Department of Bioengineering, School of Engineering, The University of Tokyo, Tokyo, Japan.
Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan.
Sci Rep. 2024 Aug 22;14(1):19533. doi: 10.1038/s41598-024-70513-4.
Due to the high affinity and specificity of antibodies toward antigens, various antibody-based applications have been developed. Recently, variable antigen-binding domains of heavy-chain antibodies (VHH) have become an attractive alternative to conventional fragment antibodies due to their unique molecular characteristics. As an antibody-generating strategy, synthetic VHH libraries (including humanized VHH libraries) have been developed using distinct strategies to constrain the diversity of amino acid sequences. In this study, we designed and constructed several novel synthetic humanized VHH libraries based on biophysical analyses conducted using the complementarity determining region-grafting method and comprehensive sequence analyses of VHHs deposited in the protein data bank. We obtained VHHs from the libraries, and hit clones exhibited considerable thermal stability. We also found that VHHs from distinct libraries tended to have different epitopes. Based on our results, we propose a strategy for generating humanized VHHs with distinct epitopes toward various antigens by utilizing our library combinations.
由于抗体对抗原具有高亲和力和特异性，因此开发了各种基于抗体的应用。最近，由于重链抗体（VHH）的可变抗原结合结构域具有独特的分子特性，因此成为传统片段抗体的替代品。作为一种抗体生成策略，已经使用不同的策略开发了合成 VHH 文库（包括人源化 VHH 文库），以限制氨基酸序列的多样性。在这项研究中，我们基于互补决定区嫁接方法进行的生物物理分析和对保存在蛋白质数据库中的 VHH 的综合序列分析，设计并构建了几种新型合成的人源化 VHH 文库。我们从文库中获得了 VHH，并且命中克隆显示出相当高的热稳定性。我们还发现，来自不同文库的 VHH 倾向于具有不同的表位。基于我们的结果，我们提出了一种通过利用我们的文库组合来生成针对各种抗原的具有不同表位的人源化 VHH 的策略。
