Antibody-dependent cellular cytotoxicity (ADCC) A431 cells were plated on a microtiter plate The following day, the cells were incubated with a ten-point 1:3 dilution titration of anti-TF antibodies or the ADCs starting at 50 nM An ADCC effector-to-target cell ratio of 8:1 was added to each well and incubated for 6 h at 37°C Luciferase Assay Reagent was added to each well to measure luminescence on an Envision plate reader Antibody-dependent cellular cytotoxicity (ADCC) reporter luminescence was evaluated after a 6-hour incubation of the reporter Jurkat cell line with TF-positive A431 cells and a titration of anti-TF antibody or ADC The ADCC reporter luminescence EC50 values for each anti-TF antibody or ADC are listed.

To evaluate ADC cytotoxicity, cells were plated in 384-well plates Anti-TF antibodies conjugated to MC-vc-PAB-MMAE were serially diluted as shown Plates were incubated for 3 days, followed by lysis in CTG assay reagent For each ADC, the IC50 and its associated 95% confidence interval (95% CI) were calculated Titrations of the TF-specific ADCs were added to A431 cells, with a 72-hour incubationThis treatment resulted in efficacious cell killing.

To evaluate ADC cytotoxicity, cells were plated in 384-well plates Anti-TF antibodies conjugated to MC-vc-PAB-MMAE were serially diluted as shown Plates were incubated for 3 days, followed by lysis in CTG assay reagent For each ADC, the IC50 and its associated 95% confidence interval (95% CI) were calculated Titrations of the TF-specific ADCs were added to A431 cells, with a 4-hour incubation followed by removal of excess ADC and culture for another 68 hours This treatment resulted in efficacious cell killing.