PDF(Pubmed)
Abstract：
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.
摘要：
Notch信号通路是治疗炎症性疾病和癌症的重要治疗靶点。我们之前创建了Notch信号的配体特异性抑制剂，该抑制剂由Fc融合到Notch1胞外域的特异性EGF样重复序列组成。叫做Notch诱饵，结合配体，阻塞的陷波信号,并显示出抗肿瘤活性，低毒性。然而,其功能的研究依赖于病毒介导的表达，这排除了剂量控制和限制临床适用性。我们改进了诱饵设计，以创建包含核心结合域的基于肽体的Notch抑制剂，Notch1或Notch4的EGF样重复10-14。这些Notch肽体显示出高分泌特性和产量，与以前的Notch诱饵相比提高了近100倍。使用表面等离子体共振光谱结合免疫共沉淀测定，我们观察到Notch1和Notch4肽体表现出对Notch配体DLL4和JAG1的强但不同的结合性质。Notch1和Notch4肽体均干扰内皮细胞中的Notch信号传导并在治疗后降低典型Notch靶标的表达。虽然以前的DLL4抑制剂会导致过度发芽，Notch1肽体在三维体外发芽测定中减少了血管生成。对新生小鼠施用Notch1肽体导致视网膜脉管系统的径向生长减少，确认抗血管生成特性。我们得出的结论是,包含EGF样重复10-14的纯化的Notch肽体结合DLL4和JAG1配体并表现出抗血管生成特性。根据它们的分泌情况,独特的Notch抑制活性,和抗血管生成特性，Notch肽体为治疗性Notch抑制提供了新的机会。
