Internalization assay

ADC internalization was detected using two methods: flow cytometry and confocal microscopy. For flow cytometry, five groups (Ctrl, BsAb, PD-L1 ADC, B7-H3 ADC, and BsADC) were prepared, and each group was further divided into 0, 1, 2, 4, and 6 hours time points. The cells were seeded in a 12-well plate at a density of 2×10⁵ cells/well. The next day, the cells were treated with 100 nM at 4°C for 1 hour, washed with PBS, and then incubated at 37°C for 0, 1, 2, 4, or 6 hours, separately. Subsequently, cells were washed, acquired, and fixed, followed by incubation with secondary antibodies (ab6854; Abcam) at 4°C for 40 min.

For confocal microscopy, cells were seeded and grown in a 24-well confocal plate at a density of 5×10⁴ cells per well. The next day, cells were treated with RPMI 1640 or BsADC (100 nM) at 4°C for 1 hour to facilitate drug-cell binding. Subsequently, cells were transferred to a 37°C incubator for 1 or 4 hours, washed with PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.5% Triton X-100 in PBS. The cells were then stained with goat anti-human IgG (Abcam, ab6854) and the cell nuclei were stained with DAPI (Solarbio, Cat# C0060). Images were captured using a confocal microscope (Olympus IXplore Spin SR).