Figure 5. The 4D12G1 mAb kills EOC cells by inducing apoptosis, CDC, and ADCP.

(A) AMHR2-OVCAR8 cells were treated with a green fluorescent dye and the 4D12G1 mAb or an isotype control mAb. Apoptosis was assessed by live imaging using the IncuCyte S3 analyzer. 4D12G1 mAb induced substantial apoptosis at 16 hours (right panel) compared to isotype control mAb (left panel). (B) AMHR2-OVCAR8 cells were treated with different concentrations of the 4D12G1 mAb for 24 hours and Western blots of the cell lysates showed detection of the intact 116 kDa PARP-1 and its 89 kDa cleaved variant, consistent with apoptosis. Immunostaining with a β-actin antibody was used to confirm normalized lysate loading. (C) AMHR2-OVCAR8 cells were incubated with the 4D12G1 mAb for different time periods at either 37° C (left column) or 4° C (right column). Clustered patterns of cytoplasmic antibody-receptor complexes became increasingly more prominent at 2 and 3 hours after treatment at 37° C, but not at 4° C, and no staining occurred in cells treated with secondary antibody alone (right column, bottom panel). (D) AMHR2-OVCAR8 cells were incubated in either 10% normal human serum or 10% heat-inactivated human serum and treated for 4 hours with varying doses of either 4D12G1 mAb or isotype control mAb. Cell lysis mediated by CDC was measured by release of LDH activity and occurred only in cells treated with the 4D12G1 mAb. (E) AMHR2-OVCAR8 target cells were labeled with a green fluorescent dye and incubated with two different concentrations of 4D12G1 mAb or isotype control mAb. The cells were mixed with effector macrophages from C57BL/6 mouse bone marrow at an effector to target cell ratio of 10:1. Live target cells were analyzed by flow cytometry 3 days later for demonstrating ADCP. All error bars indicate ±SD. All experiments are representative of three experiments yielding similar data.