Strategies for enriching and characterizing proteins with inhibitory properties on the yeast surface.

Display technologies are powerful tools for discovering antibodies and other binding proteins against a broad range of biological targets. However, it remains challenging to adapt display technologies for the discovery of proteins that inhibit the enzymatic activities of targets because the phenotypic readout during display screens is binding. The goal of this work is to investigate approaches for discovering inhibitory antibodies in yeast display format using a well-defined series of constructs and the target matrix metalloproteinase-9 (MMP-9). Three previously reported antibodies (DX-2802, M0076 and FAPB2.3.6) were used to create model libraries that are representative of protein libraries consisting of inhibitory binders, non-inhibitory binders, and non-binding constructs. Conditions that preferentially enrich for inhibitory clones were identified for both magnetic bead-based enrichments and fluorescence-activated cell sorting (FACS). Finally, we used direct titration of yeast to estimate inhibitor IC50 values with yeast-displayed and soluble constructs and found that the IC50 obtained for DX-2802 in yeast display format (20.01 ± 9.01 nM) falls within the confidence interval of the IC50 value of a soluble scFv-Fc form of DX-2802 (17.56 ± 6.16 nM). Thus, it is possible to determine IC50 values on the yeast surface, which greatly streamlines initial characterizations of the inhibitory properties of candidate clones. Overall, these well-defined constructs enabled identification of strategies for the discovery and characterization of inhibitory clones directly in yeast display format.