Figure 2

Meso-TR3 binding to MUC16-expressing cancer targets. A, FACS-analysis of OVCAR3 cells assessed for expression of MUC16 (mAb X75) and a PE-conjugated secondary Ab (red line). The secondary Ab alone served to establish the background fluorescence (black line). B, OVCAR3 cells in suspension were incubated with HEK293T-derived culture supernatant containing soluble mesothelin. Mesothelin binding was detected via anti-FLAG antibody staining (mAb M2) and a FITC-conjugated secondary Ab (green line). Cells treated with culture medium alone served as negative control (black line). C, OVCAR3 cells in suspension were incubated with HEK293T-derived culture supernatant containing Meso-TR3. To prevent binding of Meso-TR3 via TR3/death receptor interaction, Meso-TR3 was complexed with soluble DR5-Fc. Meso-TR3 binding was detected via anti-FLAG antibody staining similar to (B) using mAb M2, followed by FITC-conjugated secondary Ab (green line). Cells treated with culture medium alone served as negative control (black line). D, OVCAR3 cells were grown on 4-chamber slides and incubated the following day with Meso-TR3 complexed with DR5-Fc, similar to what has been described for (C). After washing, the cells were stained with a mixture of MUC16 pAb (red) and FLAG mAb (green), respectively. The cells were counterstained with TOPRO3 (blue, nuclei) and analyzed by confocal microscopy. The individual channels were overlaid to document co-localization of tumor marker and the targeted cancer drug (Merge). Original magnification: 63 × .