Fig. 1: CAR19 design, expression in human primary T cells, cytotoxicity in vitro, and tumor-specific cytokine response in vitro.

Schematic representation of CAR19.8 and CAR19 (A). CAR19.8 has a CD19-targeting ScFv sequence FMC63, CD8 hinge sequence, CD8 transmembrane region sequence, a co-stimulatory domain derived from 4-1BB/CD137, and a CD3ζ activation domain. CAR19 has a CD19-targeting ScFv sequence FMC63 in frame with CD8 hinge sequence, a transmembrane region sequence from TNFRSF19, a co-stimulatory domain derived from 4-1BB/CD137, and a CD3ζ activation domain. B Representative detection of anti-CD19 CARs. Target-specific cytotoxicity was evaluated by co-incubation of CAR19, or comparator CAR19.8 with target cell lines Raji (C), Reh (D), K562 (E), and 293T (F) for 18 h at effector to target ratio 5:1, 10:1, or 20:1, in triplicate, red lines represent CAR19, blue lines represent CAR19.8 and green line represents untransduced (UTD). Then, target cell lysis was analyzed by luminometry. Sample means were compared by one-way ANOVA followed by Tukey’s multiple comparisons test. IL-2 (G), IFNγ (H), and TNFα (I) cytokine secretion analysis was performed on supernatants from CAR T cells challenged overnight with Raji lymphoma cells at effector to target ratio 10:1, in triplicate, and measured by ELISA, blue bars represent Raji cells, and red bars represent CAR alone. Data are representative of three independent experiments, each performed on CAR T cells produced from a different donor, bars represent mean, error bars represent SD. Sample means were compared by One-way ANOVA followed by Tukey’s multiple comparisons test. ****p < 0.0001, **p < 0.01, ns-non-significant. Source data are provided as a Source Data File for panels c–i.