Figure 2.

IL-13Rα2 CAR T Cells Control Tumor Growth In Vivo

(A) Flow-based EGFRvIII and IL-13Rα2 expression on the D270 tumor cell line controlled with control antibodies. (B) EGFRvIII-targeting (2173BBz) and IL-13Rα2-targeting (Hu08BBz) CAR T cells co-cultured with D270 tumor cell line. The stimulation of T cells was illustrated by FITC-conjugated anti-CD69 antibody staining, the median fluorescence intensity (MFI) was quantified on CD4 and CD8 CAR-positive T cells after 24- or 48-hr co-culture, controlled with un-transduced T cells. Statistically significant differences were calculated by one-way ANOVA with post hoc Tukey test. (C) Human T cells enumerated in the spleens of D270-injected NSG mice (n = 3), 11 days after i.v. transferring equal numbers of un-transduced T cells, EGFRvIII-targeting (2173BBz), or IL-13Rα2-targeting (Hu08BBz) CAR T cells. (D) Five million CAR-positive EGFRvIII-targeting (2173BBz) or IL-13Rα2-targeting (Hu07BBz and Hu08BBz) CAR T cells or the same number of un-transduced T cells after i.v. infusion in a D270 subcutaneously implanted NSG mouse model (n = 10 per group), 7 days after tumor implantation. Tumor volume measurements (left panel) and bioluminescence imaging (middle panel) were performed to evaluate the tumor growth. Linear regression was used to test for significant differences between the experimental groups. Endpoint was predefined by the mouse hunch, inability to ambulate, or tumor reaching 2 cm in any direction, as predetermined IACUC-approved morbidity endpoint. Survival based on time to endpoint was plotted using a Kaplan-Meier curve (Prism software). Statistically significant differences were determined using log-rank test. (E) 800,000 IL-13Rα2-targeting CAR-positive (Hu08BBz) CAR T cells or the same number of un-transduced T cells were given by i.v. infusion in NSG mice (n = 8 per group) orthotopically implanted with the D270 tumor, 8 days after tumor injection. Bioluminescence imaging was repeated every 3–4 days to evaluate the tumor growth. Endpoint was predefined and statistically significant differences were determined as described in (D). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are presented as means ± SEM.