Fig. 6

Antigen-induced CAR dimerization enhances on-tumor cytotoxicity. a Viability of MM.1S-luc (upper) and RPMI 8226-luc cells (lower) co-cultured with T cells transduced with the indicated nanobody CAR constructs compared to untreated (UT) control T cells (color coding at the top of the panel) for 24 h at the effector cell: target cell (E: T) ratios of 1:1 and 4:1. n = 4 replicates per group. Unpaired two-tailed Student’s t-test was performed on each grouped sample without adjustments for multiple comparisons. b Granzyme A, granzyme B, and perforin levels in the supernatant were measured. n = 4 replicates per group. Unpaired two-tailed Student’s t-test was performed on each grouped sample without adjustments for multiple comparisons. c Representative imaging flow cytometry micrographs of tested CAR T–MM.1S interactions at ×40 magnification. d CAR T cells were assessed for mean fluorescence intensity of F-actin at the immune synapse. n = 4 replicates per group. Unpaired two-tailed Student’s t-test was performed without adjustments for multiple comparisons. e Evaluation of cell-surface BCMA levels on CHO-luc cells. Escalating amounts of BCMA RNA were delivered by electroporation and the antibody binding capacity was assessed using an anti-BCMA antibody conjugated with phycoerythrin. f Viability of CHO-luc cells electroporated with varying BCMA amounts as measured in (e) while being co-cultured with the indicated CAR T cells for 24 h at the effector cell: target cell (E: T) ratios of 4:1. The one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test or the Kruskal-Wallis test followed by Dunn’s multiple comparisons correction was performed to assess the differences among different groups, n = 3 replicates per group. All data are represented as mean ± SEM