Fig. 3: Zanidatamab binding induces HER2 capping and the formation of large HER2 clusters on the cell surface. | Nature Communications

Fig. 3: Zanidatamab binding induces HER2 capping and the formation of large HER2 clusters on the cell surface.

From: An anti-HER2 biparatopic antibody that induces unique HER2 clustering and complement-dependent cytotoxicity

Fig. 3: Zanidatamab binding induces HER2 capping and the formation of large HER2 clusters on the cell surface.

a Illustration showing zanidatamab bound in trans to ECD2 and ECD4 of two HER2 molecules along with the anti-HER2 ECD1-AF647 used to detect HER2 localization following Ab treatment. HER2 extracellular domains ECD1-4 and tyrosine kinase (TK) domains are labeled. b Graphical representation of cell surface HER2 caps vis-a-vis microclusters and representative 3D reconstructed confocal microscopy images. c, d SK-BR-3 cells were treated with 200 nM anti-HER2 or negative control (NC) Ab palivizumab for the indicated times. Cells where then imaged by confocal microscopy following HER2 detection with 74 nM anti-HER2 ECD1-AF647 OAA (fluor to Ab ratio of 8.9). Percent of cells exhibiting microclusters or caps is shown as the mean ± SEM for three independent experiments (c). Representative confocal images are shown from 30-43 images (185-351 cells) per Ab treatment condition from three independent experiments, scale bar = 10 μm (d). (e) SK-BR-3 cells were treated with 200 nM anti-HER2 Abs or control Ab for 15 min and imaged by dSTORM super-resolution microscopy. Upper row shows HER2 dSTORM localizations (detected with 148 nM anti-HER2 ECD1-AF647 OAA) in representative ROIs. Color bars = localization density. Lower row shows HER2 clusters identified by the StormGraph clustering algorithm and color-coded by cluster areas (nm2). Scale bar = 500 nm. For each Ab treatment condition, 55-124 images (ROI) from multiple cells were imaged. f The number of localizations per cluster is graphed versus cluster area and the best-fit linear regression is shown. g Percent of clusters with > 1500 localizations. Numbers above the bars are fold-difference versus NC. h The mean cluster area was determined for each ROI. The violin plot shows the distributions of mean cluster areas for each Ab treatment. Pairwise comparisons were performed using a two-sided non-paired t-test on a log10 scale and p values were corrected using the Bonferroni method. Black brackets indicate comparison and p value (zani vs. tras df = 133, p = 0.005; zani vs. pert df = 135, zani vs. tras + pert (1:1) df = 147, and zani vs. NC df = 116, p < 0.001). Source data are provided in the Source Data file.

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