Extended Data Fig. 8: Scalability, therapeutic efficacy and safety of NR4A2/IL-12 human anti-LeY CAR T cells.

a. Human anti-LeY CAR T cells engineered to express IL-12 from the NR4A2 locus were maintained in culture with IL-2 for over 11 days and the fold expansion following transduction was determined from n = 2 donors. b-c. Human anti-LeY CAR T cells engineered to express IL-12 from either the NR4A2 or PDCD1 locus were co-cultured with OVCAR-3 or MCF-7 tumor cells for 24 h before the concentrations of IL-12 (b) as well as IFNγ, TNF and IL-2 (c) in supernatants were assessed. Data represent mean ± SD of technical triplicates. d-k. Human anti-LeY CAR T cells engineered as per (b-c) were adoptively transferred into mice bearing subcutaneous OVCAR-3 tumors. d. Tumor growth curves from n = 2 donors, represented as mean ± SEM from n = 5 (NR4A2/IL-12) and 6 (Non-treated, Mock, NR4A2 KO) mice/group (Donor 1), or n = 3 (NR4A2/IL-12), 4 (Mock, NR4A2 KO) and 6 (Non-treated) mice/group (Donor 2). e. Mice with previously cured OVCAR-3 tumors following treatment with NR4A2/IL-12 human anti-LeY CAR T cells were rechallenged with OVCAR-3 tumors in the opposite flank. Tumor growth curves represented as mean ± SEM from n = 5 (NR4A2/IL-12) and 9 (Naïve) mice/group. f. Body weight of mice post treatment, represented as mean ± SEM from n = 6 mice/group. g-k. Ex vivo analysis of human CAR T cells isolated from tumors (g-h, j-k), spleens (j-k) or blood (i, k) 11-14 days post transfer. g. t-SNE plots comparing expression of indicated proteins in intratumoral Mock and NR4A2/IL-12 CD8⁺ human CAR T cells, concatenated from n = 4 mice/group. h. Quantification of IFNγ and TNF expression in intratumoral CD8⁺ human CAR T cells. Data represent mean ± SEM from n = 4 mice/group. (g-h) Tumor samples were ex vivo stimulated with PMA/Ionomycin for 3 h. i. Flow cytometry plots showing Ki67 expression in CD8⁺ human CAR T cells isolated from the blood, concatenated from n = 5 (NR4A2/IL-12) and 6 (Mock, NR4A2 KO) mice/group. j. Quantification of CAR expression in the tumor and spleen, represented as mean ± SEM from n = 4 mice/group. k. Quantification of bulk, CD8⁺ and CD4⁺ human CAR T cells isolated from tumors (left), spleens (middle) and blood (right). Left and middle: Data represent mean ± SEM from n = 4 mice/group. Right: Data represent mean ± SEM from n = 8 mice/group, representative of n = 2 donors. (b-c, d-e) Two-way ANOVA. (h, j-k) One-way ANOVA. n.s. not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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