Example 15
To further investigate the mechanism of PSMAb ADC in PSMA specific cell killing, PSMAb ADC induced apoptosis was studied in PC-3 and C4-2 cells. Briefly, PC-3 and C4-2 cells were seeded in 6 well plates at the density of 2×105/2 ml medium/well and cultured overnight. The next day, medium was changed and cells were incubated with PSMAb-ADC at the concentrations of 50 μg/ml, 20 μg/ml, 10 μg/ml, 5 μg/ml, 1 μg/ml, 0.5 μg/ml, 0.01 μg/ml, and 0.001 μg/ml in 2 ml medium. Human IgG and PSMAb at the concentration of 50 mg/ml were used as controls. After 48 hour incubation, cells were trypsinized and washed twice with PBS, and stained with Annexin-V/PI (Roche) following the manufacture's instruction. Apoptosis was detected using flow cytometry, signals with Annexin-V(+)/PI (−) indicating early apoptosis and double positive staining, i.e., Annexin-V(+)/PI (+), indicating late apoptosis. Total apoptosis is the sum of early and late apoptosis. Results showed that all the three PSMAb based ADCS, i.e., PSMAb-DM1, PSMAb-MMAE, and PSMAb-MMAF, were capable of efficient induction of both early and late apoptosis, while PSMAb-DM1 mainly induced late apoptosis and PSMAb-MMAE and PSMAb-MMAF mainly induced early apoptosis (