Talquetamab-mediated lysis is hampered by the presence of BMSCs in 2 of 3 cell lines. (A) Luciferase-transduced cell lines RPMI-8226, MM.1S, and UM9 were incubated with solvent control, serial dilutions of talquetamab (0.00128-4.0 µg/mL), or control antibodies, and with PB-MNCs obtained from healthy donors at an E:T ratio of 10:1, in the absence or presence of BMSCs (ratio of BMSCs to MM cells was 2:1). After a 48-hour incubation, MM cell lysis was assessed using a BLI-based cytotoxicity assay. Data represent mean MM lysis ± SEM of 3 to 4 independent experiments performed in duplicate. Differences in talquetamab-mediated tumor cell lysis in the presence or absence of BMSCs were calculated using nonlinear regression analysis; P values are provided. (B) The indirect or direct impact of BMSCs on talquetamab-mediated lysis was evaluated by performing transwell experiments in which MM cells (RPMI-8226 or MM.1S cells) and PB-MNCs obtained from healthy donors were placed in the lower chambers, and no cells (medium alone) or BMSCs (indirect contact) were placed in the upper chambers. To study direct cell contact, all cell types were combined in the lower chambers. Cells were incubated with serial dilutions of talquetamab (0.032-4.0 µg/mL) or control antibodies for 48 hours. Lysis of MM cells was assessed using a BLI assay. Data represent mean lysis ± SEM of 3 independent experiments, performed in duplicate or triplicate. The impact of direct or indirect contact with BMSCs was calculated using nonlinear regression analysis and 2-way analysis of variance test; P values are provided. (C) GPR5D expression on MM cells was assessed by flow cytometry after a 48-hour incubation in the presence or absence of BMSCs. Paired Student t test was used to evaluate significance between both groups.