Anti-CD79b antibodies are targeted to the MIIC. Binding and internalization of anti-CD79b antibodies and ADCs in vivo and in vitro. BJAB-luc xenografts were treated with a control mouse anti-gp120-MCC-DM1 conjugate (A) or anti-CD79b(SN8)-MCC-DM1 conjugate (B). Frozen tissue sections were stained with antibodies to human IgM (green) and murine IgG (red); nuclei were counterstained with DAPI (blue). In control-treated xenografts (A), BJAB-luc cells show membrane staining for hIgM; mIgG was clearly detected within the tumor but was not colocalized with hIgM (inset). By contrast, in tumors treated with anti-CD79b(SN8)-MCC-DM1 (B), very few cells were positive for IgM; those that were positive showed colocalization of IgM and the murine IgG in a punctate pattern, consistent with the internalization of the ADC conjugate in these cells (inset). Note the reduced and pyknotic DAPI staining compared with panel A, caused by ADC-mediated cell killing. (C and D) In vitro, Ramos cells were allowed to internalize anti-CD79b(SN8) antibody and Alexa647-transferrin for 3 hours at 37°C in the presence of lysosomal protease inhibitors, then fixed, permeabilized, and stained with Cy3-conjugated anti-mouse antibody. Uptaken anti-CD79b(SN8) (red channel in panels C and D) is efficiently delivered to lysosomes, as shown by its extensive colocalization (yellow color) with the lysosomal marker anti-LAMP1 (Alexa488-labeled, green channel in panel C). There is much less yellow overlap with Alexa647-transferrin (shown in the green channel in panel D for ease of comparison with panel C). (E,F) Cells were labeled as in panels C and D, respectively, except 20 μg/mL brefeldin A was added during the last 15 minutes of uptake to further distinguish recycling endosomes from lysosomes, confirming that uptaken anti-CD79b(SN8) is delivered to lysosomes. The image shown is representative of 7 independent experiments. Scale bar = 20 μm in panels C-E and insets in panels A-B, and 100 μm in main panels A-B.