Fig. 1.

Outline of screening strategy and data from the first step of the screening, i.e. phage binding to DU145 cells. A, Schematic of HCA screening to identify macropinocytosis-dependent antibodies. HCA instruments allow automated high throughput detection of antibody colocalization with a macropinocytosis marker. The starting materials for the screening are sublibraries generated previously by us from LCM-based phage antibody library selection (1) that are enriched for internalizing phage antibodies binding to tumor cells in situ. B, DU145 cells were incubated in 96-well plates with phage-containing supernatants for 24 h at 37 °C in complete DMEM and10% FBS. Nuclei were stained with Hoechst 33342. Bound phages were immunolabeled with anti-fd antibodies (green). Zoomed insert portrays software-based, automated cell analysis, measuring mean fluorescence intensities (MFI) of immunolabeled phages. Over 300 cells were quantified for each phage clone. C, Plot of MFI values of immunolabeled phage binding to cell for 1439 phage clones. Red horizontal line represents MFI of ∼250,000, the threshold for prioritizing clones for further internalization analysis.