Fig. 1. EGFRvIII-specific CAR-T cells control newly established orthotopic tumors but fail to control large ones.

A GL261 were transduced with a retroviral vector to express the murine version of EGFRvIII. The mutated portion of EGFRvIII was fused with the transmembrane domain of the mouse EGFR to obtain cells which express the epitope on the surface. Left panel: flow cytometry staining of wild type GL261 (blue) and GL261 transduced to express EGFRvIII (red). Right panel: immunohistochemistry staining for EGFRvIII on orthotopically implanted tumors (scale bar represents 100 μm). One representative tumor is shown of four mice. B Murine CAR construct. The C-terminal portion of murine CD34 was included as marker gene and separated by a T2A peptide from the CAR construct, which included an ScFv to graft specificity, a CD8 stalk, and CD28-CD3ζ as activation domains. MR1 was used as ScFv specific for EGFRvIII, while 4g7 was used as ScFv specific for human CD19, used as negative control CAR. C “Stress experiment”. Direct comparison of the effect on tumor control of intravenous CAR-T cell administration on day 11 or 17 post tumor implantation. D Representative MRI images (axial orientation) of a mouse receiving either TBI only or TBI followed by CAR-T cells at day 11 post tumor implantation. E Survival curves (n = 3 for TBI, n = 4 for TBI+CAR mice from one experiment, p = 0.0288 (*), Log-rank test) and F tumor volume quantification. G Representative MRI images (axial plane) of a mouse receiving either TBI only or TBI followed by CAR-T cells at day 17 post tumor implantation. H Survival curves (n = 3 for TBI, n = 4 for TBI+CAR mice from one experiment) and I tumor volume quantification. Source data are provided as a Source Data file.