Binding affinity and activation of MM CARTs in response to BCMA and TACI target antigens. (A) CARTs were incubated with fluorophore-conjugated sBCMA and sTACI for 45 minutes at 4°C. Plots indicate mean fluorescence intensity of mCherry-gated CARTs bound to BCMA or TACI at the indicated concentrations. (B-E) Luciferase-based cytotoxicity assays of CARTs against a panel of targets cocultured at various E:T ratios, as indicated on the x-axis. Luciferase activity of targets was measured after 8 hours (MM.1S, RPMI8226) or 16 hours (K562). Data points indicate the mean ± standard error of the mean (SEM) of triplicates from a representative of 3 normal donors. Degranulation (F) and activation (G) of UTDs and BCMA, APRIL, and TriPRIL CARTs stimulated with BCMA- and/or TACI-expressing target cells at a 1:1 E:T ratio. Percentage of CD3⁺ or CD3⁺mCherry⁺ cells, respectively, that express CD107a and CD69, accordingly, was measured by flow cytometry and is displayed relative to the positive control (phorbol 12-myristate 13-acetate/ionomycin). Bars and heat map show means ± SEM of 3 normal donors.