Figure 4.

IL-13Rα2 CAR T Cells Are Selectively Enhanced by In Situ-Secreted Anti-CTLA-4 Checkpoint Blockade

(A) Vector map of minibody-secreting anti-IL-13Rα2 CAR design based on the size of each components. Minibodies were simplified as PD-1, CTLA-4, and TIM-3 targeting scFvs jointing with human IgG1 spacer and CH3 domain. A self-cleaving sequence (P2A) was used to express minibodies with anti-IL-13Rα2 CAR in a same open reading frame. (B) CAR expression was detected on the minibody-secreting IL-13Rα2-targeting CAR T cells as well as the no minibody-secreting IL-13Rα2-targeting CAR T cells. (C) Supernatant of anti-PD-1 and anti-CTLA-4 minibody-secreting IL-13Rα2-targeting CAR T cells was collected and concentrated separately. A standard direct ELISA was performed to evaluate the binding ability of anti-PD-1 and anti-CTLA-4 minibodies secreted by CAR T cells to recombinant hPD-1 and hCTLA-4. Statistically significant differences were calculated by unpaired t test. (D) Un-transduced T cells, IL-13Rα2-targeting (Hu08BBz) CAR T cells, and minibody-secreting Hu08BBz CAR T cells were co-cultured with D270 tumor cell line. Median fluorescence intensity (MFI) was quantified by BV605-conjugated anti-TIM-3 antibody staining on CD4 and CD8 subgroups of CAR-positive T cells after 24- or 48-hr co-culture. Statistically significant differences were calculated by one-way ANOVA with post hoc Tukey test. (E) 800,000 IL-13Rα2-targeting (Hu08BBz) CAR T cells and minibody-secreting Hu08BBz CAR T cells or the same number of un-transduced T cells were injected i.v. 8 days after D270 subcutaneous implantation (n = 8). Tumor size was calipered and compared between each group. Statistically significant differences of tumor growth were determined by linear regression. ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are presented as means ± SEM.