ADC cytotoxicity assay

Anti-human secondary antibody–drug conjugated with the cytotoxic drugs MMAE (anti-HuFc-MMAE, cat. #AH-102AE), PNU-159682 (anti-HuFc-PNU-159682, cat. #AH-102PN), pyrrolobenzodiazepine (anti-HuFC-PBD, cat. #AH-106PB) and AAMT (Fab-anti-HuFC-AAMT, cat. #AH-205AM) were purchased from Moradec. P1C1TM was conjugated to PNU-159682 via a vc-PAB linker, to a DAR of about 4.1 (Levena Biopharma). For indirect killing assays, target cells (20,000 per well) were first incubated with P1C1TM at various concentrations for 30 min before the addition of the secondary antibodies at equimolar concentrations. For direct killing assays, target cells (20,000 per well) were incubated with P1C1TM-PNU at various concentrations. Assays were incubated for 3 days at 37 °C and viable cells were quantified with the either the CellTiter 96^© AQueous One Solution Cell Proliferation Kit (Promega) or the alamarBlue Cell Viability assay (Thermo Fisher Scientific) according to the manufacturer’s instructions. Abs was measured at 490 nM using a microplate reader (EnSpire™, Perkin Elmer). Fluorescence was read at an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Viability was calculated as Abs_treated/Abs_untreated × 100% or Fl_treated/Fl_untreated × 100%. Cytotoxicity was calculated as % cytotoxicity = (100% − %viability).