Abstract 1876: ROCKETS Science: A novel processing toolbox for light sheet microscopy reveals unknown binding sites for EpCAM-targeted antibodies

Abstract

Abstract Summary: We present a novel light sheet fluorescence microscopy (LSFM)-based imaging platform and corresponding set of procedures for greatly improved preclinical investigation of the biodistribution of novel drug candidates by the example of an EpCAM-targeting antibody. Methods: An anti-EpCAM antibody (G.8.8R) was applied intravenously to mice. Whole mice or individual organs were optically cleared following novel protocols, which we termed Rapid Optical Clearing Kit for Enhanced Tissue Scanning (ROCKETS). The procedures enabled processing of mouse organs and entire bodies for LSFM. For the GI tract, we developed 3D-Swiss rolls, a method that enables fixation, clearing and imaging of intestines in a compact form as a whole. The new procedures allowed us to investigate the biodistribution of G.8.8R in the entire mouse body and highly detailed in individual organs. Results: The ROCKETS concept achieved full transparency of any mouse organ, the entire GI tract and whole mouse bodies. All positive organs were easily identified in scans of whole mice, thereby providing excellent guidance for scanning of positive individual organs at higher magnifications. We detected G8.8R bound to all normal cuboidal and columnar epithelia, as well as in lymphoid organs and were able to reproducibly score binding levels. All reported EpCAM+ tissues in mice were accessed by G8.8R after intravenous application and binding was restricted to basolateral membranes of epithelia, as expected from published expression analyses. Moreover, we detected G8.8R in tissues that were reported EpCAM- or were not investigated, e.g. gustatory papillae of the tongue, choroid plexi in the brain or lingual mucous salivary glands. 3D-Swiss rolls of the GI tract revealed a highly heterogeneous binding pattern in the stomach, while the pattern along the small intestine was overall homogeneous. All binding was restricted to basolateral membranes of epithelial cells. We detected a general gradient of signals, decreasing from the bottom of the crypts to differentiated cells in all tissues, corresponding to described EpCAM downregulation with progressing differentiation. Furthermore, we first describe significantly increased signals at the common bile duct, major and minor duodenal papillae, as well as the mucosa in proximity of Peyer's patches (PP). Conclusions: The developed ROCKETS toolbox allowed for simple preparation of mouse organs and bodies for high-quality LSFM imaging. Our investigations of the highly heterogeneous biodistribution of G8.8R revealed previously unknown EpCAM binding locations, which may have far-reaching implications for EpCAM-targeting therapeutics in general, many of which failed in clinical studies due to dose limiting toxicities. In the future, the developed LSFM-imaging platform may contribute valuable data to preclinical drug development studies of any targeted therapeutic. Citation Format: Joerg P. Mueller, Nils O'Brien, Franz Osl, Frank Herting, Christian Klein, Pablo Umana, Sara Colombetti, Thomas Poeschinger, Andreas Beilhack. ROCKETS Science: A novel processing toolbox for light sheet microscopy reveals unknown binding sites for EpCAM-targeted antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1876.