Identification of highly selective MMP-14 inhibitory Fabs by deep sequencing

Biotechnol Bioeng. 2017 Jun;114(6):1140-1150. doi: 10.1002/bit.26248. Epub 2017 Feb 20.

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 IC50 values 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.

Keywords: bioinformatics; deep sequencing; inhibitory antibody; matrix metalloproteinase; synthetic library.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Antibodies, Monoclonal / chemistry*
  • Antibodies, Monoclonal / immunology
  • Binding Sites
  • Drug Screening Assays, Antitumor / methods*
  • Epitope Mapping / methods
  • High-Throughput Screening Assays / methods*
  • Humans
  • Immunoglobulin Fab Fragments / chemistry*
  • Immunoglobulin Fab Fragments / immunology
  • Matrix Metalloproteinase 14 / chemistry*
  • Matrix Metalloproteinase 14 / immunology
  • Matrix Metalloproteinase Inhibitors / chemistry*
  • Protein Binding
  • Sequence Analysis, Protein / methods*

Substances

  • Antibodies, Monoclonal
  • Immunoglobulin Fab Fragments
  • Matrix Metalloproteinase Inhibitors
  • Matrix Metalloproteinase 14