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Identification of highly selective MMP-14 inhibitory Fabs by deep sequencing
Tyler Lopez1, Dong Hyun Nam1, Evan Kaihara1
1Department of Chemical and Environmental Engineering, University of California, 900 University Ave, Riverside, California 92521.
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.
Insights
Next-generation sequencing identified novel antibody inhibitors for matrix metalloproteinase (MMP)-14, a key target in cancer metastasis. This deep sequencing approach overcomes limitations of traditional methods, discovering potent and selective MMP-14 inhibitors like Fab R2C7.
Area of Science:
- Biotechnology
- Immunology
- Oncology
Background:
- Matrix metalloproteinase (MMP)-14 is crucial for tumor invasion and metastasis, making it a significant cancer treatment target.
- Broad-spectrum MMP inhibitors have failed clinically, highlighting the need for selective therapies.
- Monoclonal antibodies (mAbs) offer high specificity for targeting particular MMPs.
Purpose of the Study:
- To develop a more effective method for discovering MMP-14 inhibiting mAbs.
- To identify novel, highly specific antibody inhibitors of MMP-14 using next-generation sequencing (NGS).
Main Methods:
- Phage panning was employed to enrich for MMP-14 inhibiting antibody clones.
- Next-generation sequencing (NGS) was used to analyze antibody clone enrichment profiles over three panning rounds.
- Enzyme-linked immunosorbent assays (ELISA) were used for initial screening and validation.
Main Results:
- NGS identified 20 Fab inhibitors of MMP-14 with IC50 values ranging from 10-4,000 nM.
- Fifteen of these inhibitory Fabs were not detected by conventional monoclonal phage ELISA.
- Fab R2C7 demonstrated potent inhibition (100 nM IC50) and high selectivity for MMP-14 over MMP-9.
Conclusions:
- Deep sequencing is a powerful tool for systematic discovery of functional mAbs, particularly protease inhibitors.
- NGS significantly enhances the identification of inhibitory antibody clones compared to traditional ELISA methods.
- The identified selective MMP-14 inhibitors, such as R2C7, hold promise for targeted cancer therapy.
