Molecular Targeted Photoimmunotherapy As A Treatment For Bladder Cancer

Introduction: Bladder cancer (BCa) is a common cancer and is the most expensive malignancy to treat from diagnosis to death. Although no new therapies have been introduced in the past two decades, data from ‘The Cancer Genome Atlas (TCGA)’ confirms that Epidermal Growth Factor Receptor (EGFR) family and its downstream signaling molecules are altered in almost 72% of muscle invasive bladder cancer (MIBC) cases. However, anti-EGFRs to date have failed to demonstrate any benefit over standard of care chemotherapy. We have applied a technique of employing monoclonal antibodies (mAbs) conjugated with photo-activatable compounds (IRDye 700Dx, a phthalocyanine dye) that are activated by near-infrared light (NIR). When incubated with the conjugate, exposure to NIR destroys only targeted cells. We explored photoimmunotherapy (PIT) in a panel of BCa cell lines. The purpose of this study was to study the efficacy and mechanism of action of PIT using the anti-EGFR panitumumab (Pan)-IR700 immunoconjugate as a selective therapeutic strategy for bladder cancer. Methods: Using flow cytometry, the surface expression of EGFR was profiled in several BCa cell lines. The cytotoxicity of Pan-IR700 was analyzed using LIVE/DEAD and IC50 was measured using the MTS assay. The type of cell death was examined by fluorometric caspase assay, Annexin V-PI staining, and TEM. The mechanism of cell death was elucidated using fluorometric measurement of reactive and singlet oxygen species. Results: Using flow cytometry, the surface expression of EGFR was profiled in several BCa cell lines and cell lines with increasing amounts of surface EGFR (RT4, TCCSUP, 5637 and UMUC-5) were selected for further studies. Pan-IR700 rapidly killed UMUC-5 cells (high EGFR expressions) with an IC50 of 4 nM at 4 J/cm2 NIR. In TCCSUP cells (low surface EGFR), the same IC50 of Pan-IR700 could be achieved at 64 J/cm2 of NIR. No significant cytotoxicity was observed in the presence of IR700 or NIR alone, or in cell lines without any EGFR expression. Absence of any caspases and presence of most cells in the late apoptosis/necrosis quadrant of Annexin V-PI staining suggested that PIT kills cells by necrosis. In TEM, these cells showed classic features of necrotic cell death such as swollen organelles, disintegrated plasma membrane and depleted cytoplasm and nucleoplasm. Cells undergoing PIT showed production of ROS and singlet oxygen species in large amounts immediately after exposure to NIR. However, only the singlet oxygen quencher (NaN3) and not the ROS quencher (Trolox) was able to protect the cells from PIT induced cell death indicating partial involvement of singlet oxygen in PIT-induced necrosis of cells. Conclusions: PIT is a new targeted treatment for bladder cancer. Our data demonstrate that Pan-IR700-induced PIT selectively and efficiently kills EGFR-expressing bladder cancer cells in vitro and therefore warrants further preclinical therapeutic studies in in vivo bladder cancer models. Citation Format: Reema Railkar, Quentin Li, Srinivas Vourganti, Sam J. Brancato, Peter L. Choyke, Hisataka Kobayashi, Piyush K. Agarwal. Molecular targeted photoimmunotherapy as a treatment for bladder cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3311. doi:10.1158/1538-7445.AM2015-3311