Fig. 2: Zanidatamab binds HER2 in trans and forms large Ab:HER2 complexes in solution.
a Cartoon depicts the SPR experimental set up to distinguish between cis and trans Ab:receptor binding. The dissociation constant koff for the monospecific anti-HER2 Ab control (left) is expected to be independent of Ab surface density; the same is expected for a biparatopic Ab binding in cis. For a biparatopic Ab binding in trans, crosslinking of receptor is expected to increase with increasing Ab chip densities (right), causing koff to decrease. Reduction in koff observed with increasing zanidatamab and zanidatamab precursor surface concentrations, but not for the control trastuzumab, shows ability of zanidatamab and zanidatamab precursor to bind HER2 in trans (right, see Supplementary Table 3). Data from two independent experiments are shown. b AUC results for mixtures of HER2 ECD with anti-HER2 Abs at 1-, 2- and 5-fold HER2 excess. For reference, HER2 ECD and Abs were run separately and plotted with the HER2:Ab mixtures. For zanidatamab and tras + pert, higher order complexes were detected with increasing amounts when the excess of HER2 ECD was reduced. Tras or pert formed mainly 1:1 and 2:1 HER2:Ab complexes and only trace amounts of higher order complexes were detected. Complex compositions larger than 2:1 (HER2:Ab) are approximate. c Representative cryo-EM 2D class average, 3D reconstruction at 7.6 Å; resolution (middle) showing a core HER2 molecule bound to zanidatamab Fab and scFv, and a cartoon representation of the zanidatamab:HER2 complex observed by cryo-EM (right). The fuzzy halo observed in the 2D images is likely due to the Fc domain in multiple conformations. A model of HER2 (cyan) in complex with zanidatamab Fab (green) and scFv (blue) is also shown. The distance between the C-termini of the two antigen-binding domains cannot be spanned by a single zanidatamab molecule (see Supplementary Fig. 4). Because this structural constraint prevents zanidatamab from binding to HER2 in cis, a single zanidatamab molecule can only bind in trans and crosslink two HER2 molecules by binding to the ECD2 on one and the ECD4 on another. Source data are provided in the Source Data file.
