JNJ-64407564 specifically kills GPRC5D⁺ target cells with concomitant T-cell activation. (A) JNJ-64407564 mediated cytotoxicity of GPRC5D⁺ cells (MM.1R, OPM-2, and H929) but not GPRC5D⁻ cells (NALM-6 and Daudi), when incubated with healthy purified T cells at a 5:1 E/T ratio. Cytotoxicity was calculated as the percent of remaining CFSC-labeled cells compared with PBS controls after 48-hour incubation with the DuoBody antibodies. JNJ-64407564 (black circles) efficiently killed GPRC5D⁺ cells, but not GPRC5D⁻ cells, while control antibodies GPRC5DxNull (red triangle) and NullxCD3 (blue square) had no effect. (B) T-cell activation as measured by flow cytometry. T cells were gated using the CD3 surface marker and CD25 activation marker. Percent CD25⁺ T-cell values were plotted on the y-axis. JNJ-64407564 activated T cells efficiently when incubated with GPRC5D⁺ cells, but not GPRC5D⁻ cells, while control antibodies GPRC5DxNull and NullxCD3 had no effect. (C) Supernatants from the H929 assays were evaluated for cytokine levels using the Meso Scale Discovery human proinflammatory panel 1 kit (K15049D; Meso Scale Discovery). Average EC₅₀ values from 6 different donor T cells are listed in the table.