Mechanism of action of SGN-CD19B. (A) Cell surface and intracellular localization of SGN-CD19B in WSU-DLCL2 lymphoma cells was detected by fluorescence microscopy. SGN-CD19B binds to CD19 on the cell surface following treatment of 1 hour at 4°C and is distinguishable from LAMP-1 (upper panels). Following a 4-hour incubation at 37°C (t = 4 hours), SGN-CD19B shows evidence of internalization (lower left) and colocalization with LAMP-1 (lower right). Arrows indicate areas of colocalized signal (yellow) observed in the merged image (lower right). Original magnification ×600. WSU-DLCL2 lymphoma cells were incubated with SGN-CD19B (ng/mL) or SGD-1882 (nM) for 48 or 72 hours prior to western blot analyses with antibodies specific for γ-H2AX (B) or cleaved PARP (E). Antibodies recognizing total H2AX (B) or β-actin (E) were used to confirm equivalent protein loading. (C) Caspase 3/7 activation was assessed by treating cells with SGN-CD19B (blue closed triangles) or a nonbinding control ADC (red open circles) for 72 hours prior to evaluation of caspase 3/7 activity. (D) Caspase 3/7 activation following treatment of cells with free drug (SGD-1882) at 48 hours (red open circles) and 72 hours (blue closed triangles).