Human bile duct cancer cells HuCC-T1 available from Zhejiang MeisenCTCC, human epidermal cancer cells A431 available from Procell Life Science&Technology Co.,Ltd. (Wuhan) and Chinese hamster ovary cells CHO from negative control ATCC were seeded into a 96-well plate at 2500 cells/well. It was then incubated in a 5% C02 incubator overnight at 37°C. Half of the medium was removed from the cell plate on the next day. A 2x hIgG (negative control antibody, provided by Biointron), Vatelizumab, FS002-Al-vc-MMAE, FS002-A15-vc-MMAE, FS002-A36-vc-MMAE, Vatelizumab-vc-MMAE, hIgG1-vcMMAE (negative control ADC, provided by ChemPartner) was prepared with a complete culture medium, and then diluted at a 3-fold gradient with 11 specific concentration points in total. Serial dilutions of the above molecules were added to the appropriate wells, and it was then incubated in a 5% C02 incubator for 5 days at 37°C. After the incubation, 100 uL of Cell-Titer-Glo 2.0 reagent was added to the 96-well plate. It was then well mixed on a plate shaker for 2 minutes, and finally equilibrated at room temperature for 10 minutes. Readings were taken using a microplate reader. Finally, IC50 was used to compare the biological activity of the ADC against each cell line.

Human bile duct cancer cells HuCC-T1 available from Zhejiang MeisenCTCC, human epidermal cancer cells A431 available from Procell Life Science&Technology Co.,Ltd. (Wuhan) and Chinese hamster ovary cells CHO from negative control ATCC were seeded into a 96-well plate at 2500 cells/well. It was then incubated in a 5% C02 incubator overnight at 37°C. Half of the medium was removed from the cell plate on the next day. A 2x hIgG (negative control antibody, provided by Biointron), Vatelizumab, FS002-Al-vc-MMAE, FS002-A15-vc-MMAE, FS002-A36-vc-MMAE, Vatelizumab-vc-MMAE, hIgG1-vcMMAE (negative control ADC, provided by ChemPartner) was prepared with a complete culture medium, and then diluted at a 3-fold gradient with 11 specific concentration points in total. Serial dilutions of the above molecules were added to the appropriate wells, and it was then incubated in a 5% C02 incubator for 5 days at 37°C. After the incubation, 100 uL of Cell-Titer-Glo 2.0 reagent was added to the 96-well plate. It was then well mixed on a plate shaker for 2 minutes, and finally equilibrated at room temperature for 10 minutes. Readings were taken using a microplate reader. Finally, IC50 was used to compare the biological activity of the ADC against each cell line.