Extended Data Fig. 6: Assessment of transgene expression driven by the Nr4a2 and NFAT promoters.
From: Rewiring endogenous genes in CAR T cells for tumour-restricted payload delivery
a. NR4A2/GFP OT-I cells were stimulated with anti-CD3/28 antibodies or the indicated ova-expressing tumor lines for 24 or 72 h before GFP expression was quantified. Data represent mean ± SD of technical triplicates, representative of n = 3 experiments. b-d. NR4A2/IL-12 OT-I cells were generated and assessed in vitro (b) and in vivo (c-d). b. IL-12 concentration in supernatants of Mock or NR4A2/IL-12 OT-I cells stimulated with anti-CD3/28 antibodies for 24 h, represented as mean ± SD of n = 6 technical replicates. c-d. 1 × 106 Mock, NR4A2 KO or NR4A2/IL-12 OT-I cells were adoptively transferred into mice bearing orthotopic AT-3-ova tumors. c. Tumor growth curve represented as mean ± SEM from n = 5 mice/group, representative of n = 3 experiments. d. Survival curve from n = 5 (NR4A2 KO), 9 (NR4A2/IL-12) and 10 (Non-treated, Mock) mice/group, pooled from n = 2 experiments. e-f. Murine anti-hHer2 CAR T cells were engineered to express Gfp via the Pdcd1 or Nr4a2 locus and assessed upon in vitro stimulation with anti-CD3/28, anti-CAR antibodies or the indicated tumor cell lines (e) and in mice bearing orthotopic E0771-hHer2 tumors (f). e. Concatenated flow cytometry plots (left) and quantification (right) of GFP expression in CAR T cells following a 24 h stimulation. Data represent mean ± SD of technical triplicates, representative of n = 3 experiments. f. Concatenated flow cytometry plots (left) and quantification (right) of GFP percentage in CAR T cells in the tumor and spleen harvested 7 days post transfer. Data represent mean ± SEM from n = 3 mice/group, representative of n = 2 experiments. g. Murine T cells were retrovirally transduced with an anti-hHer2 CAR and either with an NFAT-GFP construct or CRISPR-engineered to express Gfp from the Nr4a2 locus, and assessed for GFP expression in mice bearing orthotopic E0771-hHer2 tumors. Quantification of GFP percentage in CAR T cells pre-infusion and in the spleen of mice 3, 6 and 9 days post transfer, represented as mean ± SEM from n = 3 (Mock) and 4 (NR4A2/GFP, NFAT-GFP) mice/group. h-i. Human T cells were retrovirally transduced with an anti-LeY CAR and either with an NFAT-GFP construct or CRISPR-engineered to express Gfp from the NR4A2 locus, and assessed for GFP expression in mice bearing subcutaneous OVCAR-3 tumors (h-i) or non-tumor bearing mice (i). Quantification of GFP percentage in CD8+ human CAR T cells in h. the blood of tumor-bearing mice 4 and 7 days post transfer and i. the spleen of tumor- and non-tumor bearing mice 7 days post transfer, represented as mean ± SEM from n = 4 mice/group. j. Murine T cells engineered as per (g) were assessed for GFP expression in mice bearing orthotopic E0771-hHer2 tumors, E0771 parental tumors or non-tumor bearing mice. Quantification of GFP percentage in CAR T cells in the spleen of mice 3 days post transfer, represented as mean ± SEM from n = 3 (Mock) and 4 (NR4A2/GFP, NFAT-GFP) mice/group (E0771-hHer2); n = 4 mice/group (E0771 parental); n = 3 mice/group (no tumor). k. IL-12 concentration in supernatants of Mock, NR4A2 KO or NR4A2/IL-12 murine anti-hHer2 CAR T cells stimulated with anti-CD3/28 or anti-CAR antibodies, or E0771-hHer2 tumor cells for 24 h. Data represent mean ± SD of technical triplicates, representative of n = 3 experiments. l. 5 × 106 mock or NR4A2/IL-12 murine anti-hHer2 CAR T cells were adoptively transferred into mice bearing orthotopic E0771-hHer2 tumors > 30 mm2. Tumor growth curves (left) represented as mean ± SEM from n = 6 mice/group. Survival curves (right) from n = 6 mice/group. m. 5 × 106 mock, NR4A2 KO or NR4A2/IL-12 murine anti-hHer2 CAR T cells were adoptively transferred into mice bearing orthotopic E0771-hHer2 tumors (left) or subcutaneous MC38-hHer2 tumors (right). Body weights represented as mean ± SEM from n = 4 (NR4A2/IL-12) and 6 (Non-treated, Mock, NR4A2 KO) mice/group (E0771-hHer2); n = 6 mice/group (MC38-hHer2). n-o. Murine T cells were retrovirally transduced with an anti-hHer2 CAR and either with an NFAT-IL-12 construct or CRISPR-engineered to express IL-12 from the Nr4a2 locus. n. Concentration of IL-12 in supernatants upon in vitro stimulation with anti-CD3/28, anti-CAR antibodies or hHer2-expressing tumor cells. Data represent mean ± SD of technical triplicates, representative of n = 2 experiments. o. 5 × 106 mock, NR4A2/IL-12 or NFAT-IL-12 murine anti-hHer2 CAR T cells were adoptively transferred into mice bearing orthotopic E0771-hHer2 tumors. Body weights represented as mean ± SEM from n = 5 (NFAT-IL-12) and 6 (Non-treated, Mock, NR4A2/IL-12) mice/group. (a-c, e, f-h, j-k, l (left), n) Two-way ANOVA. (d, l (right)) Logrank Mantel-Cox test. (i) One-way ANOVA. n.s. not significant, **p < 0.01, ***p < 0.001, ****p < 0.0001.
