title: Identification of highly selective MMP-14 inhibitory Fabs by deep sequencing.
abstract: Matrix metalloproteinase (MMP)-14 is an important target for cancer treatment due to its critical roles in tumor invasion and metastasis. Previous failures of all compound-based broad-spectrum MMP inhibitors in clinical trials suggest that selectivity is the key for a successful therapy. With inherent high specificity, monoclonal antibodies (mAbs) therefore arise as attractive inhibitors able to target the particular MMP of interest. As a routine screening method, enzyme-linked immunosorbent assays (ELISA) have been applied to panned phage libraries for the isolation of mAbs inhibiting MMP-14. However, because of suboptimal growth conditions and insufficient antibody expression associated with monoclonal ELISA, a considerable number of potentially inhibitory clones might not be identified. Taking advantage of next-generation sequencing (NGS), we monitored enrichment profiles of millions of antibody clones along three rounds of phage panning, and identified 20 Fab inhibitors of MMP-14 with inhibition IC50values of 10-4,000 nM. Among these inhibitory Fabs, 15 were not found by monoclonal phage ELISA. Particularly, Fab R2C7 exhibited an inhibition potency of 100 nM with an excellent selectivity to MMP-14 over MMP-9. Inhibition kinetics and epitope mapping suggested that as a competitive inhibitor, R2C7 directly bound to the vicinity of the MMP-14 catalytic site. This study demonstrates that deep sequencing is a powerful tool to facilitate the systematic discovery of mAbs with protease inhibition functions. Biotechnol. Bioeng. 2017;114: 1140-1150. © 2017 Wiley Periodicals, Inc.
authors.affiliation: Department of Chemical and Environmental Engineering, University of California, 900 University Ave, Riverside, California 92521.
authors.forename: Tyler
authors.initials: T
authors.lastname: Lopez
authors.affiliation: Department of Chemical and Environmental Engineering, University of California, 900 University Ave, Riverside, California 92521.
authors.forename: Dong Hyun
authors.initials: DH
authors.lastname: Nam
authors.affiliation: Department of Chemical and Environmental Engineering, University of California, 900 University Ave, Riverside, California 92521.
authors.forename: Evan
authors.initials: E
authors.lastname: Kaihara
authors.affiliation: Department of Chemical and Environmental Engineering, University of California, 900 University Ave, Riverside, California 92521.
authors.forename: Zahid
authors.initials: Z
authors.lastname: Mustafa
authors.affiliation: Department of Chemical and Environmental Engineering, University of California, 900 University Ave, Riverside, California 92521.
authors.forename: Xin
authors.initials: X
authors.lastname: Ge
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D001665:Binding Sites; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D018604:Epitope Mapping / Q000379:methods; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000276:immunology; D053511:Matrix Metalloproteinase 14 / Q000737:chemistry* / Q000276:immunology; D061965:Matrix Metalloproteinase Inhibitors / Q000737:chemistry*; D011485:Protein Binding; D020539:Sequence Analysis, Protein / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D061965:Matrix Metalloproteinase Inhibitors; D053511:Matrix Metalloproteinase 14
keywords: bioinformatics; deep sequencing; inhibitory antibody; matrix metalloproteinase; synthetic library