Avram and Stella Goldstein-Goren Department of Biotechnology Engineering and the National Institute of Biotechnology in the Negev, Ben-Gurion University of the Negev, 1 Ben-Gurion Avenue, 8410501, Beer-Sheva, Israel.
Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
Cell Mol Life Sci. 2022 May 23;79(6):312. doi: 10.1007/s00018-022-04336-9.
The human signaling molecules Tie1 and Tie2 receptor tyrosine kinases (RTKs) play important pathophysiological roles in many diseases, including different cancers. The activity of Tie1 is mediated mainly through the downstream angiopoietin-1 (Ang1)-dependent activation of Tie2, rendering both Tie 1 and the Tie1/Tie2/Ang1 axis attractive putative targets for therapeutic intervention. However, the development of inhibitors that target Tie1 and an understanding of their effect on Tie2 and on the Tie1/Tie2/Ang1 axis remain unfulfilled tasks, due, largely, to the facts that Tie1 is an orphan receptor and is difficult to produce and use in the quantities required for immune antibody library screens. In a search for a selective inhibitor of this orphan receptor, we sought to exploit the advantages (e.g., small size that allows binding to hidden epitopes) of non-immune nanobodies and to simultaneously overcome their limitations (i.e., low expression and stability). We thus performed expression, stability, and affinity screens of yeast-surface-displayed naïve and predesigned synthetic (non-immune) nanobody libraries against the Tie1 extracellular domain. The screens yielded a nanobody with high expression and good affinity and specificity for Tie1, thereby yielding preferential binding for Tie1 over Tie2. The stability, selectivity, potency, and therapeutic potential of this synthetic nanobody were profiled using in vitro and cell-based assays. The nanobody triggered Tie1-dependent inhibition of RTK (Tie2, Akt, and Fak) phosphorylation and angiogenesis in endothelial cells, as well as suppression of human glioblastoma cell viability and migration. This study opens the way to developing nanobodies as therapeutics for different cancers associated with Tie1 activation.
人类信号分子 Tie1 和 Tie2 受体酪氨酸激酶（RTKs）在许多疾病中发挥着重要的病理生理作用，包括不同类型的癌症。Tie1 的活性主要通过下游血管生成素-1（Ang1）依赖性激活 Tie2 来介导，这使得 Tie1 和 Tie1/Tie2/Ang1 轴成为有吸引力的潜在治疗靶点。然而，开发针对 Tie1 的抑制剂以及了解它们对 Tie2 和 Tie1/Tie2/Ang1 轴的影响仍然是未完成的任务，这主要归因于以下事实：Tie1 是一个孤儿受体，难以生产且难以获得用于免疫抗体文库筛选所需的数量。在寻找这种孤儿受体的选择性抑制剂时，我们试图利用非免疫纳米抗体的优势（例如，体积小，可结合隐藏表位），同时克服其局限性（即表达和稳定性低）。因此，我们针对 Tie1 细胞外结构域进行了酵母表面展示的原始和预先设计的合成（非免疫）纳米抗体文库的表达、稳定性和亲和力筛选。筛选得到了一种对 Tie1 具有高表达和良好亲和力和特异性的纳米抗体，从而优先结合 Tie1 而不是 Tie2。通过体外和基于细胞的测定，对这种合成纳米抗体的稳定性、选择性、效力和治疗潜力进行了分析。该纳米抗体触发 Tie1 依赖性 RTK（Tie2、Akt 和 Fak）磷酸化和内皮细胞血管生成的抑制，以及抑制人胶质母细胞瘤细胞活力和迁移。这项研究为开发针对与 Tie1 激活相关的不同癌症的纳米抗体疗法开辟了道路。
