Mechanism of action of ADCT-402. (A) Merged immunofluorescent images of CD19⁺, human Burkitt lymphoma-derived Ramos cells treated with 2 μg/mL ADCT-402 for 1 hour; washed and fixed after T = 0, 1, 2, 4, or 24 hours in medium at 37°C; and stained with labeled antihuman IgG (green), anti-LAMP-1 (red), and Hoechst 33342 (blue) nuclear stain. Yellow indicates colocalization, with specific sites of colocalization indicated by stars. Original magnification ×63. (B) Time course of DNA interstrand crosslinking measured as the % reduction on Olive Tail Moment in Ramos cells treated for 2 hours with ADCT-402 (40 pM), SG3199 (10 pM), or a nontargeted ADC (40 pM) followed by postincubation in drug-free medium for the indicated time. Results are the mean ± standard deviation from at least 3 independent experiments. (C) Percentage viability of CD19⁻ Karpas-299 cells after 96 hours of exposure to media transferred from ADCT-402–treated CD19⁺ Ramos cells or CD19⁻ NCI-N87 cells for 1, 2, or 3 days. Statistical analysis was done with unpaired t test with Welch’s correction (not assuming equal standard deviations). P values are 2-tailed. *P ≤ .05, **P ≤ .01. ns, not significant.