c1F6 mediates ADCC, ADCP, and CDC activity. (A) Specific lysis of c1F6-treated (closed symbols) or nonbinding control IgG1–treated (open symbols) target cells was determined by ⁵¹Cr release assay. Target cells were mixed with NK-enriched PBMCs at an effector-target ratio of 10:1. (B) Effector cells were untreated (solid bars) or pretreated with saturating amounts of anti-CD16 blocking antibody (hatched bars) prior to incubation with c1F6-coated target cells. Open bars represent target cells that were mixed with nonbinding control IgG1. (C) Representative flow cytometry analysis and fluorescence microscopy of c1F6-mediated phagocytosis. For flow cytometry, 786-O target cells were labeled with PKH26 lipophilic dye, treated with c1F6 or nonbinding control IgG, then mixed with monocyte-derived macrophages (Mφ). Mφ were stained with AF488-conjugated anti-CD11b. Cells present in the top-right quadrant (PKH26⁺CD11b⁺) are Mφ that internalized tumor cells. For microscopy, tumor cells were labeled with PKH67 (green), and the macrophages were detected with Alexa Fluor 568–conjugated antibody specific for CD11b (red). Images were taken with a Leitz Orthoplan Research microscope (100×/1.32 NA objective lens) using a Nikon Coolpix 990 camera (Nikon, Tokyo, Japan), then processed with Adobe Photoshop Elements version 2.0 (Adobe Systems, San Jose, CA). (D) Tumor targets were treated with varying concentrations of c1F6 (solid symbols) or 2 μg/mL IgG1 (open symbols) prior to incubation with Mφ. Percent Mφ that engulfed tumor cells was determined by flow cytometry as in panel C. Internalization of tumor cells by Mφ was confirmed by fluorescence microscopy (not shown). (E) c1F6-treated (solid symbols) or control IgG–treated (open symbols) target cells were incubated at 37°C in 10% human serum. The percentage of nonviable cells was identified by flow cytometry after staining with propidium iodide. (A-B,E) Bars represent SD of triplicate samples.