Figure 2. C.TNC-CAR T cells have antitumor activity in vitro and in vivo. (A) Schematic of C.TNC-CAR design. (B) Transduction efficiency of healthy donor T cells determined via flow cytometry on day 7 (n=5, mean+SEM). Graph shows the percentage of positive cells stained for each respective antibody for CAR transgene detection. (C,D) IFN-γ production measured by ELISA after 48 hours of co-culture with (C) sarcoma or (D) brain tumor cell lines at 2:1 effector to target (E:T) ratio. Negative values were plotted as zero (mean+SEM, n=3–4 for NT and C.TNC), two-way ANOVA, ***p<0.001, ****p<0.0001. (E,F) Cytotoxicity after 72 hours at a 4:1 E:T ratio determined by a luciferase-based assay (mean+SEM, n=3), two-way ANOVA, ****p<0.0001. (G) Cytotoxicity of C.TNC-negative cell line CCRF-CEM determined by a luciferase-based assay (n=4–5, mean+SEM). (H) Schematic of experiment in 5–6-week-old female NSG mice. 1×10⁶ LM7.green fluorescent protein.firefly luciferase cells were injected intraperitoneally (i.p.), followed by 1×10⁶ T cells injected i.p. 7 days later. (I) Flux values from weekly IVIS images (n=5 per cohort). (J) Overall survival of the mice, Mantel-Cox test, **p<0.01. (K) Schematic of experiment in 10–12-week-old male NSG mice. 1×10⁶ DIPG007.YFP.ffLuc cells were injected intracranially (i.c.), followed by 2×10⁶ T cells injected i.c. 7 days later. (L) Flux values from weekly IVIS images (n=5 per cohort). (M) Overall survival of the mice, Mantel-Cox test. ANOVA, analysis of variance; CAR, chimeric antigen receptor; C.TNC, tenascin C encoding the C domain; DIPG, diffuse intrinsic pontine glioma; IFN, interferon; NT, non-transduced; scFv, single chain variable fragment.