Achieving protease substrate-specific inhibition by mAb dual functional selections

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Proteases are enzymes with broad substrate activity, making selective inhibition challenging.
• Developing safe and effective protease inhibitors requires distinguishing between pathogenic and physiological proteolysis.

Purpose of the Study:

• To establish a dual functional selection system for discovering protease inhibitory monoclonal antibodies (mAbs) with substrate specificity.
• To isolate and characterize anti-matrix metalloproteinase (MMP)-14 inhibitory mAbs that exhibit substrate-specific inhibition.

Main Methods:

• Engineered bacterial periplasmic expression systems using beta-lactamase and levansucrase as selection markers.
• Developed positive and negative selection strategies for identifying protease inhibitory mAbs.
• Isolated and characterized anti-MMP-14 mAbs for binding affinity, inhibitory potency, selectivity, and substrate specificity.

Main Results:

• Successfully established a dual functional selection system for protease inhibitor discovery.
• Isolated anti-MMP-14 inhibitory mAbs with nanomolar affinities and high stability.
• Demonstrated substrate-specific (SS) inhibition, blocking syndecan-1 cleavage while allowing macrophage chemoattractant protein 3 cleavage.
• Mechanistic studies revealed active-site competitive inhibition and substrate specificity based on protease subsites.

Conclusions:

• The developed dual functional selection is effective for discovering substrate-specific protease inhibitors.
• The isolated anti-MMP-14 mAbs represent promising therapeutic candidates due to their SS inhibition.
• This selection strategy has broad applicability for targeting other proteases with pharmaceutical significance.