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. 2021 Jun 18;12:3790. doi: 10.1038/s41467-021-23948-6

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a Layout of tetravalent biparatopic constructs in the scFv–IgG format, based on scFv–Fab fusions 841 and 641. Construct 241 lacked effect compared to 441 (see Supplementary Fig. 3b) b FRAP of 441 (50 mM) treated cells (n = 5 cells, error bar SEM) showed complete HER2 lockdown; untr untreated. c The effect of 441 on HER2 was further studied with single-molecule tracking experiments. Labeled Hela cells overexpressing a SNAP-tag HER2 fusion were analyzed with a TIRF-SMLM setup at room temperature. d The immobile fraction of HER2 was calculated to be 60% when analyzing single-molecule localizations of HER2 with the adapted DBSCAN algorithm. Two-sided unpaired t test with Welch’s correction demonstrated a significant difference from untreated cells (PBS only). e The additional antiproliferative activity of 441 over its parental antibody combination is seen across different cell lines and tissues (mean of n = 3), with relevant mutations indicated. The inhibitory effect of TZB, hA21G, and their combination on proliferation was only strong on certain HER2-dependent cell lines. 441, however, showed stronger reduction of viability throughout and thus a more universal response, hence indicating a benefit over the combination treatment. Source data are available in the Source Data file.