FIGURE 4.

Rate-limiting toxicity step measured in rGel is similarly observed in scFv- and Fn3-targeted immunotoxins using antigen-dependent internalization. A, the internalization of immunotoxins by antigen-positive cells was measured by the quantitative internalization flow cytometry assay at varying times and concentrations. All HT-29 cells treatments were made at 30 nm, as were the C7rGel and E4rGel treatments on HT-1080(CEA) and A431 cells, respectively, whereas the 3ErGel and FErGel treatments on HT-1080(CEA) cells were made at 10 nm. For all treatments, strictly antigen-dependent internalization is reported by subtracting signal from cells blocked with an unlabeled competitor. B, concentration- and exposure-matched cytotoxicity was measured using the WST-1 assay. C, data from A and B were combined and plotted to show the dependence of cytotoxicity on the number of internal immunotoxins, resulting in the determination of the TN₅₀ near 3 × 10⁶ for all species.