The superior function of CD28-based CAR T cells can be confirmed in a PDX mouse model using human T cells. (A) IFNG and IL2 cytokine secretion after 24 h co-culture of untransduced T cells or different L1CAM-specific CAR T cell constructs with SK-N-BE(2) target cells in an effector to target (E:T) ratio of 1:5 assessed by ELISA (n = 3); Error bars represent SD. (B) Cytolytic activity of the four L1CAM-specific CAR T cell subgroups against SK-N-BE(2) neuroblastoma cells determined by luciferase-based killing assay following a co-culture for 24, 48 and 72 h at an E:T ratio of 1:5. Data shown here, depict the mean cytolytic activity of 3 independent experiments. Error bars represent SD. (C) Quantification of apoptosis of CAR T cells by annexin V staining before and after co-culture with SK-N-BE(2) cells for 48h in an E:T of 1:5. by two tailed paired t test. (D) NOG mice with subcutaneous neuroblastoma PDXs were treated by intravenous injection of 1 × 107 L1CAM-specific SS-4-1BB/ζ (n = 5), SS-28/ζ (n = 5) CAR or untransduced (n = 5) T cells on three consecutive days. Each line represents change in tumor volume of an individual mouse over the period of the experiment. (E) Kaplan–Meier survival analysis of mice shown in (D). ns = not statistically significant; ms = median survival, *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.005, Kaplan–Meier survival analysis with log-rank statistics.