Proteases play critical roles in numerous physiological processes thus represent one of the largest families of potential pharmaceutical targets. Previous failure of broad-spectrum small molecule inhibitors targeting tumorigenic metalloproteinases in clinical trials emphasizes that selectivity is the key for a successful protease-inhibition therapy. With exquisite specificity, antibody-based inhibitors are emerging as very promising therapeutics. However, current selection technologies relying on affinity have little controls on epitope or inhibition. Here, we report the development of function-based ultra-sensitive FRET inhibition assays able to directly identify inhibitory mAbs present in periplasmic fraction preparation without antibody purification. Affinity clones with or without inhibitory function can be easily discriminated with a satisfactory Z factor suitable for high-throughput applications. Combined with ELISA, this method allows a fast and semi-quantitative estimation of IC50 values. Design of antibody libraries carrying unique inhibition-prone paratopes together with this selection method greatly facilitated the generation of inhibitory antibodies for cancer therapy.

Function based periplasmic FRET assays for metalloproteinases inhibitory antibody selection

245th National Meeting (2013)

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