Directed evolution of an angiopoietin-2 ligand trap by somatic hypermutation and cell surface display.
Directed evolution of an angiopoietin-2 ligand trap by somatic hypermutation and cell surface display.
复制标题
DOI：
10.1074/jbc.m113.510578
复制
发表时间：
2013-11-15
期刊：
影响因子：
--
通讯作者：
Steele KH
中科院分区：
文献类型：
--
作者：
Brindle NP;Sale JE;Arakawa H;Buerstedde JM;Nuamchit T;Sharma S;Steele KH
背景：配体血管生成素2参与血管疾病。结果：利用一种新的定向进化方法，从非特异性血管生成素受体中构建了一个特异性血管生成素2结合蛋白。结论：受体结合特异性可以通过三个残基的改变而改变。意义：新蛋白具有治疗潜力，定向进化方法对于进化哺乳动物蛋白具有优势。Tie2是一种受体酪氨酸激酶，通过结合可溶性配体血管生成素1（Ang1）和2（Ang2）对血管的发育和维持至关重要。血管生成素1由血管周围细胞组成性产生，对成人血管系统具有保护作用。Ang2在血管形成中起重要作用，并且在发育过程中正常表达。然而，其在疾病状态（包括癌症和脓毒症）中的重新表达导致血管的不稳定，从而促成这些病症的病理学。因此Ang2是一个有吸引力的治疗靶点。在这里，我们报告了通过利用B细胞体细胞超突变机制并将其与Tie2胞外域的可选择细胞表面展示偶联来定向进化Ang2的配体陷阱。定向进化产生了意想不到的突变组合，导致Ang1结合丧失但Ang2结合维持。可溶形式的进化胞外域结合Ang2，但不结合Ang1。此外，可溶性进化胞外域阻断Ang2对内皮细胞的作用，而不干扰Ang1活性。我们的研究创造了一种新的Ang2配体陷阱，并为结合B细胞中的表面展示和外源基因多样化以进化非免疫球蛋白靶标提供了概念证明。
Background: The ligand angiopoietin2 contributes to vascular diseases. Results: A new directed evolution method was used to create a specific angiopoietin2-binding protein from a nonspecific angiopoietin receptor. Conclusion: The receptor binding specificity can be switched with just three residue changes. Significance: The new protein has therapeutic potential, and the directed evolution method has advantages for evolving mammalian proteins. Tie2 is a receptor tyrosine kinase that is essential for the development and maintenance of blood vessels through binding the soluble ligands angiopoietin 1 (Ang1) and 2 (Ang2). Ang1 is constitutively produced by perivascular cells and is protective of the adult vasculature. Ang2 plays an important role in blood vessel formation and is normally expressed during development. However, its re-expression in disease states, including cancer and sepsis, results in destabilization of blood vessels contributing to the pathology of these conditions. Ang2 is thus an attractive therapeutic target. Here we report the directed evolution of a ligand trap for Ang2 by harnessing the B cell somatic hypermutation machinery and coupling this to selectable cell surface display of a Tie2 ectodomain. Directed evolution produced an unexpected combination of mutations resulting in loss of Ang1 binding but maintenance of Ang2 binding. A soluble form of the evolved ectodomain binds Ang2 but not Ang1. Furthermore, the soluble evolved ectodomain blocks Ang2 effects on endothelial cells without interfering with Ang1 activity. Our study has created a novel Ang2 ligand trap and provided proof of concept for combining surface display and exogenous gene diversification in B cells for evolution of a non-immunoglobulin target.