Fig. 2

Target-specific phototoxicity of AvIR-mediated PIT. a TAA-specific binding of AvIR was assessed by flow cytometry. CHO cells were first incubated with unlabeled mAb or BioAb and then stained with AvIR. b CHO-CEA and CHO-EpCAM cells were incubated with the indicated BioAb (5 µg/ml) or DPBS for 30 min and further incubated with AvIR (5 µg/ml) for 30 min. Subsequently, the cells were irradiated with NIR light (3 J/cm²). After NIR light exposure, the cells were stained using LIVE/DEAD cell imaging kit, and the images were acquired using fluorescence microscope to determine whether they were alive (green) or dead (red). c LIVE/DEAD cell images of AvIR-PIT-treated co-culture of CHO-CEA and CHO-EpCAM cells. On the day before PIT treatment, the CHO-CEA cells were pre-stained with CellTracker Blue and then co-cultured with unstained CHO-EpCAM cells. The cells were targeted by incubation with the indicated BioAb(s), and AvIR-PIT was performed. The magenta cells in the overlaid images indicate dead CHO-CEA cells. Note that the CHO-EpCAM cells, even in contact with CHO-CEA cells (arrowheads in the bright-field image of top row) were alive. Data in the rightmost panels show the percentages of live cells that survived PIT-treatment measured by counting the green fluorescent cells on five randomly selected fields under the microscope. One hundred percent represents the total CHO-CEA or CHO-EpCAM cells per field. The data are the means ± SEM (n = 3)