文章基本信息

标题：More than 370-Fold Increase in Antibody Affinity to Estradiol-17β by Exploring Substitutions in the VH-CDR3
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作者：Izumi Morita ; Yuki Kiguchi ; Saya Nakamura 等
期刊名称：Biological and Pharmaceutical Bulletin
印刷版ISSN：0918-6158
电子版ISSN：1347-5215
出版年度：2022
卷号：45
期号：7
页码：851-855
DOI：10.1248/bpb.b22-00187
语种：English
出版社：The Pharmaceutical Society of Japan
摘要：Antibodies that specifically target biomarkers are essential in clinical diagnosis. Genetic engineering has assisted in designing novel antibodies that offer greater antigen-binding affinities, thus providing more sensitive immunoassays. We have succeeded in generating a single-chain Fv fragment (scFv) targeted estradiol-17β (E2) with more than 370-fold improved affinity, based on a strategy focusing the complementarity-determining region 3 in the VH domain (VH-CDR3). Systematic exploration of amino acid substitutions therein, using a clonal array profiling, revealed a cluster of four substitutions, containing H99P and a serial substitution E100eN–I100fA–L100gQ that lead to a 90-fold increase in E2-binding affinity. This substitution quartet in the VH-CDR3, combined with the substitution cluster I29V/L36M/S77G in the VL domain, resulted in a scFv fragment with a further increase in the affinity (Ka, 3.2 × 1010 M−1). This enabled a highly sensitive enzyme-linked immunosorbent assay capable of detecting up to 0.78 pg/assay. The current study has, thus, focused on the significance of reevaluating the potential of mutagenesis targeting the VH-CDR3, and encouraging the production and use of engineered antibodies that enable enhanced sensitivities as next-generation diagnostic tools.
关键词：antibody;single-chain Fv fragment (scFv);antibody engineering;affinity maturation;mutagenesis;estradiol-17β