As extremely important signaling molecules, proteases precisely control a wide variety of physiological processes, and thus serve as one of the largest families of pharmaceutical targets. Most therapeutic protease inhibitors currently in clinical use or under developments are peptides or their chemical compound mimics. Considering that 500-600 proteases have been identified in human genome, specificity is highly desired for any protease inhibition therapy. However, the reaction mechanism is highly conserved among the same class or family of proteases, thus achieving target specificity has been recognized as the main hurdle for protease inhibitor developments. The crucial importance of specificity has been highlighted by the extensive studies on inhibition of matrix metalloproteinases (MMPs) using zinc-chelating compounds as a strategy for treating cancer. Although pre-clinical results were promising, previous attempts focusing on development of chemical compound inhibitors, e.g., hydroxamates, targeting broad-spectrum MMPs all failed in clinical trials due to severe side effects. With these aspects, antibody-based inhibitors are emerging as very attractive therapeutic agents due to their exclusive specificity given by a large antigen-antibody contact surface.
Inspired by the fact that a large proportion of antibodies isolated from camels and llamas can bind to active pockets and inhibit enzymatic reactions, we analyzed the sequences and known structures of inhibitory antibodies, and hypothesized that convex shaped paratopes encoding by ultra long CDRs are inhibition-prone. Large synthetic human antibody libraries (>109 variants) carrying extended CDR-H3 ranging from 23 to 27 aa were constructed, and subjected for isolation of antibodies targeting MMP-14. Notable, dozens of inhibitory antibodies have been isolated, which accounted for > 50% of identified binding clones. CDR-H3 sequences of these inhibitory clones showed dramatic patterns with central histidines flanked by arginine and lysine rich sequences. These residues presumably contribute to chelate the catalytic zinc and interact with negatively charged surface of active sites of MMP-14, respectively. Among isolated high affinity inhibitory clones, several showed exclusive selectivity toward MMP-14 over MMP-2/-9. Particularly, 3A2 Fab was a competitive inhibitor exhibiting a binding affinity of 4.85 nM by SPR analysis and an inhibition potency of 25 - 50 nM. Gelatin zymography and type-I collagen degradation using fibrosarcoma cell lines demonstrated its inhibitory function at physiological environments. We believe our novel inhibitory antibody discovery approach has great potentials to be applied for many biomedically important but challenging targets for conventional antibody techniques