In vitro CAR-37 generation and expansion. (A) Two anti-CD37 second-generation chimeric antigen receptors were constructed with different orientations of a humanized murine antibody-derived single-chain variable fragment: the light-to-heavy orientation (CAR-37 L-H, top) and the heavy-to-light (CAR-37 H-L, bottom). (B) Representative flow plots of primary human T cells transduction efficiency after 10 days of activation with CD3/CD28 beads. (C) Expanded T cells from 3 healthy donors included variable CAR-37 expression with a mean of 38% (L-H) and 75% (H-L). (D) Ex vivo expansion of CD3/CD28 bead-activated and target-stimulated T-cells using static culture conditions in 3 healthy donors for 38 days. Each arrow represents antigen stimulation with K562 cells transduced to express CD37 and CD19. (E) Activation of Jurkat reporter (NFAT-Luc) T cells transduced with different CAR constructs and cocultured with tumor cells. Luciferase activity was measured after 16 hours. (CD3-CD28 beads: positive control). (F) Number of carboxyfluorescein succinimidyl ester–labeled, unstimulated target T cells measured by flow cytometry after 24 hours of coculture at indicated E:T ratio with CAR-37H-L, CAR-19, or UTD T cells. (G) Number of carboxyfluorescein succinimidyl ester–labeled T cells stimulated with PMA/ionomycin for 6 hours, then measured by flow cytometry after 24 hours of coculture at indicated E:T ratio with CAR-37H-L, CAR-19, or UTD T cells. (H) Number of Jeko-1 CBG⁻GFP cells measured by flow cytometry after 24 hours of coculture at indicated E:T ratio with CAR-37H-L, CAR-19 or untransduced T cells. Bars indicate mean ± standard error of the mean (SEM) count of triplicates from 1 normal donor, representative of 3 normal donors. (I) CD107a and IFN-γ production relative to media by CAR-37H-L, CAR-19 T cells incubated with primary immune cells for 6 hours at 1:1 E:T ratio was analyzed by flow cytometry. Bars show mean ± SEM percentage of the 3 normal donors analyzed.