(7e) Directed Evolution of Metalloproteinase Inhibitors Targeting ADAM-17

The metalloproteinase (MP) family of proteases, including matrix metalloproteinases (MMPs) and a disintegrin and metalloproteases (ADAMs), plays a crucial role in the extracellular matrix (ECM) remodeling and degradation activities. ADAMs, in particular ADAM-17, play an integral role in cell regulation, through the activation or inhibition of cytokines, growth factors, and their receptors, as well as adhesion molecules via cleavage of ectodomains. ADAM-17’s integral role in cell regulation also makes it a top contender for various human diseases, such as heart disease, cancer, Alzheimer’s disease, and diabetes. The use of small molecule therapeutics targeting ADAM-17 have failed in clinical trials due to non-specific targeting of MPs, which led to severe side effects in patients. Protein-based therapeutics such as antibodies or tissue inhibitors of metalloproteinases offer higher binding selectivity.

We used directed evolution and yeast surface display to screen a synthetic single chain antibody (anti-fluorescein scFv) library, previously engineered to reduce non-specific binding¹, for improved binding to the ADAM-17 catalytic domain using fluorescent-activated cell sorting (FACS). DNA sequence analysis from each round of FACS, conducted via DNA sequence alignment bioinformatic tools, indicated that frequent antibody mutations within non-complementarity-determining regions (CDRs), light chain CDRs, and heavy chain CDR-2 are responsible for improving ADAM-17 binding. The individual scFv clones with improved binding affinity for ADAM-17 were further tested for MP binding affinities. This study set the stage for further engineering and design of protein scaffolds targeting MPs with high affinity and selectivity using directed evolution and rational design.

1. Kelly, R. L., Le, D., Zhao, J., & Wittrup, K. D. (2018). Reduction of nonspecificity motifs in synthetic antibody libraries. Journal of Molecular Biology, 430(1), 119-130. doi:10.1016/j.jmb.2017.11.008