Fig. 8

Transwell-based extravasation assay predicts in vivo extravasation behavior. a Transwell experiment design modeling tumor extravasation. b Control experiments with fluorescently labeled dextrans with the hydrodynamic radius of an IgG show enhanced transport in the presence of TNF-α, suggesting passive paracellular diffusion as an important transport route. c As expected, transendothelial electrical resistance (TEER) is reduced by TNF-α, but is not significantly affected by the presence of antibodies. Additional charge-patch variants (AbNeg-2, AbPos-2), engineered in the same manner as AbNeg, AbBal, and AbPos, were included as additional controls. d In agreement with the behavior observed in vivo, transendothelial transport in presence of TNF-α trends (Spearman’s ρ=0.6263; p=0.0712 (two-tailed)) with the presence of positive charge patches. Black symbols represent additional charge-patch variants, engineered in the same manner as AbNeg, AbBal, and AbPos. e Transport across the endothelial barrier seems to be independent of FcRn binding, since it is neither changed by FcRn nonbinding (AAA) nor binding-enhanced (YTE) mutations. AbPos-3 wild-type data shown here are also included in panel d. Horizontal lines indicate the arithmetic mean of duplicates and error bars the standard deviation