further unfolding. The disappearance of trimeric TNF furthermore corresponded to the disappearance of activity measured in a TNF ELISA (Fig. 3D).


Therapeutic TNF Inhibitors can Differentially Stabilize Trimeric TNF by Inhibiting Monomer Exchange
Stability of trimeric TNF.(A) TNF-488 (DOL 0.6; 3 ng/mL) was incubated at 37 °C and samples were analyzed by HP-SEC. Column was calibrated using a sample of IgG and Fab fragments (resp. 150 and 50 kDa; grey line, detected at 215 nm). Monomeric TNF was only partially recovered. (B) Disappearance of trimeric TNF-488 (3 ng/mL) in time. (C) TNF-488 (3 ng/mL) was incubated alone or in the presence of 27 or 297 ng/mL of unlabeled TNF for 72 hrs and recovery of trimeric TNF-488 was evaluated as in (A). (D) TNF or TNF-488 (3 ng/mL) was incubated at 37 °C for various times and analyzed using an ELISA that only measures trimeric TNF. (A–C) representative of n = 2; D average of triplicate experiment, error bars indicate SEM. F = fluorescence.
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Related In: Results  -  Collection

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f3: Stability of trimeric TNF.(A) TNF-488 (DOL 0.6; 3 ng/mL) was incubated at 37 °C and samples were analyzed by HP-SEC. Column was calibrated using a sample of IgG and Fab fragments (resp. 150 and 50 kDa; grey line, detected at 215 nm). Monomeric TNF was only partially recovered. (B) Disappearance of trimeric TNF-488 (3 ng/mL) in time. (C) TNF-488 (3 ng/mL) was incubated alone or in the presence of 27 or 297 ng/mL of unlabeled TNF for 72 hrs and recovery of trimeric TNF-488 was evaluated as in (A). (D) TNF or TNF-488 (3 ng/mL) was incubated at 37 °C for various times and analyzed using an ELISA that only measures trimeric TNF. (A–C) representative of n = 2; D average of triplicate experiment, error bars indicate SEM. F = fluorescence.
Mentions: Next, we investigated the dissociation of TNF trimers. TNF-488 was incubated at 3 ng/mL for various times at 37 °C and analyzed using high-performance size-exclusion chromatography (HP-SEC), monitoring the elution of TNF by in-line fluorescence detection. Disappearance of trimeric TNF in time was observed, accompanied by the appearance of monomeric TNF (Fig. 3A). However, recovery of total TNF was not complete, indicating that monomeric TNF may have precipitated, or otherwise, remained on the column. The time-course of dissociation roughly corresponded to that observed for the monomer exchange (Fig. 3B). In the presence of increasing amounts of (unlabeled) TNF, the dissociation was diminished (Fig. 3C). This is consistent with a dynamic process of monomer exchange, with faster re-association at higher TNF concentrations preventing progressive loss of trimeric and monomeric TNF by further unfolding. The disappearance of trimeric TNF furthermore corresponded to the disappearance of activity measured in a TNF ELISA (Fig. 3D).

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.