The glycosylation profile of 2F8 does not influence antigen binding and Fab-mediated inhibition of EGF-R signaling.(A) Binding of 2F8-C (●) and 2F8-H (○), or a human IgG1 control antibody (Δ), to EGF-R on A431 cells was analyzed by flow cytometry. A431 cells were incubated with various concentrations (0.016-100 μg/mL) of 2F8 and stained with polyclonal fluorescein isothiocyanate-conjugated rabbit anti–human IgG serum. Each data point represents the mean plus or minus SEM of triplicates (error bars are smaller than symbols and therefore not visible in the graph). The capacity of 2F8-C (●) and 2F8-H (○) to inhibit tumor cell growth (B) or EGF-induced EGF-R phosphorylation (C) was evaluated. (B) A431 cells were seeded in the presence of various concentrations of antibody (0.002-30 μg/mL). After 5 days, vital cell mass was analyzed by measuring fluorescence of reduced AlamarBlue. Each data point represents the mean of duplicate wells. (C) A431 cells were seeded in the presence of various concentrations of antibodies (0.04-2.5 μg/mL) and subsequently stimulated with 50 ng/mL EGF. EGF-R phosphorylation was measured in cell lysates of treated cells using a phospho-EGF-R–specific ELISA and time-resolved fluorescence (TRF). Each data point represents the mean plus or minus SEM of triplicates (error bars are smaller than symbols). Every experiment was performed at least 2 times.
