CAR T-cell design and characterization. (A) Diagram of lentiviral cassette design for CD19-targeted CARs including CD19 scFv (FMC63, VH4vκ1, and 4D5), immunoglobulin G4 hinge, and CH3 connected by a 10-amino-acid glycine and serine (GS) linker (ch2Δ), CD4 transmembrane domain, cytoplasmic 4-1BB costimulatory domain, and cytoplasmic CD3ζ stimulatory domain. A non-signaling truncated EGFR is separated from the CAR by a T2A ribosome skip sequence for tracking transduction efficiency. (B) CD19 CARs were expressed in the healthy donor (HD) Tn/mem cells by lentiviral transduction. CAR expression is measured as % EGFR⁺. Representative histograms (top) and dot plots (bottom) are shown (n = 3). (C) Murine and humanized CAR T cells were characterized by analyzing the expressions of CD27, CCR7, CD45RA, CD28, programmed cell death protein 1 (PD1), TIM3, and LAG3 (n = 4). Data are shown as mean ± standard error of the mean (SEM).