Apoptosis-inducing anti-HER2 agents operate through oligomerization-induced receptor immobilization

ARTICLE https://doi.org/10.1038/s42003-021-02253-4 OPEN Apoptosis-inducing anti-HER2 agents operate through oligomerization-induced receptor immobilization 1234567890():,; Jakob C. Stüber 1,3,5, Christian P. Richter2,5, Junel Sotolongo Bellón2, Martin Schwill Benjamin Schuler 1, Jacob Piehler 2 ✉ & Andreas Plückthun 1 ✉ 1, Iwo König 1,4, Overexpression 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. 1 Department of Biochemistry, University of Zurich, Zurich, Switzerland. 2 Department of Biology/Chemistry and Center for Cellular Nanoanalytics, Osnabrück University, Osnabrück, Germany. 3Present address: Roche Pharma Research & Early Development, Large Molecule Research, Roche Innovation Center Munich, Penzberg, Germany. 4Present address: Roche Diagnostics Int. AG, Rotkreuz, Switzerland. 5These authors contributed equally: Jakob C. Stüber, Christian P. Richter. ✉email: ; COMMUNICATIONS BIOLOGY | (2021)4:762 | https://doi.org/10.1038/s42003-021-02253-4 | www.nature.com/commsbio 1 ARTICLE H COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-021-02253-4 uman epidermal growth factor receptor 2 (HER2/ErbB2/ neu) has gained tremendous importance as a biomarker in diagnostics and a target in the therapy of breast cancers, but also in gastric and gastroesophageal cancers, over the past decade1. This receptor tyrosine kinase has a distinct role within the ErbB family of receptors, in that it has no ligand. Deduced from the rigidity seen in all crystal structures, in molecular dynamics, and binding experiments, HER2 has therefore been assumed to always be in a dimerization-competent state2. Under physiological conditions, HER2 likely does not homodimerize, and thus its role is restricted to that of a signalamplifying co-receptor for other, ligand-activated members of its family3. Upon malignant overexpression, however, spontaneous formation of homodimers and heterodimers, even in the absence of ligands of the other ErbB family member, enables sustained proliferation and survival signaling4. This stimulus, in turn, frequently transforms the cancer cell signaling architecture towards singular dependence on HER2 signaling5. Such HER2-addicted cancers are more aggressive but, at the same time, also susceptible to targeted therapies. Therefore, HER2 overexpression, previously merely a correlate of bad prognosis, nowadays generally also implies the availability of several targeted treatment options. Among these, monoclonal antibodies (mAbs) have been successfully developed that bind to the HER2 extracellular domain and thus block activity. Prominent examples that have been approved for therapy are the humanized mouse mAbs trastuzumab (TZB) and pertuzumab (PZB)1. Interestingly, the mechanisms of HER2 inhibition by TZB and PZB are complementary (targeting complexes with the non-liganded and liganded states of HER3, respectively), and therefore most efficacious therapies employ the combination of both mAbs6,7. Of note, subtle changes in the relative orientation of the Fab domains can transform F (ab’)2-like molecules derived from TZB into active proproliferative agents, underlining the importance of binding geometry for the modulation of HER2 activity by affinity reagents8. Recently, we have reported a novel strategy based on designed ankyrin repeat proteins (DARPins)9 to inhibit HER2 activity in breast cancer cells10,11. To this end, we engineered biparatopic antitumor DARPins (bipDARPins) consisting of two binding moieties, which recognize the extracellular subdomains I and IV of HER2, respectively (Fig. 1a). These binding moieties are connected by a short linker (see Supplementary Fig. 1 for a detailed overview), with the aim to trap and stabilize an inactive conformation11. Compared to TZB and PZB, bipDARPins much more efficaciously promote apoptosis in HER2-dependent tumor cell lines10 by dephosphorylating HER2 (and not only HER3) and subsequently preventing re-activation of phosphoinositide-3 kinase through RAS10. While the signaling pathways underlying this effect have been characterized in detail10, the exact mechanism responsible for the potent inhibitory function of bipDARPins, in contrast to less active molecules, has so far remained largely unclear. Less active molecules not only include the antibodies TZB and PZB but also the same DARPin units with longer linker in between or in different orientation10,11. X-ray structures of single DARPin moieties in complex with the isolated, soluble extracellular subdomains of HER211 led to the conclusion that the most active bipDARPins cannot bind both epitopes in an intramolecular fashion, implying that rather two neighboring HER2 molecules are engaged (Fig. 1c, d). In contrast, intramolecular trapping would be possible for the less active molecules with longer linkers. Possible mechanisms of action considered had initially included locking an inactive HER2 conformation similar to the tethered conformation seen in the other ErbB receptors, but we have shown subsequently that the extracellular domain is very rigid, making such a conformation highly unlikely2. This suggests that inhibition is instead achieved via restrained dimerization (Fig.1c, d), in which interactions between the cytosolic tyrosine kinase domains required for allosteric activation are prevented12. However, this binding mode also may allow elongation into daisy chain-like arrangements (Fig.1d), which we have previously proposed to cause efficient inhibition of HER2 signaling11. To delineate the mechanism responsible for HER2 inhibition by bipDARPins, we explored here their effects on the spatiotemporal organization and dynamics of HER2 in the plasma membrane. Chemical crosslinking in combination with immunoprecipitation revealed oligomerization induced by active bipDARPins, but not by TZB and PZB. Making use of cell surface-specific posttranslational labeling techniques, we quantified the diffusion properties of HER2 in the plasma membr (...truncated)