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

Fig. 1. Antibody-derived biparatopic binders achieve high antiproliferative activity, induce apoptosis and partial lockdown of HER2.

Fig. 1

a Structural design of four closely related scFv–Fab fusions (641, 641i, 841, and 841i). They follow the blueprint of the biparatopic 6L1G, by targeting domains 1 and 4 of HER2 with either the scFv or Fab fragment. These are derived from TZB (domain 4 binder) and A21 (domain-1 binder), with the color codes indicated. b Proliferation inhibition (XTT assay) of HER2-addicted cancer cell lines BT474 (top row) and HCC1419 (bottom row (n = 3 biological replicates, mean plus error bar SD) upon treatment with the scFv–Fab variants from (a) was similarly strong but not equal to the DARPin–DARPin fusion 6L1G. The more active variants 641 and 841 surpassed the clinically used mAb combination TZB + PZB. All data are from the same experiment, but distributed over two plots to avoid overcrowding; 6L1G is shown again as a dotted line as reference. c 641 and 841 treatment (50 nM) of BT474 cells led to marked decrease of phosphorylation of key players in the HER2-dependent signaling pathways and induction of apoptosis, as seen by PARP cleavage already after 24 h in immunoblots. d Mobility reduction of surface HER2 analyzed with FRAP on cells (n = 5 cells, error bar SEM) was strong, while an incomplete “lockdown” was seen upon treatment with apoptotic agents 641 and 841 compared to DARPin 6L1G (dotted line as reference) and mAb co-treatment (TZB + PZB) (all agents 50 nM); untr. Untreated. Source data are available in the Source Data file.