FIG. 3.

Equivalent function of second- and third-generation anti-EGFRvIII CAR vectors. Using anti-EGFRvIII human mAb 139, a third-generation CAR vector was assembled using CD28, 4-1BB, and CD3ζ T-cell signaling domains (139-28BBZ, diagram at top of figure). This vector was compared with the original vector design containing CD28 and CD3ζ T-cell signaling elements (139-28Z, diagram at top of figure). Primary human T cells were transduced with both vectors and evaluated for IFN-γ cytokine production following overnight coculture (imbedded tables) and in 4-hr ⁵¹Cr release assays to determine cell lysis activity (shown as percent lysis at the indicated E:T). Target cells included: U87 cells transduced with a GFP vector (GFP), wild-type EGFR (EGFRwt), or EGFR variant III (EGFRvIII); and U251 cells transduced with wild-type EGFR (EGFRwt) or EGFR variant III (EGFRvIII). A vector expressing an anti-ERBB2 CAR (ERBB2) was used as a control along with GFP and untransduced (UnTd) cells. Data are representative of three independent experiments. IFN-γ values were determined by ELISA (shown in pg/mL, mean of triplicate determinations), and percent lysis was plotted as the mean of quadruplicate determinations. Differences between recognition of EGFRvIII-transduced versus control lines were statistically significant (p<0.05, Student's t test).