Abstract 4243: ChAcNLS-A14-B105, a novel fluorescence imaging agent for enhanced detection of muscle invasive bladder cancer

Abstract

Abstract Fluorescence imaging (FI) provides increased sensitivity for the detection of bladder tumors compared to white light cystoscopy. However, most advanced dyes used in FI suffer from nonspecific cellular accumulation resulting in increased false-positives. In addition, these dyes have not made significant progress in detecting the most aggressive bladder tumors such as muscle invasive bladder cancer (MIBC). Our objective is to develop an antibody-conjugate (AC) PDI agent against interleukin-5 receptor α-subunit (IL-5Rα)-positive MIBC cells. IL-5Rα is overexpressed on MIBC cells relative to superficial bladder cancer or healthy bladder cells. We selected a class of fluorescent dyes termed BODIPY for its availability, facile coupling to proteins, and easy chemical modifications to accommodate required absorption and fluorescence properties. BODIPY was attached to an AC composed of the antibody A14 functionalized with a moiety termed ChAcNLS, which we have shown enables it to escape endosome entrapment and redirect its trafficking to the nucleus. This novel approach results in increased intracellular accumulation of attached molecular payloads in MIBC cells. We aim to determine if ChAcNLS-A14 will increase intracellular accumulation of BODIPY to enhance fluorescence detection of MIBC by probe-based confocal laser endomicroscopy (pCLE). In addition, we aim to develop in an improved orthotopic rat bladder tumor model of human MIBC for testing. Methods We have prepared a carboxyl BODIPY derivative (B105) that allows for facile coupling to A14-ChAcNLS using routine procedures and reagents. The number of B105 and ChAcNLS per A14 was determined by fluorescence spectroscopy and SDS-PAGE, respectively. Intracellular accumulation of B105 was determined by treating HT1376 MIBC cells with A14-B105 and ChAcNLS-A14-B105. Cells were then imaged by fluorescence confocal microscopy. For the in vivo model of MIBC, RNU rats were anesthetized and their bladder washed in mild acid to disrupt the endothelium. HT1376 cells marked with the fluorescent marker DiL and instilled in the rat bladder. Intravesical pCLE was performed 2 weeks later. Results ChAcNLS-A14-B105 conjugate contained 7 molecules of B105 and 3 ChAcNLS peptides per A14. Confocal microscopy revealed the level of B105 intracellular accumulation in IL-5Rα-positive MIBC cells was increase ≥10-fold with ChAcNLS-A14-B105 relative to A14-B105. pCLE revealed HT1376 cells survived and colonized the rat bladder urothelium. Conclusion ChAcNLS-A14-B105 was successfully constructed and shown to significantly enhance B105 intracellular accumulation in target IL-5Rα-positive MIBC cells. A novel orthotopic rat bladder tumor model of human MIBC was also generated. Future testing of ChAcNLS-A14-B105 administered intravesically in RNU rat bladders bearing orthotopic tumors of human MIBC will be performed to determine the potential clinical translation of this new FI agent. Citation Format: Laurent Fafard-Couture, Simon Beaudoin, Samira Osati, Véronique Dumoulon-Perreault, Réjean Lebel, Brigitte Guérin, Johan E. van Lier, Jeffrey Leyton. ChAcNLS-A14-B105, a novel fluorescence imaging agent for enhanced detection of muscle invasive bladder cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4243.