Cross-domain inhibition of TACE ectodomain

Abstract

Proteolytic release from the cell surface is an essential activation event for many growth factors and cytokines. TNF-α converting enzyme (TACE) is a membrane-bound metalloprotease responsible for solubilizing many pathologically significant membrane substrates and is an attractive therapeutic target for the treatment of cancer and arthritis. Prior attempts to antagonize cell-surface TACE activity have focused on small-molecule inhibition of the metalloprotease active site. Given the highly conserved nature of metalloprotease active sites, this paradigm has failed to produce a truly specific TACE inhibitor and continues to obstruct the clinical investigation of TACE activity. We report the bespoke development of a specific TACE inhibitory human antibody using "two-step" phage display. This approach combines calculated selection conditions with antibody variable-domain exchange to direct individual antibody variable domains to desired epitopes. The resulting "cross-domain" human antibody is a previously undescribed selective TACE antagonist and provides a unique alternative to smallmolecule metalloprotease inhibition. ADAM17 | antibody phage display | cancer therapeuticsT NF-α converting enzyme (TACE) [a disintegrin and metalloprotease 17 (ADAM17)], is a membrane-bound metalloprotease responsible for cleaving a variety of pathologically significant substrates (1). Initially identified as the enzyme responsible for solubilizing membrane-associated pro-TNF-α (2, 3)-a process subsequently termed "ectodomain shedding," TACE has since proved capable of cleaving epidermal growth factor receptor (EGFR) ligands (4), extracellular Notch1 (5), cell-surface receptors (6), and adhesion molecules (7). As proteolytic cleavage is an indispensable activation event for many of these substrates, TACE has emerged as an attractive therapeutic target for the treatment of cancer (8) and rheumatoid arthritis (9).Preceding the current clinical interest in TACE, members of the related matrix metalloprotease (MMP) family were also considered viable therapeutic targets (10). Despite sound preclinical rational for antagonizing MMPs, early trials of smallmolecule inhibitors (SMIs) failed due to poor inhibitor specificity profiles (11,12). Metalloprotease SMIs exclusively target the catalytic site. This paradigm treats the raw proteolytic capacity of the catalytic site as the only significant target-often with no regard to noncatalytic domains. This catalytic focus forces the selectivity of SMIs to rely exclusively on modest biophysical differences surrounding individual metalloprotease catalytic sites and typically yields inhibitors simply with a bias toward a particular metalloprotease. Macromolecular metalloprotease inhibitors [e.g., tissue inhibitor of metalloproteases (TIMPs) and metalloprotease prodomains] also focus on binding the catalytic site and suffer comparable specificity limitations. The unfortunate simplification of multidomain extracellular proteases to spatially isolated catalytic sites has ignored the potential for noncatal...

Shedding of Endogenous Interleukin-6 Receptor (IL-6R) Is Governed by A Disintegrin and Metalloproteinase (ADAM) Proteases while a Full-length IL-6R Isoform Localizes to Circulating Microvesicles

“…Small molecule inhibitors tend to have off-target effects and indeed both GI and GW have been reported to additionally inhibit certain matrix metalloproteases (29). Therefore, we sought to additionally validate our results and took advantage of a recently developed ADAM17 inhibitory antibody, which due to its "cross-domain" architec-ture is highly specific for human ADAM17 (19). IL-6R shedding after PMA stimulation was significantly reduced in the presence of the antagonistic ADAM17 antibody D1(A12) confirming that cleavage of the endogenous human receptor is in fact relying on ADAM17 (Fig.…”

“…Cells were stimulated with PMA or LPS from Escherichia coli O111:B4 (both Sigma) as depicted in the figure legends. Generation of neutralizing, bivalent ADAM17 antibody D1(A12) and recombinant murine ADAM10 prodomain were described previously (19,20). The hydroxamate-based ADAM inhibitors GI254023X and GW280264X were synthesized by Iris Biotech (Marktredwitz, Germany).…”

NK cell CD16 surface expression and function is regulated by a disintegrin and metalloprotease-17 (ADAM17)

“…Cells were stained with antibodies against extracellular antigens and were then fixed and permeabilized before intracellular staining to detect IFN-g. A highly selective ADAM17 inhibitor (BMS566394) from Bristol-Myers Squibb (referred to as inhibitor 32) 23 was used at a 10 mM concentration, as previously described, 24 and was added to 1 3 10 6 /mL purified NK cells 30 minutes before testing. We also used a specific ADAM17 inhibitory monoclonal antibody (D1[A12], 6 mg/mL) generated by phage display that contains individual antibody variable domains to 2 distinct ADAM17-specific epitopes 25 to confirm ADAM17 specificity (kindly provided by Dr Gillian Murphy, University of Cambridge, UK).…”

A Monoclonal Antibody to ADAM17 Inhibits Tumor Growth by Inhibiting EGFR and Non–EGFR-Mediated Pathways

“…Although the molecular basis for this discrepancy remains unresolved, active cellular ADAM17 is known to undergo a structural transition allowing accessibility of its active site to small-molecule inhibitors (15). To some extent, these challenges have been overcome in the recent report of a human ADAM17 antibody capable of inhibiting ligand shedding in cells with an IC 50 of approximately 10 nmol/L (16). Nevertheless this antibody does not inhibit mouse ADAM17 and therefore cannot be used to determine the requirement for ADAM17 in either syngeneic mouse tumor models or xenograft models with a significant component of stromal-mediated ligand production.…”