PDF(Pubmed)
Abstract：
Using conventional immunoglobulin G (IgG) molecules as therapeutic agents presents several well-known disadvantages owing to their large size and structural complexity, negatively impacting development and production efficiency. Single-domain antibodies (sdAbs) are the smallest functional antibody format (~ 15 kDa) and represent a viable alternative to IgG in many applications. However, unlike natural single-domain antibodies, such as camelid VHH, the variable domains of conventional antibodies show poor physicochemical properties when expressed as sdAbs. This report identified stable sdAb variants of human VH3-23 from a framework region 2-randomized human VH library by phage display selection under thermal challenge. Synthetic complementarity determining region diversity was introduced to one of the selected variants with high thermal stability, expression level, and monomeric content to construct a human VH sdAb library. The library was validated by panning against a panel of antigens, and target-specific binders were identified and characterized for their affinity and biophysical properties. The results of this study suggest that a synthetic sdAb library based on a stability-engineered human VH scaffold could be a facile source of high-quality sdAb for many practical applications.
摘要：
使用常规的免疫球蛋白G(IgG)分子作为治疗剂,由于它们的大尺寸和结构复杂性,存在几个众所周知的缺点。对发展和生产效率产生负面影响。单结构域抗体(sdAb)是最小的功能性抗体形式(〜15kDa),并且在许多应用中代表IgG的可行替代物。然而,与天然的单结构域抗体不同,比如骆驼VHH，当表达为sdAb时,常规抗体的可变结构域显示差的物理化学性质。该报告通过热攻击下的噬菌体展示选择从框架区2随机化的人VH文库中鉴定了人VH3-23的稳定sdAb变体。将合成的互补决定区多样性引入到具有高热稳定性的选定变体之一中，表达水平，和单体内容物以构建人VHsdAb文库。通过对一组抗原进行淘选来验证该文库，和靶特异性结合剂被鉴定并表征其亲和力和生物物理特性。这项研究的结果表明，基于稳定性工程化的人VH支架的合成sdAb文库可能是许多实际应用的高质量sdAb的容易来源。
