Talquetamab-mediated lysis of cell lines. (A) GPRC5D protein expression on cell lines was assessed by flow cytometry. Representative overlay histograms depict GPRC5D cell surface expression (red histogram) on 4 luciferase-transduced (LUC⁺) MM cell lines (U266, RPMI-8226, MM.1S, UM9) and the LUC⁺ Burkitt lymphoma cell line Raji, compared with isotype control (gray histogram). GPRC5D MFI is provided. (B) These cell lines were incubated with solvent control, serial dilutions of talquetamab (0.00128-4.0 µg/mL), or control antibodies, together with PB-MNCs obtained from healthy donors as effector cells, at an E:T ratio of 10:1 for 48 hours. Tumor cell lysis was assessed by using a BLI-based cytotoxicity assay. Data represent mean MM lysis ± standard error of mean (SEM) of 3 to 5 independent experiments, performed in duplicate. Half maximal effective concentration (EC₅₀) values are provided. (C) LUC⁺ RPMI-8226 cells were incubated for 48 hours with solvent control or serial dilutions of talquetamab (0.00128-4.0 µg/mL), and PB-MNCs obtained from healthy donors as effector cells at different E:T ratios (10:1, 3:1, 1:1, 1:3, and 1:10). MM cell lysis was determined by using a BLI-based cytotoxicity assay. Data represent mean MM lysis ± SEM of 3 independent experiments, performed in duplicate. (D) Talquetamab-mediated activation and degranulation of healthy CD4⁺ and CD8⁺ T cells were assessed by flow cytometric analysis of CD25 and CD107a cell-surface expression, respectively (n = 3). Representative flow cytometry histogram overlays depict cell surface expression of CD25 and CD107a on CD4⁺ (green) and CD8⁺ (purple) T cells treated with talquetamab (4.0 µg/mL) or solvent control (gray). (E) Pro-inflammatory cytokines and granzyme B were measured in cell-culture supernatants from RPMI-8226 cells treated with talquetamab (0.032-4.0 μg/mL) in the presence of PB-MNCs obtained from healthy donors for 48 hours, by using a flow cytometry-based assay and an enzyme-linked immunosorbent assay, respectively. Bars represent mean ± SEM of 3 independent experiments, performed in triplicate. (F) RPMI-8226 cells were incubated with solvent control or serial dilutions of talquetamab (0.00128-4.0 µg/mL), and PB-MNCs derived from 4 healthy donors or from 9 RRMM patients (median of 3 prior lines of therapy, 66% refractory for both IMiD and PI; PB was obtained at the time of progression during last line of therapy) at an E:T ratio of 10:1. After a 48 hour-incubation, lysis of RPMI-8826 cells was determined by BLI. Data represent mean MM lysis ± SEM; each individual experiment was performed in duplicate. Differences in talquetamab-mediated tumor cell lysis in the presence of PB-MNCs from healthy donors or MM patients were calculated using nonlinear regression analysis.