DARPins as Bispecific Receptor Antagonists Analyzed for Immunoglobulin E Receptor Blockage
DARPins as Bispecific Receptor Antagonists Analyzed for Immunoglobulin E Receptor Blockage
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DOI：
10.1016/j.jmb.2009.08.014
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发表时间：
2009-10-30
影响因子：
5.6
通讯作者：
Vogel, Monique
中科院分区：
文献类型：
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作者：
Eggel, Alexander;Baumann, Michael J.;Vogel, Monique
多特异性抗体的概念具有很高的治疗意义，但由于这些分子的生物物理性质差而未能产生药物产品。在这里，我们提出了一种替代和简单的方法来产生双特异性结合分子使用设计锚蛋白重复蛋白（DARPins）。为此目的，选择针对人免疫球蛋白E（IgE）的高亲和力受体（Fc β RI α）的α链的具有不同表位特异性的单价DARPin。干扰IgE与受体结合的两种分离的结合剂通过柔性蛋白质接头彼此连接或自身连接。使用稳定转染有人Fc β RI α的大鼠嗜碱性白血病细胞测试所得二价和双特异性DARPin防止变应原诱导的细胞脱粒的能力。双特异性DARPin构建体是最有效的构建体，其有效地阻断IgE-Fc β RI相互作用并防止促炎介质的释放。值得注意的是，多价和多特异性DARPin构建体未显示单价亲本DARPin的有益生物物理性质的任何改变。因此，双特异性DARPin可用于产生同时靶向同一分子上的不同表位的受体拮抗剂。此外，它们容易克服限制性免疫球蛋白结合范例（一个结合分子=一个表位），从而在免疫球蛋白支架不合适的情况下代表单克隆抗体的替代方案。(c)2009爱思唯尔有限公司保留所有权利。
The concept of multispecific antibodies is of high therapeutic interest but has failed to produce pharmaceutical products due to the poor biophysical properties of such molecules. Here, we propose an alternative and simple way to generate bispecific binding molecules using designed ankyrin repeat proteins (DARPins). For this purpose, monovalent DARPins with different epitope specificities were selected against the alpha chain of the high-affinity receptor for human immunoglobulin E (IgE) (Fc epsilon RI alpha). Two of the isolated binders interfering with IgE binding to the receptor were joined to each other or to themselves via a flexible protein linker. The resulting bivalent and bispecific DARPins were tested for their ability to prevent allergen-induced cell degranulation using rat basophilic leukemia cells stably transfected with human Fc epsilon RI alpha. The bispecific DARPin construct was the most potent one, efficiently blocking the IgE-Fc epsilon RI interaction and preventing the release of proinflammatory mediators. Noteworthy, the multivalent and multispecific DARPin construct did not show any alteration of the beneficial biophysical properties of the monovalent parental DARPins. Hence, bispecific DARPins may be used to generate receptor antagonists simultaneously targeting different epitopes on the same molecule. Moreover, they easily overcome the limiting immunoglobulin binding paradigm (one binding molecule=one epitope) and thereby represent an alternative to monoclonal antibodies in cases where the immunoglobulin scaffold is unsuitable. (c) 2009 Elsevier Ltd. All rights reserved.