Novel fluorogenic indicators of antibody and biologic trafficking and lysosomal catabolism 2670

Abstract Description The fundamental cellular mechanisms of internalization and trafficking are crucial to many areas of cell biology, and especially to the proper function of therapeutic antibodies. Antibodies intended for use as antibody-drug conjugates (ADCs) or targeted protein degraders should specifically bind to target cells and rapidly internalize via endocytosis. Those biologics designed for checkpoint blockade, immune cell recruitment, or direct cell death should remain bound to their target antigen except when undergoing FcRn-mediated transcytosis. However, the ability to study these processes has historically been limited by the lack of tools that directly monitor the internalization and subsequent catabolism of extracellular material. Here we demonstrate sensitive, rapid, high-throughput, and no-wash methods to screen for antibody internalization and lysosomal degradation. These fluorescence-based reagents can be conjugated to antibodies or proteins using lysine, cysteine, or site-specific Fc conjugation. Additionally, we present techniques to directly visualize the internalization of therapeutic antibodies and ADCs in live cells for structure-activity relationship studies. We also demonstrate how the insights provided by these approaches have the potential to accelerate biotherapeutic lead generation and development. Topic Categories Technological Innovations in Immunology (TECH)