Discovery Biologics, Merck & Co., Inc., Boston, MA 02115, USA.
Computational and Structural Chemistry, Merck & Co., Inc., West Point, PA 19486, USA.
Protein Eng Des Sel. 2020 Sep 14;33. doi: 10.1093/protein/gzaa028.
Single-domain antibody fragments known as VHH 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 VHH is via animal immunization, which can be costly and time-consuming. Here we describe the development of a synthetic VHH 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 VHH 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.
单域抗体片段，又称 VHH，已在制药行业崭露头角，成为有用的生物疗法。这些分子是由骆驼天然产生的，与传统的人类免疫球蛋白具有相同的高亲和力和特异性特征，而只由一个重链组成。目前，产生 VHH 的最常见方法是通过动物免疫，这可能既昂贵又耗时。在这里，我们描述了一种用于体外选择单域结合物的合成 VHH 文库的开发。我们结合基于结构的设计和下一代测序分析，构建了一个具有与天然库密切相似特征的文库。为了验证我们合成文库的性能，我们分离了针对三种不同大小和特征的模型抗原（可溶性小鼠 PD-1 胞外域、淀粉样β肽和 MrgX1 GPCR）的 VHH。我们能够分离出针对所有三个靶标具有高亲和力（高达 5 nM）的针对不同表位的多样化结合物。然后，我们证明了抗 mPD-1 结合物在受体阻断测定中具有功能活性。
