Extended Data Fig. 10: Characterization of RGS16/IL-2 human anti-LeY and murine anti-hHer2 CAR T cells.
From: Rewiring endogenous genes in CAR T cells for tumour-restricted payload delivery
a-b. Human anti-LeY CAR T cells were engineered to express IL-2 from either the NR4A2, PDCD1 or RGS16 locus and co-cultured with OVCAR-3 or MCF-7 tumor cells for 24 and 72 h before the concentration of IL-2 in supernatants was assessed. a. Concentration of IL-2 post 24 h of stimulation with OVCAR-3 or MCF-7 tumor cells. b. Concentration of IL-2 post 24 and 72 h of stimulation with OVCAR-3 tumor cells. (a-b) Data represent mean ± SD of technical triplicates. c-e. Human anti-LeY CAR T cells engineered as per (a-b) were adoptively transferred into mice bearing subcutaneous OVCAR-3 tumors. c. Body weight of mice post treatment, represented as mean ± SEM from n = 6 mice/group. d. Flow cytometry plots of Ki67 expression in intratumoral CD8+ human CAR T cells following ex vivo stimulation with PMA/Ionomycin for 3 h, concatenated from n = 4 mice/group. e. Quantification of CD8+ and CD4+ human CAR T cells in tumors (left), spleens (middle) and blood (right) 14-15 days post transfer. Left and middle: Data represent mean ± SEM from n = 4 mice/group. Right: Data represent mean ± SEM from n = 6 mice/group, representative of n = 2 donors. f-g. 5 × 106 mock, RGS16 KO or RGS16/IL-2 murine anti-hHer2 CAR T cells were adoptively transferred into mice bearing orthotopic E0771-hHer2 tumors. f. Tumor growth curves represented as mean ± SEM from n = 4 (RGS16 KO), 5 (Mock) and 6 (Non-treated, RGS16/IL-2) mice/group. g. Quantification of intratumoral Tregs (CD4+ CD25+ FoxP3+) 9 days post transfer. Data represent mean ± SEM from n = 4 mice/group. (a-b, f) Two-way ANOVA. (e, g) One-way ANOVA. n.s. not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
