XmAb5592 has significantly improved ADCC and ADCP activity against MM cells. (A) ADCC was measured with an LDH release assay using PBMCs from healthy donors as effector cells, and MM target cells. MM cells, opsonized with serial dilutions of antibodies, were mixed with PMBCs at an effector/target (E/T) ratio of 50:1. Percentage specific lysis was calculated from data obtained in triplicate, and presented as mean ± SD. XmAb5592 significantly augmented ADCC relative to the IgG1 analog against all cell lines, in contrast to XmAb isotype control inducing no cell lysis. (B) The EC₅₀ and maximum specific lysis were calculated from data obtained from 3 independent experiments using 3 separate donor PBMCs. XmAb5592 potency was enhanced up to 9 fold, and efficacy improved up to 15 fold compared with the anti-HM1.24 IgG1 analog. (C-D) Autologous ADCC activity against CD138⁺ patient MM cells was determined using purified NK cells from the same patient. Calcein-AM labeled patient MM cells were incubated with CD56⁺ NK cells at an E/T ratio of 10:1. (C) XmAb5592 enhanced potency and efficacy against patient MM cells compared with the IgG1 analog. (D) Specific XmAb5592-induced (open symbol) autologous MM cell lysis was determined in 2 additional patients (MM1 & MM2) with XmAb isotype control (solid symbol) showing no activity. (E) Antibody dependent cellular phagocytosis (ADCP) was determined by flow cytometry using MM cells as targets, and monocyte-derived macrophages (MDM) as effector cells. MM cells were fluorescently labeled with PKH67, opsonized with antibodies, and cocultured with MDMs at an E/T ratio of 4:1 for 4 hours. Percent phagocytosis was determined as the number of triple positive (CD14⁺/CD11B⁺/PKH67⁺) cells divided by the total number of PKH67⁺ cells. Data were obtained in triplicate and represent the mean ± SD.