Intrinsic selectivity of Notch 1 for Delta-like 4 over Delta-like 1.

Notch signaling makes critical contributions to cell fate determination in all metazoan organisms, yet remarkably little is known about the binding affinity of the four mammalian Notch receptors for their three Delta-like and two Jagged family ligands. Here, we utilized signaling assays and biochemical studies of purified recombinant ligand and receptor molecules to investigate the differences in signaling behavior and intrinsic affinity between Notch1-Dll1 and Notch1-Dll4 complexes. Systematic deletion mutagenesis of the human Notch1 ectodomain revealed that epidermal growth factor (EGF) repeats 6-15 are sufficient to maintain signaling in a reporter assay at levels comparable with the full-length receptor, and identified important contributions from EGF repeats 8-10 in conveying an activating signal in response to either Dll1 or Dll4. Truncation studies of the Dll1 and Dll4 ectodomains showed that the MNNL-EGF3 region was both necessary and sufficient for full activation. Plate-based and cell binding assays revealed a specific, calcium-dependent interaction between cell-surface and recombinant Notch receptors and ligand molecules. Finally, direct measurement of the binding affinity of Notch1 EGF repeats 6-15 for Dll1 and Dll4 revealed that Dll4 binds with at least an order of magnitude higher affinity than Dll1. Together, these studies give new insights into the features of ligand recognition by Notch1, and highlight how intrinsic differences in the biochemical behavior of receptor-ligand complexes can influence receptor-mediated responses of developmental signaling pathways.

Characterization of Notch1 antibodies that inhibit signaling of both normal and mutated Notch1 receptors.

BACKGROUND: Notch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signaling is associated with many different types of cancer as well as tumor angiogenesis, making Notch1 a potential therapeutic target. PRINCIPAL FINDINGS: Here we report the in vitro activities of inhibitory Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Two classes of antibodies were found, one directed against the EGF-repeat region that encompasses the ligand-binding domain (LBD), and the second directed against the activation switch of the receptor, the Notch negative regulatory region (NRR). The antibodies are selective for Notch1, inhibiting Jag2-dependent signaling by Notch1 but not by Notch 2 and 3 in reporter gene assays, with EC(50) values as low as 5+/-3 nM and 0.13+/-0.09 nM for the LBD and NRR antibodies, respectively, and fail to recognize Notch4. While more potent, NRR antibodies are incomplete antagonists of Notch1 signaling. The antagonistic activity of LBD, but not NRR, antibodies is strongly dependent on the activating ligand. Both LBD and NRR antibodies bind to Notch1 on human tumor cell lines and inhibit the expression of sentinel Notch target genes, including HES1, HES5, and DTX1. NRR antibodies also strongly inhibit ligand-independent signaling in heterologous cells transiently expressing Notch1 receptors with diverse NRR "class I" point mutations, the most common type of mutation found in human T-cell acute lymphoblastic leukemia (T-ALL). In contrast, NRR antibodies failed to antagonize Notch1 receptors bearing rare "class II" or "class III" mutations, in which amino acid insertions generate a duplicated or constitutively sensitive metalloprotease cleavage site. Signaling in T-ALL cell lines bearing class I mutations is partially refractory to inhibitory antibodies as compared to cell-penetrating gamma-secretase inhibitors. CONCLUSIONS/SIGNIFICANCE: Antibodies that compete with Notch1 ligand binding or that bind to the negative regulatory region can act as potent inhibitors of Notch1 signaling. These antibodies may have clinical utility for conditions in which inhibition of signaling by wild-type Notch1 is desired, but are likely to be of limited value for treatment of T-ALLs associated with aberrant Notch1 activation.