Cytotoxic synergy of 5F11 in combination with bortezomib. (A-D) XTT viability assays of various cell lines after 48-hour exposure to subtoxic concentrations of cross-linked 5F11 (5 μg/mL 5F11; 25 μg/mL GaH-IgG), increasing concentrations of bortezomib (○), and the combination of both (•). The cell viability after exposure to 5 μg/mL 5F11 (▵), 25 μg GaH (▿), and the same amounts of 5F11 plus GaH (♦) is indicated. The mAb 5F11 and GaH-IgG were distributed in 100-μL aliquots in 96-well plates with target cells (2 × 10⁴; L540, Karpas299, L428, and REH), and the plates were incubated for 30 minutes before addition of bortezomib. A synergistic increase of cytotoxicity is observed for the CD30-expressing cell lines (A-C). The synergy is not seen upon preincubation with bortezomib before 5F11/GaH addition (C, ⋄) and for the CD30^- cell line REH (D). Means and standard deviation of 3 independent experiments are given. (E) Depiction of the XTT data for subtoxic concentrations of cross-linked 5F11 (5F11/GaH: 5/25 μg/mL for each cell line), subtoxic concentrations of bortezomib (L540: 7.5 μg/mL; Karpas299: 2.5 μg/mL; L428: 50 μg/mL; REH: 5 μg/mL), and the combination of both. Error bars indicate mean and SD of 3 independent experiments. (F) Effect of 5F11 and bortezomib on the tumor growth of subcutaneous L540Cy Hodgkin tumors in SCID mice. One of 2 independent experiments is shown, and the standard error of the mean is given. (G) Statistical analysis of the tumor volumes measured on day 47 (see dashed box in panel F, and an independent series, each group with n = 4). The differences between 5F11, bortezomib, and the combination versus the control are significant (the P values are estimated with the paired t test using GraphPadPrism software; GraphPad Software, San Diego, CA).