GoKawiila. CAR expression in AAVS1 and GNAS-KO CD19-28z CAR T cells from 2 human donors before transfer into NSG mice. b. Tumour growth (left) and survival (right) of NSG mice bearing subcutaneous CD19-A375 melanoma tumours treated with CD19-28z CAR T cells. n = 6 mice per group. Data are from one donor; the second donor is shown in Fig. 5a. c. Absolute number of CAR+ T cells per gram of WT-A375 or CD19-A375 tumours from the rechallenge experiment shown in Fig. 5b. n = 4 mice. d-f. Absolute tumour weight (d), CD4/CD8 proportion in CAR+ cells (e) and CAR+ CD4 and CD8 cell number per gram of tumour (f) from the experiment shown in Fig. 5c. n = 5 mice per group. g. MES-SA tumour growth upon tumour rechallenges. Naïve NSG mice were newly challenged and surviving mice from Fig. 5g that experienced complete responses were rechallenged with MES-SA tumour cells on day 70 post initial B7H3-BBz CAR T cell infusion. n = 5 naïve mice and 7 surviving GNAS KO-treated mice. h-i. Tumour growth (h) and survival (i) of NSG mice bearing MES-SA uterine sarcoma tumours treated with the indicated doses of AAVS1 or GNAS-KO B7H3-BBz CAR T cells. n = 6 mice for TRAC KO and 7 mice for all other groups. Highest-dose data are also shown in Fig. 5g. j-l. Transcriptomic profiling of B7H3-BBz CAR T cells recovered from MES-SA tumours. Heatmap showing differentially expressed genes between AAVS1 control and GNAS-KO CAR T cells (j, n = 6 replicates from 3 donors per group; each replicate represents pooled TIL from 2 mice). GSEA with Benjamini–Hochberg multiple test correction showing reduced enrichment of Tex signature genes in GNAS KO CAR T cells compared to AAVS1 controls (k). FPKM + 1 values of selected inhibitory molecules, lines connecting paired samples from the same donor (l), n = 3 donors per group. m-p. 1.5 × 106 OE19 esophageal adenocarcinoma cells were subcutaneously engrafted into NSG mice at day 0, followed by intravenous infusion of 0.5 × 106 (low dose) CLDN18.2 CAR T cells at day 8. Tumour growth curves (m), absolute number of CLDN18.2 CAR T cells per gram of tumour (n), CD4/CD8 proportions among CAR T cells (o) and CD39 expression in tumour-infiltrating CAR T cells (p) were analyzed on day 30 post CAR T cell infusion. n = 5 mice per group. q-x. Lung metastasis model using luciferase-A549 NSCLC cells and B7H3-BBz CAR T cells from an additional donor. Shown are: tumour growth measured by IVIS (q), lung masses and lung images along with representative lung H&E staining at day 90 after CAR T cell infusion (r); CAR+ frequencies (s), CD4/CD8 proportions (t), activation marker expression (u), ki-67 levels (v), cytokine production (w), and CD39 expression (x) in T cells isolated from lung. n = 6 mice per group. P values determined using two-way ANOVA in (b, g, h, m, q) for tumour growth analysis, Log-rank (Mantel-Cox) test in (b, i) for mice survival analysis, and two-tailed unpaired Student’s t-test in (c–f, n–p, r–x) and two-tailed paired Student’s t-test in (l). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data are mean ± s.e.m. (c, h). Box plots show the median (centre line), 25th–75th percentiles (box bounds), minimum and maximum values (whiskers), and all individual data points (d-f, n-p, r, s-x). Dashed lines, individual mice; solid, group mean (b, g, m, q).
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