(A) Schematic of 2nd generation EGFR806-CAR extracellular domain variants. Spacer variants: Short (S) – IgG4-hinge; Medium (M) – IgG4-hinge-CH3; Long (L) – IgG4-hinge-CH2-CH3. (B) EGFRt expression by flow cytometry showing CAR expression in CD8+ TCM transduced with short, medium or long spacer 2nd generation EGFR806-CAR lentivirus and selected for EGFRt expression. Mock transduced cells were unselected. Representative data for one donor are shown, and all donors (N = 3) yielded >90% EGFRt positive T cells after the selection step. (C) Protein-L was used to label the scFv portion of the CAR, demonstrating surface expression. Representative data from one donor. (D) EGFR806-CAR T cells kill EGFRvIII-expressing Raji (Raji-vIII) cells but not untransduced Raji cells in a 4-hour chromium release assay. The x-axis shows the ratio of effector: target cells. (E) An EGFR aa. 287-302 peptide, which encompasses the mAb binding epitope, inhibits short-spacer EGFR806-CAR T cell lysis of Raji-vIII cells. The x-axis shows the ratio of effector: target cells. (F) Cytokine levels in supernatants obtained from 24hr co-cultures of EGFR806-CAR T cells expressing extracellular spacer variants and Raji-vIII cells at a 2:1 ratio. Cytokine data from three independent experiments was analyzed by Student t test. Error bars represent SEM. All panels: S, short spacer; M, medium spacer; L, long spacer. **
P < 0.01; ***
P < 0.01; ****
P.001.