| dc.description.abstract | The past decade has seen an enormous increase in FcRn-targeted engineering strategies to generate antibodies with therapeutic and diagnostic implications. The neonatal Fc receptor, FcRn, is a multifunctional receptor expressed abundantly throughout the body. The trafficking of engineered antibodies in the body is a multiscale process, taking place at subcellular, intracellular, and organ/tissue levels. In this study, various advanced imaging modalities are employed to capitulate the dynamics of Fc-engineered antibodies at different scales. The study investigates the fate of antibody-opsonized tumor cells after phagocytosis in macrophages, an intracellular/micrometer-scale process, using microscopy techniques. Results show a vacuole-like structure associated with the phagosomes exhibiting distinct characteristics. They are lysosomal in nature and impermeable to certain solutes, as seen using fluorescence microscopy analyses. The identification of this vacuole-like compartment has implications for understanding the subsequent processes involved in the degradation of antibody-opsonized tumor cells.
The use of advanced imaging approaches to study subcellular dynamics provides mechanistic insight with excellent spatiotemporal resolution. We imaged the 3D dynamics of two engineered FcRn-inhibitors at the subcellular/nanometer level, using an advanced imaging platform- rMUM. rMUM is capable of imaging the dynamics of the single molecule and the cellular organelles they interact with, in 3D. This enables compensation of the motion of a single molecule with the organelle's movement, thus giving a real snapshot of the dynamics. To that end, we utilized this multi-dimensional, nanometer resolution microscopy technique to image two engineered FcRn-inhibitors in endosomes in cells.
At the macro level, we investigate the effective clearance of antigens by an engineered Fc-antigen fusion protein (HER2-Seldeg), consequently improving contrast during whole-body imaging in mice. HER2-Seldeg is designed to selectively capture anti HER2 antibodies, bind to FcRn with enhanced affinity, and direct them to degradative lysosomes in FcRn-expressing cells. Positron emission tomography (PET) imaging of HER2-Seldeg revealed rapid clearance of radiolabeled antibodies from the systemic circulation following tumor localization and consequently improved contrast. | en |
