Figure 5. Treatment of mice bearing EGFRvIII^POS tumors with mCARs provides long-term protection against EGFRvIII^NEG tumor.

(A) To evaluate whether treatment with EGFRvIII mCAR T cells could elicit de novo priming against antigen-loss variants, long-term survivors (greater than 60 days after treatment) of subcutaneous SMA560vIII tumors by EGFRvIII mCAR therapy were rechallenged with 5×10⁵ EGFRvIII^NEG SMA560 cells on the contralateral flank and their survival was monitored. (B) SMA560 (EGFRvIII^NEG) gliomas were maintained in vitro or passaged in a mouse subcutaneously for 14 days, then harvested and both were stained for EGFRvIII expression using L8A4 mAb and analyzed by flow cytometry. Total numbers and percentages of mCAR T cells in blood was evaluated in (C) tumor-naïve mice, and (D) SMA 560vIII (EGFRvIII^POS) tumor-cured mice before and 7 days after subcutaneous challenge with SMA560 (EGFRvIII^NEG) tumors. Statistical significance was determined using a paired t test. Horizontal bar represents a statistical significance of P < 0.05 (C-E) Tumor naïve VM/Dk mice were either left untreated, received vector control, or EGFRvIII mCAR T cells 1 day after receiving a lymphodepletive conditioning regimen by 5Gy TBI. All mice were challenged subcutaneously with SMA560 (EGFRvIII^NEG) tumors 28 days after adoptive-T-cell transfer. Additionally, VM/Dk mice that were previously inoculated with 5×10⁵ SMA560vIII (EGFRvIII^POS) tumor cells and were cured of tumor after receiving 5Gy TBI and EGFRvIII mCAR T cells (tumor cured) were rechallenged subcutaneously with 5×10⁵ SMA560 (EGFRvIII^NEG) tumor cells at the same time. (E) Tumor growth was monitored in two dimensions. Mice were examined for morbidity end points approved by the Duke University IACUC and sacrificed when end points were met. Experiment is representative of two independent repeats with similar results. Statistical significance of P < 0.0001was determined using two-way ANOVA.