MDA-MB-468 at 100uL/well (1000 to 10000 cells), 0.9mg/ml ADCs (10, 3.3, 1.1, 0.37, 0.12, 0.041, 0.014, 0.0046, 0.0015, and 0.00051 nM) were added and incubated for 72 to 120 h, then 100 L of CellTiter Glo reagent was added into each well, a shaker shook for 2 min, followed by standing in a dark place at rt for 10 min, and the relative light unit (RLU) was measured with a Cytation 3 microplate reader.

SK-BR-3 at 100uL/well (1000 to 10000 cells), 0.9mg/ml ADCs (10, 3.3, 1.1, 0.37, 0.12, 0.041, 0.014, 0.0046, 0.0015, and 0.00051 nM) were added and incubated for 72 to 120 h, then 100 L of CellTiter Glo reagent was added into each well, a shaker shook for 2 min, followed by standing in a dark place at rt for 10 min, and the relative light unit (RLU) was measured with a Cytation 3 microplate reader.

HCC1954 at 100uL/well (1000 to 10000 cells), 0.9mg/ml ADCs (10, 3.3, 1.1, 0.37, 0.12, 0.041, 0.014, 0.0046, 0.0015, and 0.00051 nM) were added and incubated for 72 to 120 h, then 100 L of CellTiter Glo reagent was added into each well, a shaker shook for 2 min, followed by standing in a dark place at rt for 10 min, and the relative light unit (RLU) was measured with a Cytation 3 microplate reader.

Ntera-2 cell at 625 cells/25 pL were seeded in a 384-well plat. ADCs were formulated at 2x starting concentration. Filter sterilized samples were serial diluted (1:3) under sterile conditions and added onto cells in triplicates. For cell viability measurement, 30 microliter of Cell Titer-Glo@ reagent (Promega Corp, Madison, WI) was added into each well, and plates processed as per product instructions.

MCF-7 cells at 625 cells/25 pL were seeded in a 384-well plat. ADCs were formulated at 2x starting concentration. Filter sterilized samples were serial diluted (1:3) under sterile conditions and added onto cells in triplicates. For cell viability measurement, 30 microliter of Cell Titer-Glo@ reagent (Promega Corp, Madison, WI) was added into each well, and plates processed as per product instructions.