Fig 1: Nr2f6-modified CAR-T cells confer superior anti-tumor immunity, achieving complete remission in a preclinical Panc02-EpCAM mouse model of solid tumors in vivo.A Experimental CAR structure. scFv = single chain variable fragment. h hinge. TM transmembrane. IC intracellular domain. Created in BioRender. Klepsch, V. (https://BioRender.com/1s7z4s9). B Experimental setup and timeline. Immunocompetent wild type mice were injected subcutaneously with 1 × 106 Panc02-EpCAM cells (mosaic: 71% EpCAM positive, 29% EpCAM negative) and treated 2 days later with 3.6 × 106 genetically modified 28ζ CAR-T cells using CRISPR/Cas9 (Non-targeting control/NTC vs Nr2f6crispr−/−.g04). Created in BioRender. Humer, D. (https://BioRender.com/egrvdww). Average tumor growth curve (C) and survival curve (D) and tumor growth in single mice (E). Tumor area was compared to untreated tumor-bearing mice (PBS) and measured by caliper. 5 of 9 in the Nr2f6crispr−/−.g04 CAR-T receiving cohort (n = 9 mice) and 1 of 11 in the NTC CAR-T receiving cohort (n = 11 mice) showed complete responses. 1 of 9 in the Nr2f6crispr−/−.g04 CAR-T receiving cohort relapsed and had to be killed on day 100. PBS (n = 9 mice), pooled from two independent experiments. F–J FACS analysis of NTC and Nr2f6crispr−/− CAR+ T cells d3 after ACT in Panc02-EpCAM tumor-bearing mice. F Percentage of CAR T cells from total tumor digested cells. G gMFI of TCF-1 protein in CAR T cells in the draining lymph node. H Representative plot for TCF-1 protein expression. I, J CAR T cells from spleens were restimulated ex-vivo with Panc02-EpCAM cells and analyzed for intracellular cytokine (INFγ + GzmB) production by FACS. The experiments were done as two (C–E) biological replicates / one (F–J) biological replicate. F–J n = 5 mice per genotype. Two-way ANOVA (C), Log-Rank test (D), two-tailed unpaired Student’s t-test [F–J]. Data shown as mean ± SEM.