Figure 4.

Characterization Qualification of DC internalization assay. (A) The internalization rate represents the internalization efficiency as calculated by the slope of the mean fluorescence intensity (MFI) values of antibodies coupled to a pH sensitive fluorophore into the acidic lysosome of CD11c+ moDCs from 4 human healthy blood donors (color coded) at 120 min, normalized to the fluorescence of the antibody dosing solution to account for differences in labeling efficiency between antibodies. (B) The relative internalization rate uses the slope of an internal control antibody to normalize the donor specific internalization rate (according to the Material and Methods section). A one-sided paired t-test was applied for the comparison of antibodies sharing the same target (displayed at the top, p< 0.01**; p< 0.05*). A one sample two-sided paired T-test between each group (antibody) and the internal control antibody has been performed (corrected for multiple testing, displayed at the bottom, p< 0.01**; p< 0.05*). (C) Comparison of the contribution of donor, compound and residual error on the total variance for the non-normalized (A) vs normalized internalization data (B). (D) Correlation plot between the normalized MAPPs score (see Material and Methods for the equation used and Supplementary Figure 3 for the heatmap of the detected peptide clusters) and the normalized and relative (to var1) DC internalization rate. Normalization for each treatment has been achieved by subtracting the corresponding assay dataset mean and dividing it by the corresponding standard deviation.