(584ar) Generation of Inhibitory Monoclonal Antibodies Targeting Membrane-Type Matrix Metalloproteinase By Motif Grafting

MMP-14, a membrane-bound zinc endopeptidase, plays an important role in tumor growth, invasion and neovascularization, therefore, it has been recognized as an attractive therapeutic target. However, previous studies of broad-spectrum small molecule inhibitors toward tumorigenic metalloproteinases all failed in clinical trials. It implies that selectivity is the key for a successful proteases inhibition. With exquisite specificity, antibody-based inhibitors are emerging as promising therapeutics. Recently, a cyclic peptide (CFSIAHEC) specifically inhibiting MMP-14 but not other MMPs has been reported. Unfortunately, this peptide inhibitor has a very low affinity of 150 μM, preventing its further applications as a potent therapeutic. We hypothesize that grafting this inhibitory peptide into the CDR-H3 region of antibody scaffolds will confer binding specificity and inhibition function to the antibody. The Fab library carrying the inhibitory motif in CDR-H3 and diversities at other five CDRs was generated by gene assembly. After four rounds of phage panning, several inhibitory Fab clones were identified. Biochemical and enzymatic studies with synthetic substrates were performed to characterize those antibodies. This grafting approach can convert a low affinity cyclic peptide to a more stable and potent therapeutic agent specifically inhibiting MMP-14.