Simple SummaryNear-infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that uses photoreactive agents and light irradiation, while photodynamic therapy (PDT) is an existing phototherapy using photosensitizers and light irradiation as well as NIR-PIT. The current study investigated the properties of these therapies and their differences with a focus on their cellular binding/uptake specificity and cytotoxicity in vitro. NIR-PIT showed molecule-selective responses and cytotoxicity, whereas PDT showed non-selective cell type responses and cytotoxicity. Additionally, NIR-PIT and PDT induced damage-associated molecular patterns (DAMPs), a surrogate marker of immunogenic cell death, although PDT had different sensitivity between cell lines. Therefore, molecule-specific NIR-PIT may have advantages compared with PDT in terms of efficiency in tumor visualization and induction of DAMPs.AbstractNear-infrared photoimmunotherapy (NIR-PIT) is a new phototherapy that utilizes a monoclonal antibody (mAb) against cancer antigens and a phthalocyanine dye, IRDye700DX (IR700) conjugate (mAb-IR700). Photodynamic therapy (PDT) is a combination therapy that utilizes photoreactive agents and light irradiation as well as NIR-PIT. In the present study, we compared these therapies in vitro. The characterization of cellular binding/uptake specificity and cytotoxicity were examined using two mAb-IR700 forms and a conventional PDT agent, talaporfin sodium, in three cell lines. As designed, mAb-IR700 had high molecular selectivity and visualized target molecule-positive cells at the lowest concentration examined. NIR-PIT induced necrosis and damage-associated molecular patterns (DAMPs), a surrogate maker of immunogenic cell death. In contrast, talaporfin sodium was taken up by cells regardless of cell type, and its uptake was enhanced in a concentration-dependent manner. PDT induced cell death, with the pattern of cell death shifting from apoptosis to necrosis depending on the concentration of the photosensitizer. Induction of DAMPs was observed at the highest concentration, but their sensitivity differed among cell lines. Overall, our data suggest that molecule-specific NIR-PIT may have potential advantages compared with PDT in terms of the efficiency of tumor visualization and induction of DAMPs.