ATRA reverses resistance to daratumumab-mediated CDC by increasing CD38 expression and reducing expression levels of complement-inhibitory proteins on MM cells. (A) Ex vivo CDC and ADCC assays were performed with BM aspirates from 15 patients, who were subsequently treated in the GEN501 study. One-hour CDC and 48-hour ADCC assays were performed as described in Materials and methods. Shown are means ± SEM of CDC and ADCC mediated by 10 µg/mL daratumumab according to response achieved in the GEN501 study. P values between the indicated groups were calculated using a Student t test; *P < .05. (B) Serial BM aspirates were obtained from 5 patients both before start of daratumumab and at the time of progression during daratumumab. One-hour CDC and 48-hour ADCC assays were performed as described in Materials and methods with pretreatment samples and samples obtained at the time of progression. We expect that at the time of progression MM cells have already daratumumab, given to the patient via intravenous infusion, bound to their cell surface; therefore, these graphs show the effect of freshly added daratumumab. To evaluate the effect of possible prebound daratumumab in CDC assays, we analyzed the effect of the control antibody IgG1-b12 (no CDC activity). IgG1-b12 in the presence of heat-inactivated serum or native human serum did not induce CDC against MM cells obtained at the time of progression (lysis: 0.81% and −1.14%). This indicates that prebound daratumumab does also not induce CDC at the time of progression. Dose-response curves for ADCC and CDC were constructed according to treatment status. Data are presented as mean ± SEM. P values between the indicated groups were calculated using a paired Student t test; *P < .05, **P < .01. (C) BM-MNCs were obtained from 8 patients before the first daratumumab infusion and at the time of progression (PD). BM-MNCs obtained at the time of progression were subsequently incubated with solvent control or with 10 nM ATRA for 48 hours. Cells were then collected to determine CD38, CD55, and CD59 expression levels by flow cytometry. HuMax-003-FITC was used to detect CD38 expression. Data are presented as mean ± SEM. P values between the indicated groups were calculated using a paired Student t test; *P < .05, ns, not significant. (D) Pooled results of 1-hour CDC and 48-hour ADCC assays, using BM-MNCs of 6 patients. BM-MNCs were pretreated for 48 hours with solvent control or 10 nM ATRA, followed by incubation with IgG1-b12 control antibody or daratumumab. Pooled human serum (10%) was used as a source of complement. The survival of primary CD138⁺ MM cells in the BM-MNCs was determined by flow cytometry. Percentage lysis of MM cells was calculated as indicated in Materials and methods. Data are presented as mean ± SEM. P values between the indicated groups were calculated using a paired Student t test; *P < .05.