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...
The metalloproteinase ADAM17 activates ErbB signalling by releasing ligands from the cell surface, a key step underlying epithelial development, growth, and tumour progression. However, mechanisms acutely controlling ADAM17 cell-surface availability to modulate the extent of ErbB ligand release are poorly understood. Here, through a functional genome-wide siRNA screen, we identify the sorting protein PACS-2 as a regulator of ADAM17 trafficking and ErbB signalling. PACS-2 loss reduces ADAM17 cell-surface levels and ADAM17-dependent ErbB ligand shedding, without apparent effects on related proteases. PACS-2 co-localizes with ADAM17 on early endosomes and PACS-2 knockdown decreases the recycling and stability of internalized ADAM17. Hence, PACS-2 sustains ADAM17 cell-surface activity by diverting ADAM17 away from degradative pathways. Interestingly, Pacs2-deficient mice display significantly reduced levels of phosphorylated EGFR and intestinal proliferation. We suggest that this mechanism controlling ADAM17 cell-surface availability and EGFR signalling may play a role in intestinal homeostasis, with potential implications for cancer biology.
Purpose: BRCA1 and BRCA2 are key tumor suppressors with a role in cellular DNA repair, genomic stability, and checkpoint control. Mutations in BRCA1 and BRCA2 often cause hereditary breast and ovarian cancer; however, missense polymorphisms in these genes pose a problem in genetic counseling, as their impact on risk of breast and ovarian cancer is unclear. Experimental Design: We resequenced BRCA1 and BRCA2 in 194 women with a familial history of breast and/or ovarian cancer and identified nine possibly biologically relevant polymorphisms (BRCA1 Gln356Arg, Pro871Leu, Glu1038Gly, Ser1613Gly, and Met1652Ile. BRCA2 Asn289His, Asn372His, Asp1420Tyr, and Tyr1915Met). We evaluated risk of breast and/or ovarian cancer by these polymorphisms in a prospective study of 5,743 women from the general population followed for 39 years and in a casecontrol study of 1,201 breast cancer cases and 4,120 controls. Results: We found no association between heterozygosity or homozygosity for any of the nine polymorphisms and risk of breast and/or ovarian cancer in either study. We had 80% power to exclude hazard/odds ratios for heterozygotes and/or homozygotes for all nine missense polymorphisms above 1.3 to 3.3 in the prospective study, and above 1.2 to 3.2 in the case-control study. Conclusions: Heterozygosity and homozygosity of any of the examined nine BRCA1 and BRCA2 missense polymorphisms cannot explain the increased risk of breast and/or ovarian cancer observed in families with hereditary breast and/or ovarian cancer. Therefore, genetic counseling of such families safely can disregard findings of these missense polymorphisms. (Cancer Epidemiol Biomarkers Prev 2009;18(8):2339-42)
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