Selected EGFRt⁺ CD19CAR⁺ T cells can be expanded with maintenance of effector phenotype. (A) Schematic of the immunomagnetic huEGFRt selection procedure. (B) After 1 rapid expansion cycle, nonselected (Unslxd) and huEGFRt-selected (Slxd) T cells were phenotyped for surface EGFR (ie, EGFRt, with biotinylated cetuximab), Fc (ie, CAR), and effector T-cell markers TCRαβ, CD3, CD4, CD8, CD28, or intracellular granzyme A (black histogram) versus isotype control Ab (gray histogram) by flow cytometry. Percentage of positive staining is indicated in each histogram. (C) Fold expansion of nontransduced (EGFRt-) and transduced (EGFRt+) T cells in unselected cultures of 4 different transduced cell lines was determined after OKT3-mediated stimulation. (D) huEGFRt expression of selected T cells was determined by flow cytometry after each of 3 rounds of OKT3-mediated stimulation (Stim 1, 2, and 3). Percentage of cetuximab-mediated staining (black) compared with that of SA-PE alone (gray) is indicated in each histogram. (E) Nonselected (Unslxd) and huEGFRt-selected (Slxd) T cells were incubated for 4 hours with ⁵¹Cr-labled CD19⁺ LCL or SupB15 cells, or OKT3-epressing LCL cells as targets at the indicated effector to target ratios. Percentage cytotoxicity (mean ± SE) of triplicate wells is depicted. (F) Nonselected (Unslxd) and EGFRt-selected (Slxd) T cells were incubated with CD19⁺ LCL or SupB15 cells, or OKT3-epressing LCL cells and supernatants were analyzed by Bioplex. IFN-γ and TNF-α levels (mean ± SE) are shown.