(C) Internalized anti-HER2-Bs-AF647 (red) remained colocalized with HER2 receptors (green) and appeared as yellow puncta. (C,F) Anti-HER2-Bs-AF647 and trastuzumab-AF647 mediated strikingly different trafficking of HER2 receptors at 2 h. Consistent with the previous figures, the internalization of anti-HER2-Bs-AF647 is more rapid compared to trastuzumab-AF647 (compare D to G). (B,E) At 30 min, the antibody-HER2 complexes (appearing as yellow puncta) were internalized into the cells. (A,D) Before the initiation of internalization, both anti-HER2-Bs-AF647 and trastuzumab-AF647 (red) were colocalized with HER2 receptors on the cell surface (green) and appeared yellow. Internalization time courses of anti-HER2-Bs-AF647 (A-C) and trastuzumab-AF647 (D-F) are shown. Scale bar is 10 µm.Īnti-HER2-Bs and trastuzumab mediated distinct intracellular trafficking of internalized HER2 receptors. At 2 h, the internalization of both anti-HER2-Bs-AF647 (red) and trastuzumab-AF647 (red) was inhibited in the presence of Dyngo 4a (compare B, E to A, D for anti-HER2-Bs compare H, K to G, J for trastuzumab) or Pitstop 2 (compare C, F to A, D for anti-HER2-Bs compare I, L to G, J for trastuzumab). At T = 0, anti-HER2-Bs-AF647 (A,B,C) or trastuzumab-AF647 (G,H,I) was bound on the surface of the treated cells. Overlays of anti-HER2-Bs-AF647 (A-F) and trastuzumab-AF647 (G-L) with the cytoplasm dye (green) in BT-474 cells treated with either DMSO (A,D,G,J), dynamin inhibitor Dyngo 4a (B,E,H,K), or clathrin light chain inhibitor Pitstop 2 (C,F,I,L) are shown. Effect of dynamin and clathrin inhibitors on anti-HER2-Bs and trastuzumab internalization. Collectively, these results enable a better understanding of the mechanism of action of anti-Her2-Bs and can guide the rational design of anti-HER2 therapeutics as well as other bispecific molecules.Įffect of dynamin and clathrin inhibitors on anti-HER2-Bs and trastuzumab internalization. Consistent with routing HER2 to degradation, anti-HER2-Bs significantly reduced HER2 shedding and altered its exosomal export. Trastuzumab dissociated from HER2 in 2 h, enabling the receptor to recycle, whereas anti-HER2-Bs stayed associated with the receptor throughout the entire endocytic pathway, promoting receptor ubiquitination, trafficking to the lysosomes, and efficient degradation. Cellular uptake of anti-HER2-Bs and parental arm antibodies occurred via clathrin-dependent endocytosis however, inside the cells antibodies directed different trafficking pathways. The results demonstrated that concurrent engagement of both epitopes was necessary for rapid anti-HER2-Bs internalization. Using quantitative fluorescent imaging, we compared the internalization kinetics of anti-HER2-Bs and its parental arm antibodies, alone or in combinations and under various internalization-promoting conditions. In this study, we investigated the molecular mechanism of this antibody-induced rapid HER2 internalization and intracellular trafficking. A bispecific tetravalent anti-HER2 antibody (anti-HER2-Bs), targeting two non-overlapping epitopes on HER2 in domain IV (trastuzumab) and domain II (39S), has been reported to induce rapid internalization and efficient degradation of HER2 receptors. Amplification and overexpression of HER2 (human epidermal growth factor receptor 2), an ErbB2 receptor tyrosine kinase, have been implicated in human cancer and metastasis.
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