Development of tandem CARs. (A) Schematic diagram of the recombinant lentiviral vectors encoding the prototype anti-CD19 CAR (CD19 CAR), anti-CD20 CAR (CD20 CAR), and TanCARs generated in this study. (B) Flow cytometry analysis showing the expression levels of CARs encoded by the indicated constructs. Graphs are representative of 3 independent experiments. (C) Cytotoxic activity of TanCAR, CD19 CAR, and CD20 CAR T cells using 24-hour or 48-hour CytoTox 96 Non-Radioactive Cytotoxicity Assays with Raji cells or K562 cells at an E:T ratio of 1:1. *P < .05, TanCAR7 vs CD19 CAR/TanCAR8; P < .01, TanCAR7 vs TanCAR2/TanCAR4; P < .001, TanCAR7 vs TanCAR1/TanCAR3/TanCAR5/TanCAR6; P < .0001, TanCAR7 vs CD20 CAR (upper panel). *P < .05, TanCAR7 vs CD19 CAR/TanCAR3; P < .01, TanCAR7 vs TanCAR2/TanCAR4/TanCAR5/TanCAR6; P < .001, TanCAR7 vs CD20 CAR/TanCAR1 (lower panel). (D) Cytokine production by CD19 CAR, CD20 CAR, and TanCAR7 T cells cocultured with Raji or K562 cells at an E:T ratio of 1:1, as measured by enzyme-linked immunosorbent assay. All data are mean ± standard deviation. Experiments were repeated with 3 pools of donor-derived T cells (n = 3). 2-tailed unpaired 2-sample Student t test. NT, control nontransduced T cells. IFN-γ, interferon-γ; NT, nontransduced; TNF-α, tumor necrosis factor-α.