A general approach to discover conformational epitope-directed binders for selective inhibition of protein proteolytic activation

Abstract Directing antibodies to a particular epitope among many possible on a target protein is a significant challenge. Here we present a simple and general method for epitope-directed selection (EDS) using a differential phage selection strategy. This involves engineering the protein of interest (POI) with the epitope of interest (EOI) mutated using a systematic bioinformatics algorithm to guide the local design of an EOI decoy variant. Using several alternating rounds of negative selection with the EOI decoy variant followed by positive selection on the wild-type (WT) POI, we were able to identify highly specific and potent antibodies to five different EOI antigens that bind and functionally block known sites of proteolysis. Among these we developed highly specific antibodies that target the proteolytic site on the CUB domain containing protein 1 (CDCP1) to prevent its proteolysis allowing us to study the cellular maturation of this event that triggers malignancy. We generated antibodies that recognize the junction between the pro and catalytic domains for four different matrix metalloproteases (MMPs), such as MMP1, MMP3, and MMP9, that selectively block activation of each of these enzymes and impairs cell migration. We targeted a proteolytic epitope on the cell surface receptor, EPH Receptor A2, that is known to transform it from a tumor suppressor to an oncoprotein. We believe the EDS method greatly facilitates the generation antibodies to specific EOIs on a wide range of proteins and enzymes for broad therapeutic and diagnostic applications. Significance We have developed a highly efficient platform to facilitate the directed selection in vitro of antibodies to a wide range of functional epitopes on proteins. This method uses a bioinformatic program to guide mutations in the local site of interest to create a decoy antigen that can effectively remove antibodies not binding the site of interest by negative selection, followed by positive selection with the WT antigen to identify antibodies to the epitope of interest. We demonstrate the generality and versatility of this method by successfully producing functional antibodies to block specific proteolytically sensitive epitopes on five different proteins including enzymes important in cancer. The epitope-directed selection (EDS) approach greatly facilitates the identification of binders to specific sites of interest on proteins to probe function and as potential immunotherapeutics.