Single-domain antibody fragments known as V_HH have emerged in the pharmaceutical industry as useful biotherapeutics. These molecules, which are naturally produced by camelids, share the characteristics of high affinity and specificity with traditional human immunoglobulins, while consisting of only a single heavy chain. Currently, the most common method for generating V_HH is via animal immunization, which can be costly and time-consuming. Here we describe the development of a synthetic V_HH library for in vitro selection of single domain binders. We combine structure-based design and next-generation sequencing analysis to build a library with characteristics that closely mimic the natural repertoire. To validate the performance of our synthetic library, we isolated V_HH against three model antigens (soluble mouse PD-1 ectodomain, amyloid-β peptide, and MrgX1 GPCR) of different sizes and characteristics. We were able to isolate diverse binders targeting different epitopes with high affinity (as high as 5 nM) against all three targets. We then show that anti-mPD-1 binders have functional activity in a receptor blocking assay.

中文翻译：

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合成单域抗体文库的基于结构和序列的设计

被称为V _H H的单结构域抗体片段已在制药工业中作为有用的生物治疗剂出现。这些由骆驼科动物天然产生的分子与传统的人免疫球蛋白具有高亲和力和特异性的特征，而仅由一条重链组成。当前，最常见的产生V _H H的方法是通过动物免疫，这既昂贵又费时。在这里，我们描述了体外合成V _H H库的开发单域结合剂的选择。我们将基于结构的设计与下一代测序分析相结合，以构建一个具有与自然曲目完全相似的特性的文库。为了验证我们的合成文库的性能，我们分离V _{H ^} H ^针对不同尺寸和特性的三个模型抗原（可溶性小鼠PD-1胞外域，淀粉样蛋白β肽和MrgX1 GPCR）。我们能够针对所有三个靶标以高亲和力（高达5 nM）分离靶向不同表位的不同结合物。然后，我们显示抗mPD-1结合剂在受体阻断试验中具有功能活性。