Figure 3

A. Internalization of unpurified, bacterially produced antibodies into SKBR3 cells. SKBR3 cells were co-incubated with supernatants of E. coli expressing anti-EGFR-scFv-C10-(His)6, the non-internalizing anti-ErbB2-scFv-C10-(His)6, anti-ErbB2-scFv-F5-(His)6, or no scFv along with fluorescently labeled Ni-NTAliposomes (500 μM, Ni-NTA = 2% of phospholipid content). The fluorescence represents the amount of internalized antibody. B. Tumor cell profiling of EGFR with anti-EGFR-scFv-C10-(His)6 and Ni-NTAliposomes. Anti-EGFR-scFv-C10-(His)6 was co-incubated with fluorescently labeled Ni-NTA-liposomes and cell lines expressing varying amounts of EGFR: SKBR3, SKOV3, BT474, MCF7, MD-MBA 453, MD-MDA 468, CHO-EGFR, or CHO. The fluorescence represents the uptake of labeled liposomes. The uptake of fluorescently labeled liposomes was normalized to total cellular protein. C. Comparison of ErbB2 expression levels and uptake of anti-EGFR-scFv-F5-(His)6-conjugated immunoliposomes or anti-EGFR-scFv-F5-(His)6 chelated liposomes (anti-EGFR-scFv-F5-(His)6(+Ni-NTA-liposomes)). ErbB2 expression levels on cells were determined using fluorescently labeled anti-EGFR-scFv-F5-(His)6 and flow cytometry (top panel). Alternatively, live tumor cells were incubated with anti-ErbB2-scFv-F5-(His)6 covalently coupled to fluorescently labeled liposomes (anti-ErbB2-scFv-F5-(His)6-conjugated liposomes) (middle panel). The CLIA assay was performed by co-incubation of anti-ErbB2-scFv-F5-(His)6 and fluorescently labeled Ni-NTA liposomes (bottom panel). Liposome fluorescence was read in a microfluorimeter. The fluorescence scale in all 3 panels is indicated as % of SKBR3 signal which was set to 100 %.