Salvatore Santamaria, Kazuhiro Yamamoto, Kenneth Botkjaer, Christopher Tape, Michael R. Dyson, John McCafferty, Gillian Murphy, Hideaki Nagase

ADAMTS-5 is the multi-domain metalloprotease that most potently degrades aggrecan proteoglycan in the cartilage and its activity is implicated in the development of osteoarthritis. To generate specific exosite inhibitors for it, we screened a phage display antibody library in the presence of the zinc-chelating active site-directed inhibitor GM6001 and isolated four highly selective inhibitory antibodies. Two antibodies were mapped to react with exosites in the catalytic/disintegrin domains of the enzyme, one in the thrombospondin domain, and one in the spacer domain. The antibody reacting with the spacer (Sp) domain blocked the enzyme action only when aggrecan or the E. coli -expressed aggrecan core protein were substrates, but not against a peptide substrate. The study with this antibody revealed the importance of the spacer domain for effective aggrecanolytic activity of ADAMTS-5, and that this domain does not interact with sulfated glycosaminoglycans but with the protein moiety of the proteoglycan. An antibody directed against the catalytic/disintegrin domain of ADAMTS-5 was effective in a cell-based model of aggrecan degradation; however, the anti-Sp antibody was ineffective. Western blot analysis of endogenous ADAMTS-5 expressed by human chondrocytes showed the presence of largely of truncated forms of ADAMTS-5, thus explaining the lack of efficacy of the anti-Sp antibody. The possibility of ADAMTS-5 truncation must then be taken into account when considering developing anti-ancillary domain antibodies for therapeutic purposes.