In vitro cytotoxicity assay

Cytotoxicity assays shown in Fig. 5A-C were carried out as previously described.⁴³ For screening of ADCs against Antigen C, human cancer cells expressing Antigen C were cultured in EMEM medium (ATCC, Manassas, VA) supplemented with 10% fetal bovine serum, 10 µg/mL insulin (Sigma, St. Louis, MO), penicillin and streptomycin (Mediatech, Herndon, VA). Cells were plated on 384-well plates (Grenier Bio-One #781091, Monroe, NC), at a dilution of 400 cells in 22.5 µL growth medium. The cells were incubated for 16-24 h at 37°C, 5% CO₂. ADCs were diluted 1:100 in growth medium, then diluted serially 1:4 and 2.5 µL added to cells in triplicate with a Beckman FX automated liquid handler. Plates were sealed with gas permeable plate seals (Thermo Fisher Scientific #241205, Waltham, MA) and cultured for 5 d at 37°C, 5% CO₂. Cell viability was measured by addition of 12.5 µL CellTiter-Glo reagent (Promega #7573, Madison, WI) followed by shaking in the dark for 30 min. The luminescence intensity of the plates read on an Enspire 2300 multimode plate reader (Perkin Elmer, Shelton CT).