<i>In vitro</i> validation of VH411 phage binding.

<p>(A) Representative epifluorescence photomicrographs of CHO-hTfR-EGFP and CHO-hLDLR-EGFP cells (green) incubated with 1.10¹⁰ VH411 fd phage immunodetected with an anti-PVIII antibody diluted at 1/3000 (red). Cell nuclei are labeled with Hoechst#33258 at 0.5μg/mL(blue). Co-labeling appears in yellow/orange in the merged pictures. fd phage do not bind on cells that express the hTfR-EGFP. (B) LDLR expression and binding of phage with affinity for LDLR in the absence or presence of LDL at 0.5 mg/mL was evaluated by FACS on HUVEC for 20 min at 4°C in order to avoid endocytosis. In the present example, the cells were incubated with the anti-PVIII antibody (1/1000) detected with an allophycocyanine (APC) secondary antibody (1/800) (vertical axis) (B1, B3, B4, B5, B6) and with the anti-LDLR antibody (1/50) detected with a secondary FITC-labeled antibody (1/800) (horizontal axis) (B2, B3, B4, B5, B6). Phage VH549 (linear) and VH411 (cyclic) were added to cells in the absence (B3, B5 respectively) or presence of LDL (B4, B6 respectively). The number of positive cells was standardized with 5,000 events for each test. The results were expressed in arbitrary units of fluorescence. LDL strongly decreases the binding of VH549 but has no impact on VH411 binding. (C) Quantification of the fluorescence signal in zone Q2 of the graphs in B. No shift is measured in Q2 for the VH411 phage in the presence of LDL while a 56% reduction in Q2 signal is measured for VH549 phage with LDL indicating competition for the LDL binding site. Statistical analysis was performed using an analysis of variance, followed by Student’s test. Values represent the mean of 3 independent experiments; ***p ≤ 0.001.