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

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.

Figure 1. Structure of the different TNF inhibitors.

Adalimumab (ADL) is a fully human IgG1 kappa antibody, as is golimumab (GOL). Certolizumab pegol (CZP) is a pegylated humanized Fab’ fragment, infliximab (IFX) a chimeric antibody, and etanercept (ETN) a receptor-Fc construct. Human origin is shown in grey, murine origin in black.

Figure 2. Kinetics of monomer exchange of TNF.

(A,C,E) TNF (0.5 μg/mL), fluorescently labeled with DyLight-488 (TNF-488) or DyLight-594 (TNF-594), both with a degree of labeling of ca. 2, will exchange monomeric units resulting in trimeric TNF molecules that contain both 488- and 594-labeled monomers (A). These molecules will generate a FRET signal at 620 nm following excitation at 488 nm, which can be monitored in real-time (C). The process was also monitored for extended times by consecutive measurements (E). (B,D,F) TNF trimers that acquired monomeric subunits with both types of fluorescent label as described in A will generate a FRET signal (B). If an excess of unlabeled TNF is added, the fluorescently labeled monomers will ‘dilute’ over many TNF trimers resulting in a loss of FRET signal in time (D,F). This also represents the process of monomer exchange. Blue line represents control (ctrl) where no unlabeled TNF is added. (C,D) representative examples of triplicate experiments, (E,F) average of duplicate experiments; error bars indicate SEM. F_rel = F₆₂₀/F₅₈₈. Ex = excitation, Em = emission.

Figure 3. 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.

Figure 4. Influence of TNF inhibitors on TNF stability.

(A,B) Mixed TNF-488/594 (0.5 μg/mL, incubated at 37 °C for 7 days) was incubated at 37 °C with 3 μg/mL unlabeled TNF as described in Fig. 2B, and monomer exchange was monitored as the disappearance of FRET signal either in real-time to cover the initial kinetics (A) or by consecutive measurements for the slower kinetics (B). Different TNF inhibitors were added at 100 μg/mL. Adalimumab (ADL), etanercept (ETN), and infliximab (IFX) blocked monomer exchange to undetectable levels, whereas golimumab (GOL), and certolizumab pegol (CZP) reduced the rate of monomer exchange. In the control experiment (ctrl), no TNF was added. Shown in (A) are representative examples, in (B) the average of duplicate experiments. (C) TNF-488 (0.3 ng/mL) was incubated for up to 5 days at 37 °C and analyzed as described in Fig. 3A (n = 2). Addition of 300 ng/mL unlabeled TNF largely prevented loss of trimer. The addition of 1 μg/mL of adalimumab or etanercept to 0.3 ng/ml TNF-488 resulted in the formation of TNF-containing complexes that remained stable during the 5-day incubation. However, complexes formed with 1 μg/mL of certolizumab pegol slowly degraded. F_rel = F₆₂₀/F₅₈₈. F = fluorescence.

Figure 5. Affinities of TNF inhibitors in fluid phase.

(A) Example of fluid-phase affinity measurement of TNF-anti-TNF. DyLight-488 labeled adalimumab Fab’ is incubated with different concentrations of TNF and analyzed by HP-SEC, monitoring the elution of bound and free adalimumab Fab’-488 by in-line fluorescence detection. (B) Free Fab-488 is plotted vs the concentration of TNF (molar concentration of binding sites) and a 1:1 Langmuir binding model is fitted, thus assuming independent association of anti-TNF Fab’s to each binding site on TNF. (C) TNF-488 was incubated with different concentrations of etanercept and analyzed analogously to B, using the molar concentration of etanercept in the calculations. Representative of n = 3. F = fluorescence.

Figure 6. Influence of time, temperature and the TNF inhibitor adalimumab on TNF bioactivity.

(A) Bioactivity of a titration of TNF was measured immediately or after three days of incubation at 4 °C or 37 °C using a TNF sensitive WEHI bioassay. (B) Effect of adalimumab Fab’ on TNF activity, determined immediately or after three days of incubation at 37 °C. (C,D) Effect of adalimumab on TNF activity, measured immediately or after three days of incubation at 4 °C (C) or 37 °C (D). All graphs show representative data of two (A–C) or four (D) experiments.

Figure 7

Models of TNF in complex with (A) adalimumab Fab and (B) infliximab Fab (top view). Adalimumab binds an epitope comprising two TNF monomers. Infliximab binds an epitope comprising a single TNF monomer. Based on PDB structures 3WD5 and 4G3Y.