Anti-tmTNF-α antibody effectively kills leukemia cells via CDC and ADCC. (A) Expression of tmTNF-α by 3 leukemia cells lines was determined by FCM. The FCM plots are representative of 3 independent experiments. Mouse IgG1 was used as an isotype control. (B-C) The CDC and ADCC activity of C1 and infliximab. For the CDC assay (B), leukemia cells were incubated with 2 μg/mL C1, infliximab, or isotype control IgG1 in the presence of either guinea pig complement (for C1 or IgG1) or human complement (for infliximab) at 37°C for 2 hours. For the ADCC assay (C), leukemia cells were incubated with 2 μg/mL of the indicated antibodies in the presence of either murine macrophages (for C1 or IgG1) or human PBMCs (for infliximab) at 37°C for 4 hours. Data are expressed as means ± SEM (n = 3); *P < .05; **P < .01; ***P < .001 vs the isotype control group. (D-E) The CDC and ADCC activities of C1 and infliximab were determined in the presence of increasing concentrations of sTNF-α. The CDC (D) and ADCC (E) activity of infliximab, but not C1, could be significantly neutralized by the addition of sTNF-α. (F) Expression of tmTNF-α on 2 primary AML cells was determined by FCM. As an isotype control, mouse IgG1 was used. (G-H) Leukemia cells were incubated with 2 μg/mL C1 or isotype control IgG1 in the presence of guinea pig complement for 2 hours, followed by treatment with either cytarabine (160 μmol/L) or daunorubicin (50 nmol/L) at 37°C for an additional 24 hours. The apoptosis was determined in triplicate by staining with Annexin V and PI. Data are expressed as means ± SEM; *P < .05; **P < .01 vs the isotype control group.