Institute for Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany.
Antibody-Drug Conjugates and Targeted NBE Therapeutics, Merck KGaA, Darmstadt, Germany.
Methods Mol Biol. 2023;2702:227-243. doi: 10.1007/978-1-0716-3381-6_11.
The adaptive immune system of sharks comprises a unique heavy chain-only antibody isotype, termed immunoglobulin new antigen receptor (IgNAR), in which antigen binding is mediated by a single variable domain, referred to as vNAR. In recent years, efforts were made to harness these domains for biomedical and biotechnological applications particularly due to their high affinity and specificity combined with a small size and high stability. Herein, we describe protocols for the construction of semisynthetic, CDR3-randomized vNAR libraries for the isolation of target-specific paratopes by yeast surface display. Additionally, we provide guidance for affinity maturation of a panel of antigen-enriched vNAR domains through CDR1 diversification of the FACS-selected, antigen-enriched population and sublibrary establishment.
鲨鱼的适应性免疫系统包含一种独特的重链仅有抗体同种型，称为免疫球蛋白新抗原受体（IgNAR），其中抗原结合由单个可变结构域介导，称为 vNAR。近年来，由于其高亲和力和特异性以及小尺寸和高稳定性，人们努力利用这些结构域进行生物医学和生物技术应用。在此，我们描述了用于构建半合成、CDR3 随机化 vNAR 文库的方案，用于通过酵母表面展示分离靶特异性表位。此外，我们还提供了通过 FACS 选择的抗原富集群体和亚文库建立对 CDR1 多样化的指导，以实现抗原富集 vNAR 结构域的亲和力成熟。
