Structure- and sequence-based design of synthetic single-domain antibody libraries. (21st December 2020)

• Record Type: Journal Article
• Authors: Sevy, Alexander M; Chen, Ming-Tang; Castor, Michelle; Sylvia, Tyler; Krishnamurthy, Harini; Ishchenko, Andrii; Hsieh, Chung-Ming
• Is Part Of: Protein engineering, design & selection. Volume 33(2020)
• Publication Date: 2020-12-21
• Subjects: antibody discovery -- next-generation sequencing -- single-domain binders -- synthetic library -- yeast display; Protein engineering -- Periodicals; 660.63
• DOI: 10.1093/protein/gzaa028
• Languages: English
• ISSNs: 1741-0126

Abstract

Abstract: Single-domain antibody fragments known as VH H 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 VH H is via animal immunization, which can be costly and time-consuming. Here we describe the development of a synthetic VH H 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 VH H 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.