Figure 6.
Clinical-scale manufactured CAR T cells with PD-1/TIGIT downregulation showed a superior in vivo functionality against leukemia and lymphoma tumor models
(A and B) NSG mice were injected intravenously with 1 × 106 Nalm-6-GL-PD-L1 cells. 5 days after tumor growth, mock, 19BBz-nt, and 19PTBBz-nt cells were intravenously injected at the indicated doses. Tumor burden was monitored based on the bioluminescence intensity from the IVIS imaging system. Data are from n = 4 mock mice and n = 6 mice for all CAR T cell treatment groups. (C) Kaplan-Meier survival analysis with log-rank test comparing the CAR T cell-treated mice at 0.25 × 106 dose from (A) and (B). (D) The number of CAR T cells in the blood and spleen was determined 43 days after CAR T cell injection at a 0.5 × 106 dose. Data are the mean ± SD from six mice per group. (E) Differentiation state of T cells in the spleen of mice 14 days after 0.5 × 106 CAR T infusion. The differentiation state of CD4+ and CD8+ CAR T cells was determined by flow cytometry for CCR7 and CD45RA expression. Data are the mean ± SD from 19BBz-nt-treated mice (n = 6) and 19PTBBz-nt-treated mice (n = 6). (F) IncuCyte-based cytotoxicity kinetics of CAR T cells in the spleen of mice 43 days after CAR T cell injection against Nalm-6-GL-PD-L1 cells at a 1:3 E:T ratio. Data are the mean ± SD from two mice per group performed in duplicate. (G) NSG mice were injected intravenously with 1 × 106 Nalm-6-GL-PD-L1 leukemia cells. 5 days later, 1 × 106 19BBz-nt or 19PTBBz-nt cells were injected intravenously. 3 days after CAR T cell infusion, a plasma was obtained from blood and used to measure GM-CSF levels. Data are from n = 2 (mock) and n = 4 (19BBz-nt and 19PTBBz) mice. Data are mean ± SD from each group. (H and I) NOG mice were injected subcutaneously on the right flank with 5 × 106 Raji-GL-PD-L1 lymphoma cells. When the mean tumor volume reached approximately 100 mm3, CAR T cells were injected intravenously at the indicated doses. Tumor burden was monitored based on the bioluminescence intensity from the IVIS imaging system. Statistical analysis for (D), (E), and (F) by an unpaired two-tailed t test and for (G) by a one-way ANOVA. ∗∗p < 0.01, ∗∗∗p < 0.0001, ∗∗∗∗p < 0.0001; ns, not significant.