Notch信号通路是治疗炎性疾病和癌症的重要治疗靶点。我们之前创建了特异性配体抑制剂，即Notch诱饵（Notch decoys），它由具有一定特异性的Notch1细胞外领域EGF样重复序列的Fc融合构建而成。这种诱饵能够结合配体，阻止Notch信号通路，并显示出低毒性的抗肿瘤活性。然而，这些诱饵的研究依赖于病毒介导的表达，阻碍了剂量控制，并限制了其临床应用性。我们改进了诱饵的设计，创建了基于peptibody的Notch抑制剂，其中包括Notch1或Notch4的核心结合域，即EGF类似重复10-14。这些Notch抑制剂(peptibodies)显示出高分泌性和产量，相比之前的Notch诱饵，其产量提高了近100倍。使用表面等离子体共振光谱技术结合共免疫沉淀实验，我们观察到Notch1和Notch4 peptibody对Notch配体DLL4和JAG1表现出强烈但不同的结合性质。两种peptibodies均干扰内皮细胞中的Notch信号，并在治疗后降低了典型的Notch靶标表达。虽然之前的DLL4抑制剂会导致过度生长，但Notch1 peptibody在三维离体生长试验中减少了血管生成。向新生小鼠注射Notch1 peptibody后，视网膜血管的放射状生长减少，证实了其抗血管生成性能。我们得出结论，纯化的Notch peptibodies包括EGF样重复序列10-14能够结合DLL4和JAG1配体，并表现出抗血管生成性能。基于其分泌特性、独特的Notch抑制活性和抗血管生成性能，Notch peptibodies为治疗Notch抑制提供了新的机会。 © 2022.作者。

The Notch signaling pathway is an important therapeutic target for the treatment of inflammatory diseases and cancer. We previously created ligand-specific inhibitors of Notch signaling comprised of Fc fusions to specific EGF-like repeats of the Notch1 extracellular domain, called Notch decoys, which bound ligands, blocked Notch signaling, and showed anti-tumor activity with low toxicity. However, the study of their function depended on virally mediated expression, which precluded dosage control and limited clinical applicability. We have refined the decoy design to create peptibody-based Notch inhibitors comprising the core binding domains, EGF-like repeats 10-14, of either Notch1 or Notch4. These Notch peptibodies showed high secretion properties and production yields that were improved by nearly 100-fold compared to previous Notch decoys. Using surface plasmon resonance spectroscopy coupled with co-immunoprecipitation assays, we observed that Notch1 and Notch4 peptibodies demonstrate strong but distinct binding properties to Notch ligands DLL4 and JAG1. Both Notch1 and Notch4 peptibodies interfere with Notch signaling in endothelial cells and reduce expression of canonical Notch targets after treatment. While prior DLL4 inhibitors cause hyper-sprouting, the Notch1 peptibody reduced angiogenesis in a 3-dimensional in vitro sprouting assay. Administration of Notch1 peptibodies to neonate mice resulted in reduced radial outgrowth of retinal vasculature, confirming anti-angiogenic properties. We conclude that purified Notch peptibodies comprising EGF-like repeats 10-14 bind to both DLL4 and JAG1 ligands and exhibit anti-angiogenic properties. Based on their secretion profile, unique Notch inhibitory activities, and anti-angiogenic properties, Notch peptibodies present new opportunities for therapeutic Notch inhibition.© 2022. The Author(s).