Identification and characterization of a novel Sso7d scaffold-based binder against Notch1

Notch signaling has important functions in regulating cell growth and development, misregulation of which has been implicated in various cancers. Monoclonal antibodies (mAbs) targeting Notch protein activity have already moved into clinical trials. However due to the limitations associated with cost and productivity of mAbs, there has been a surge in the development of complementary approaches that are based on non-antibody scaffolds. Non-antibody scaffolds are small proteins that are stable and can be engineered to develop high-affinity binders against specific targets of interest. Here we describe the isolation and characterization of a novel Notch1-binding protein, N9, obtained by screening of a combinatorial library based on the ultra-stable Sso7d scaffold. N9 targets the extracellular EGF-like repeats (ELR) 11-13 in Notch1, and therefore serves as a competitive inhibitor for Notch ligands to decrease expression of Notch target genes. We demonstrate that N9 recognizes surface expression of Notch1 on the plasma membrane and binds preferentially to cell lines misexpressing Notch1. Although N9 was selected against Notch1, we also observe cross-reactivity against other Notch receptors, including Notch2/3. Finally, we demonstrate that N9 inhibits proliferation and generation of tumorspheres in Notch expressing cancer cell lines, suggesting its potential as a therapeutic agent in Notch-associated malignancies.