AbstractOverexpression of the receptor tyrosine kinase HER2 plays a critical role in the development of various tumors. Biparatopic designed ankyrin repeat proteins (bipDARPins) potently induce apoptosis in HER2-addicted breast cancer cell lines. Here, we have investigated how the spatiotemporal receptor organization at the cell surface is modulated by these agents and is distinguished from other molecules, which do not elicit apoptosis. Binding of conventional antibodies is accompanied by moderate reduction of receptor mobility, in agreement with HER2 being dimerized by the bivalent IgG. In contrast, the most potent apoptosis-inducing bipDARPins lead to a dramatic arrest of HER2. Dual-color single-molecule tracking revealed that the HER2 “lockdown” by these bipDARPins is caused by the formation of HER2-DARPin oligomer chains, which are trapped in nanoscopic membrane domains. Our findings establish that efficient neutralization of receptor tyrosine kinase signaling can be achieved through intermolecular bipDARPin crosslinking alone, resulting in inactivated, locked-down bipDARPin-HER2 complexes.