HexaBody-DR5/DR5 mediates extrinsic apoptotic pathway activation and induces low levels of ADCC in MM cell lines. (A) Representative dose-response curve of HexaBody-DR5/DR5 in RPMI8226 (left panel), NCI-H929 (middle panel), and MM.1s (right panel) cell lines after a 24-hour incubation compared with IgG1-Ctrl (20 µg/mL) (n = 3 or 4). (B) Time-lapse representation of apoptosis induction in NCI-H929 cells by HexaBody-DR5/DR5 and IgG1-Ctrl (20 µg/mL). NCI-H929 cells were labeled with caspase-3 green label, and images were taken using Incucyte for 24 hours. Each point represents the average total green object area per image of 3 replicates. (C) Expression of extrinsic apoptotic pathway–related (cleaved) proteins for RPMI8226, NCI-H929, and MM.1s cell lines, as detected by western blot after a 6-hour incubation with HexaBody-DR5/DR5 (1 µg/mL [Hx1] and 4 µg/mL [Hx4]). Actin was incorporated as loading control. A representative image is shown of 2 independent experiments. (D) Kill (%) of RPMI8226, NCI-H929, and MM.1s cell lines with HexaBody-DR5/DR5 (20 µg/mL) after a 24-hour incubation in the presence of healthy donor PBMCs in a 40:1 effector-to-target ratio. Each point represents a single donor. Data are shown as mean and SD. **P = .009, paired Student t test. (E) Kill (%) of NCI-H929 (left panel) and MM.1s (right panel) cell lines by HexaBody-DR5/DR5 and IgG1-Ctrl in the absence of PBMCs, as well as in the presence of paraformaldehyde-fixed or nonfixed PBMCs in a 40:1 effector-to-target ratio. IgG1-DR5/DR5 and the anti-CD38 antibody daratumumab (Dara) are incorporated to control for cytotoxicity that is dependent on FcγR-mediated crosslinking and cytotoxicity that is dependent on ADCC, respectively. Data are shown as mean and standard deviation of 2 (NCI-H929) or 3 (MM.1s) independent experiments. *P < .05, **P < .01, 1-way analysis of variance and Tukey’s multiple-comparisons test.