Figure 3. Comparison of CD30/CD16A TandAb and diabody, anti-CD30 IgG and Fc-enhanced anti-CD30 IgG in cytotoxicity assays. (A) Kinetics of antibody-mediated target cell lysis. 1 × 10⁴ calcein-labeled KARPAS-299 target cells were incubated for the indicated time periods with increasing concentrations of CD30/CD16A TandAb, CD30/CD16A diabody, anti-CD30 IgG and Fc-enhanced anti-CD30 IgG together with freshly isolated human NK cells at an E:T ratio of 5:1. Percent specific target cell lysis was calculated from the fluorescent calcein released into the cell culture supernatant from apoptotic target cells. EC₅₀ values (potency) and maximal target cell lysis (efficacy) were determined by nonlinear regression for all antibodies and plotted. (B) Residual cytotoxicity of pre-opsonized effector cells to measure antibodies’ retentions. Freshly isolated human NK cells were incubated with increasing concentrations of CD30/CD16A TandAb, diabody and Fc-enhanced anti-CD30 IgG for 15 min at 37 °C, washed twice and then incubated for 1 h at 37 °C to allow dissociation of the bound antibodies. After an additional washing step, NK cells were used as effector cells in a 3 h cytotoxicity assay with calcein-labeled KARPAS-299 target cells at an E:T ratio of 4.4:1 (pre-loaded – 1 h dissociation). As a control, NK cells were pre-incubated in the absence of antibodies before they were used as effector cells together with added antibodies in the same cytotoxicity assay (directly added - control). Mean values and SD from duplicates of one out of two independent experiments are shown. (C) Potency of TandAb, diabody and IgG in relation to the CD16A 158 polymorphism. The EC₅₀ values for TandAb, diabody and anti-CD30 IgG were determined in several independent 3 h cytotoxicity assays on KARPAS-299 target cells with NK cells as effector cells isolated from unrelated donors as described and plotted in the diagram together with the mean values shown as bars. The CD16A 158 phenotype of the NK cells was assessed by flow cytometry after staining of the NK cells with the CD16 158V-specific mAb MEM-154. NK cells were rated as CD16A 158F/F when the fluorescence signal was at background level. The asterisk (*) indicates statistical significance (P < 0.05). (D) Cytotoxic potency of the TandAb against a panel of five CD30⁺ cell lines. The EC₅₀ values of the TandAb were determined in independent 3 h cytotoxicity assays on target CD30⁺ cells, with NK cells as effectors, isolated from independent donors, at a 1:5 ratio. Mean values for each cell line are shown as horizontal bars.