Studying the Dynamics of Fc-Engineered Antibodies Using Advanced Imaging Methods
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.
Subject
Engineered antibodies, Multifocal plane microscopy, Phagocytosis, FcRn, Fluorescence microscopyCitation
Ramakrishnan, Sreevidhya (2021). Studying the Dynamics of Fc-Engineered Antibodies Using Advanced Imaging Methods. Doctoral dissertation, Texas A&M University. Available electronically from https : / /hdl .handle .net /1969 .1 /195216.