UC Riverside

Mounting evidence suggests that MMP-14 (Matrix Metalloproteinase-14) plays an important role in cancer proliferation, invasion and migration to other healthy tissues through its extracellular matrix degradation activity. Toward the discovery of MMP-14 inhibitors, antibodies are emerging as a very attractive approach, due to their high specificity, low side effects and excellent in vivo stability. However, most current antibody selection methods are essentially binding assays with little control on inhibition function. Here, we report the development of a functional high-throughput screening method, which is able to identify inhibitory clones from non-specific or non-inhibitory clones. This novel method is based on antibody yeast display and dual color fluorescence-activated cell sorting (FACS). More specifically, the catalytic domain of MMP-14 and its native inhibitor N-TIMP-2 (N-terminal-domain of Tissue inhibitor of metalloproteinases-2) were successfully cloned, expressed, purified and refolded to their active formats, then conjugated with fluorophores Alexa488 and Alexa647 respectively. Yeast cells displaying various antibody clones were subjected to incubate with Alexa488-MMP-14 and Alexa647-N-TIMP-2. Dual Color FACS scanning results clearly demonstrated that the inhibitory antibody cells (DX-2400, 1F8) exhibited high Alexa488 fluorescence signals and low Alexa647 fluorescence signals, while the specific but non-inhibitory antibody cells (3B1, 2A10) exhibited high signals on both Alexa488 and Alexa647, and non-specific antibody cells (1H11, 2D9, 2F9, 1B11, b12, M18) exhibited low signals on both fluorophores. This proof-of-concept study paves the way for utilizing this functional screening method to isolate inhibitory antibodies from affinity maturation and synthetic antibody libraries in future researches.