Increased Potency of a Bi-specific TL1A-ADAM17 (TACE) Inhibitor by Cell Surface Targeting

Abstract

Inflammatory bowel disease (IBD) is a multifactorial disease characterized by the dysregulated activity of many pro-inflammatory factors. Thus, bi-specific inhibitors for the simultaneous inhibition of two pro-inflammatory factors can exhibit high therapeutic potential. Here, we developed a novel bi-specific inhibitor targeting the TL1A cytokine and ADAM17/TACE metalloprotease. Biochemical analysis of the bi-specific inhibitor revealed high TL1A binding and TACE inhibition that is similar to the two respective mono-specific inhibitors. Interestingly, cell based assays for TL1A inhibition revealed strong synergism between the inhibitory domains showing an up to 80-fold increase in potency of the bi-specific inhibitor. The dramatic increase in potency is associated with binding to cell membranes through the TACE inhibitory domain leading to increased concentration of the inhibitor on the cell surface. Our study highlights the high potential of the simultaneous targeting of cell surface metalloprotease (TACE) and soluble pro-inflammatory cytokine (TL1A) as a potential therapeutic approach in IBD.

Keywords: ADAM17; TACE; TL1A; bi-specific inhibitors; metalloproteases; pro-inflammatory cytokines; protein engineering.

Figure 1

Schematic representation of the bi-specific soluble DR3 and pTACE inhibitor targeting TL1A and TACE. H3 is an engineered variant of soluble DR3 exhibiting high affinity and stability relative to the WT (Levin et al., 2017).

Figure 2

The A2 bi-specific inhibitor exhibits similar biochemical activity as the two mono-specific H3 (DR3) and pTACE inhibitors. (A) Binding of A2 to TL1A in comparison to the mono-specific H3 protein. Binding analysis was performed by ELISA using plates that were pre-coated with TL1A (see Section Materials and Methods for detailed description). (B) Inhibition of TACE by A2 in comparison to the mono-specific pTACE inhibitor. Inhibition of TACE activity was assessed using a fluorogenic peptide substrate DEVD-AMC.

Figure 3

Similar inhibition level of TNF-α release from macrophages following incubation with A2 and the mono-specific pTACE inhibitor. The levels of TNF-α secretion from mouse peritoneal macrophages were determined following 3 h of LPS stimulation at a concentration of 2.5 ng/ml with or without 1 μM of A2 or pTACE. Media supernatant was analyzed by ELISA for detection of TNF-α levels, the TNF-α values were calculated according to TNF-α calibration curve, ^*P < 0.05.

Figure 4

Increased potency of A2 vs. H3 (DR3) in inhibiting TL1A induced IFN-γ secretion and apoptosis in PBL and TF-1 cell line, respectively. (A) Inhibition of TL1A-induced secretion of IFN-γ in human PBL by increased concentration of A2 and H3. Cells were incubated for 72 h with 200 ng/ml TL1A, 20 ng/ml IL-12 and 50 ng/ml IL-18, and different concentrations of A2 and H3 inhibitors. The 1:10 diluted cell supernatant was analyzed by ELISA for detection of IFN-γ levels. The IFN-γ values were calculated according to IFN-γ calibration curve. (B) Inhibition of TL1A-induced apoptosis in TF-1 cells by increased concentration of A2 and H3. Cells were incubated for 6 h with 8 μg/ml of cyclohexamide (CHX) and 75 ng/ml of TL1A and the indicated concentration of A2 and H3 receptors. Following incubation, lysis buffer containing the caspase-3 fluorescent substrate DEVD-AMC was added and enzyme activity was monitored for 10 min. The data presented in the PBL and TF-1 experiments is the average of three independent repeats of each experiment and the error bars represent the standard deviation from the average.

Figure 5

High binding of the A2 bi-specific inhibitor to TF-1 cells through the pTACE domain. (A) Flow cytometry histogram analysis of gated TF-1 cell population incubated with A2 (blue), H3 (green) and antibody control (without the addition of the A2 or H3, red). Binding to the cell membrane was analyzed following incubation with allophycocyanin (APC) fluorescent anti-human Fc. (B) A2 binds cells through the pTACE domain. Shown is a competition experiment where 100 nM A2 was incubated with TF-1 cells in the presence or absence of 15 μM pTACE inhibitor that lacks an Fc region. A decrease in cell labeled population is observed in the presence of pTACE suggesting that A2 binds cells through endogenous membrane TACE. (C) A model for TL1A inhibition by the mono-specific soluble DR3 (H3) and the A2 bi-specific DR3 (H3)-pTACE. Left, binding of H3 to TL1A leads to depletion of free TL1A due to a competition with the endogenous DR3 membrane receptor. Right, the bi-specific H3-pTACE is bound to cell surface TACE located on the cell membrane leading to a high local concentration of the inhibitor on the cell membrane. This cell targeting leads to increased potency of inhibition of TL1A induced apoptosis in TF-1 cells by up to ~80-fold (Figure 4).