Figure 5.

EGFRvIII/CD3 tandem diabody (TandAb) does not facilitate activation of human T-cells in the absence of EGFRvIII-positive target cells. (A) No PBMC proliferation is induced by EGFRvIII^A6/CD3_x TandAb despite bivalent binding sites for CD3 after cultivation for 5 days and analyzed by BrdU incorporation assay. Control antibody OKT3 induces proliferation. Control cells were cultured in the absence of antibodies (w/o). Mean absorbance, and SD of triplicates, measured at 450 nm are plotted. (B) T-cell activation marker analysis by FACS: primary human T-cells were cultured for 24 h with or without 10 µg/mL of EGFRvIII^Li3G30/CD3_x TandAb in the presence or absence of F98^EGFRvIII target cells at an E:T ratio of 1:1 before T-cells were analyzed for expression of the activation markers by flow cytometry. As a control, cells were cultured in the absence of antibodies and in the absence of target cells (gray histograms). Activation markers are induced on T-cells only when both, target-positive cells and the TandAb are present.