HeLa cells are cultured in DMEM complete medium (Corning, 10-013-CVR) containing 10% FBS (Gibco, 10099-141) and 100 U/mL penicillin-streptomycin mixture (Thermofisher, 15140122). When the cell coverage in the culture container reaches 80-90%, TrypLE (Thermofisher, 12604-013) is used to digest the HeLa cells from the culture container, and they are seeded in a 96-well cell culture plate (Greiner, 655098) at a density of 3.3X10³ cells/mL, with 500 cells per well (145uL complete medium),and the 96-well plate is incubated at 37°C overnight. The next day, (ADC) is diluted in complete medium in a 4-fold serial dilution, with 9 concentration gradient points. Then, 5uL of dilution of the ADC molecule is added to each well in the 96-well cell culture plate, gently mixed, and the 96-well plate is incubated at 37°C for 6 days. Finally, 75 uL of CTG is added to each well, gently mixed, and the 96-well plate is incubated at room temperature for 10 minutes. Then the luminescence signal is detected using a multimode microplate reader (PerkinElmer Envision 2105). A dose-inhibition curve is generated, and the half-maximal inhibitory concentration (IC50) is calculated using non-linear regression with GraphPad software.

HeLa L1CAM-KO cells are cultured in DMEM complete medium (Corning, 10-013-CVR) containing 10% FBS (Gibco, 10099-141) and 100 U/mL penicillin-streptomycin mixture (Thermofisher, 15140122). When the cell coverage in the culture container reaches 80-90%, TrypLE (Thermofisher, 12604-013) is used to digest the HeLa L1CAM-KO cells from the culture container, and they are seeded in a 96-well cell culture plate (Greiner, 655098) at a density of 3.3X10³ cells/mL, with 500 cells per well (145uL complete medium),and the 96-well plate is incubated at 37°C overnight. The next day, (ADC) is diluted in complete medium in a 4-fold serial dilution, with 9 concentration gradient points. Then, 5uL of dilution of the ADC molecule is added to each well in the 96-well cell culture plate, gently mixed, and the 96-well plate is incubated at 37°C for 6 days. Finally, 75 uL of CTG is added to each well, gently mixed, and the 96-well plate is incubated at room temperature for 10 minutes. Then the luminescence signal is detected using a multimode microplate reader (PerkinElmer Envision 2105). A dose-inhibition curve is generated, and the half-maximal inhibitory concentration (IC50) is calculated using non-linear regression with GraphPad software.