In vitro and in vivo efficacy of CD19-CD22 bicistronic CAR constructs harboring CD28 and 4-1BB costimulatory domains. (A) Schematic representation of the initial bivalent CD19.22.BBζ CAR construct and the newly generated bicistronic constructs, harboring the m971 human anti-CD22 scFv and murine FMC63 anti-CD19 scFv under the control of the EF1α promoter. Constructs differ in the CD28 and 4-1BB costimulatory domains with all hinge-transmembrane domains derived from CD28 in the former and CD8 in the latter. (B) Cytokine production induced by coculture of T cells harboring 1 of the 4 bicistronic CARs or the bivalent CD19.22.BBζ CAR (purple) was evaluated by coculture with CD19⁺CD22⁺, CD19⁺CD22⁻, CD19⁺CD22^High, CD19⁻CD22⁺, and CD19⁻CD22⁻ NALM6 lines. Cocultures were performed at a 1:1 effector/target ratio and cytokines monitored at 1-, 3-, 6-, 12-, 18-, 24-, 30-, 36-, 42-, 48-, 60-, and 72-hour time points on a TECAN EVO 100 robotic system. Results are representative of data obtained in 3 individual T-cell donors. (C) Luciferase-transduced NALM6 cells (1e6) were injected IV into NSG mice on day 0, and the indicated bicistronic CAR T cells were injected at day 3. Leukemia growth was evaluated at the indicated time points by bioluminescent imaging. Quantification of bioluminescence at each time point is shown for each individual mouse (bottom graphs).