Abstract 3196: Investigation of mechanism on photoimmunotherapy focused on cell membrane damage
Authors: Ando Kanta , Nakajima Kohei , Takakura Hideo , Ogawa Mikako 
Published: August 2018

Objectives: Photoimmunotherapy (PIT) is a new type of cancer treatment using IR700, a hydrophilic near-infrared (NIR) photosensitizer, conjugated to monoclonal antibodies. However, the mechanism of the phototoxicity has not been clarified well. Previous study showed that the major cytotoxic mechanism of PIT is different from conventional photo-therapies which require singlet oxygen, and the cells were swelled in the process of cell death induced by PIT. We hypothesized that cell plasma membrane damage should be an important trigger to cause cell death in PIT. In order to verify the hypothesis, we investigated the plasma membrane permeability during PIT and photoresponse property of PIT agent.

Methods: To evaluate the permeability of the plasma membrane during PIT, inflow or outflow of various sized compounds, such as Na+ (diameter 0.1 nm), 111In3+ (0.09 nm), 111In-DTPA (1.0 nm), calcein (1.4 nm), or EthD-1 (2.6 nm) was investigated after NIR light irradiation to NIH3T3/HER2 cells treated with IR700 conjugated to trastuzumab, an anti-HER2 antibody. We also investigated the permeability under high osmotic pressure conditions by small or large molecules such as NaCl or dextran (3.7 nm), respectively. To evaluate the photoresponse property of PIT agent under various conditions, IR700 was irradiated by the NIR light in PBS in the presence of electron donors or accepters with or without oxygen. The irradiated solution was analyzed by HPLC and the photolytes were characterized.

Results: Na+ flowed into cells immediately after NIR light irradiation although intracellular calcein did not flow out from the cells. Calcein was flowed out within 3 min after the irradiation and EthD-1 gradually flowed into cells at 30 min. The uptake of 111In3+ was almost the same at 3 min and 60 min after the irradiation although that of 111In-DTPA increased at 60 min compared to 3 min. The uptakes of these compounds were not observed under the high osmotic pressure condition by dextran. HPLC analyses of the irradiated solution with electron donors showed that the photolytes were lipophilic. In high concentration of IR700, hydrophobic precipitations were observed after NIR light irradiation. IR700 was photodegradable in the presence of electron donors, and the degradation was promoted under oxygen depletion condition.

Discussion: We found that the size of initial damage on the cell membrane immediately after NIR irradiation was tiny that only small ions flowed into cells. Then, the size gradually increased with time and finally was larger than macromolecules, indicating that the tiny cell membrane damage should be the initial step of cell death induced by PIT. We also found that the hydrophilicity/hydrophobicity of IR700 was drastically changed. We considered the change of physiochemical property must be related to cell membrane damage.

Citation Format: Kanta Ando, Kohei Nakajima, Hideo Takakura, Mikako Ogawa. Investigation of mechanism on photoimmunotherapy focused on cell membrane damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3196.