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. 2023 Feb 28;14:947. doi: 10.1038/s41467-023-36496-y

Fig. 1. Endocytosis of HER2-specific biparatopic antibody BS4 is not dependent on clathrin and dynamin but requires F-actin.

Fig. 1

a Schematic representation of anti-HER2 biparatopic antibody BS4. The Trastuzumab (Tz) single-chain variable fragment (scFv) is attached to the N terminus of the heavy chain of 39 S IgG1 resulting in four antigen binding sites per molecule. Mutations in the Fc region (*) reduce binding to Fc gamma receptor. b, c Surface binding and endocytosis of dylight650-labelled monotopic antibodies Tz and 39 S and biparatopic BS4 (all at 3 µg/ml) after 1 h of uptake by confocal microscopy (b) and over a period of 6 h by flow cytometry (c), (means ± SD, n = 3 independent experiments). d, e Endocytosis of BS4 is independent of clathrin and dynamin. SkBr3 cells transfected with dominant-negative AP180ct (for clathrin-mediated endocytosis) and DynaminS45N N-terminally GFP-tagged expression constructs were incubated with dylight650-labelled BS4 and AlexaFluor546-transferrin for indicated times over a period of 4 h and analysed by flow cytometry. Endocytosis of transferrin and BS4 in cells from the same well expressing or not dominant-negative constructs (AP180ct+ AP180ct-, DynS45N+, DynS45N− in (d) and (e)), (means ± SD, n = 4 independent experiments). f BS4 uptake requires actin polymerisation. SkBr3 cells endocytosing pHrodo-labelled BS4 were treated with 10 µM CytoD, which was subsequently washed out as indicated, (means ± SEM, n = 6 from three independent experiments, two replicate wells each). Scale bars: 10 µm. Source data are provided as a Source Data file.