Immunohistochemical analysis of the subcellular localization of hBU12 and hBU12-vcMMAE in lymphoma cell lines. Twenty-four hours after incubation at 37°C, cells were fixed, permeabilized, and stained with the LAMP-1 lysosomal antibody (red) and/or antibodies binding to hBU12 (left panels) and hBU12-vcMMAE (right panels, green). Control reagents included a vcMMAE or mAb that did not internalize or a nonbinding molecule, which both failed to localize to the lysosomes (see Figure S1). (A) Immunohistochemical analysis of CD21^low Ramos cells and CD21^high Raji and Daudi cells. The hBU12 conjugates mostly localized within the lysosomal compartment, as indicated by the yellow staining pattern. Arrows show colocalization of ADCs with lysosomes. We were unable to identify differences in the proportion of compounds localizing to the lysosomal compartment between CD21^high and CD21^low cells. (B) Amounts of total free drug released over time from CD21^low Ramos, CD21^high Daudi, and rituximab-resistant Ramos cells. (C) Levels of free drug released over time in CD21^high, parental Raji-RP cells, and rituximab-resistant Raji-2R and Raji-4RH cells. Reduced amounts of free MMAE were observed in Raji-2R, but not Raji-4RH cells, relative to the parental Raji-RP cells.