Phototoxicity in near-infrared photoimmunotherapy is influenced by the subcellular localization of antibody-IR700

Background: Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer phototherapy that utilizes monoclonal antibody-IRDye700DX conjugates (mAb-IR700) and NIR light. We previously reported that mAb-IR700 aggregated on the plasma membrane and induced physical damage within the membrane, leading to necrotic/immunogenic cancer cell death. However, cytotoxic effects caused by internalized mAb-IR700, which is localized in lysosomes after endocytosis, remain unclear. Thus, in this study, we investigated how internalized mAb-IR700 influences phototoxicity. Methods: Cytotoxicity depending on the subcellular localization of mAb-IR700 was examined by varying the incubation time after washing. The influence of a singlet oxygen (O-1(2)) was examined by cell viability assay in the presence of O-1(2) quencher. The type of cell death was analyzed by flow cytometry with Annexin V/propidium iodide. Furthermore, IR700 fluorescence in cells was observed by fluorescence microscopy. Results: mAb-IR700 in lysosomes induced cytotoxicity, which was weaker than that induced by mAb-IR700 on plasma membranes. Cellular damage caused by mAb-IR700 in lysosomes was completely inhibited by an O-1(2) quencher. mAb-IR700 on plasma membranes and in lysosomes induced necrotic, but not apoptotic, cell death. IR700 was localized in lysosomes before light irradiation but then diffused into the cytosol immediately after irradiation. Conclusions: Although the main cytotoxic trigger in NIR-PIT is plasma membrane damage as previously reported, mAb-IR700 in lysosomes also induces necrotic cell death. The internalized mAb-IR700 caused O-1(2)-mediated damage, leading to the marked leakage of lysosomal contents into the cytosol. The mechanism of NIR-PIT depends on the subcellular localization of mAb-IR700.