AbstractDue 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.

摘要由于抗体对抗原的高亲和力和特异性，已经开发了各种基于抗体的应用。最近，重链抗体（VHH）的可变抗原结合结构域由于其独特的分子特征而成为常规片段抗体的有吸引力的替代品。

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.

作为抗体产生策略，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，我们基于使用互补决定区移植方法进行的生物物理分析和蛋白质数据库中保存的VHH的综合序列分析，设计并构建了几种新型合成人源化VHH文库。

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..

我们从文库中获得了VHH，命中克隆表现出相当大的热稳定性。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，我们提出了一种策略，通过利用我们的文库组合产生针对各种抗原具有不同表位的人源化VHH。。

IntroductionAn antibody recognizes a unique target molecule called an antigen. Due to the high affinity and specificity of antibodies toward antigens, various antibody-based applications have been developed in the biotechnological and medical fields, such as detection reagents for the target molecule, diagnostic tools, and antibody-based drugs for human therapy.

引言抗体识别称为抗原的独特靶分子。由于抗体对抗原的高亲和力和特异性，已经在生物技术和医学领域开发了各种基于抗体的应用，例如用于靶分子的检测试剂，诊断工具和用于人类治疗的基于抗体的药物。

Antibody-based proteins have become an important class of biologic therapeutics1. Currently, nearly 1200 antibody therapeutics are in clinical studies, and ~ 175 therapeutics are in regulatory review or have been approved2.In 1993, functional antibodies devoid of light chains and composed of only the heavy chain were discovered in the serum of Camelus dromedarius3.

基于抗体的蛋白质已成为一类重要的生物疗法1。目前，近1200种抗体疗法正在进行临床研究，约175种疗法正在进行监管审查或已获得批准2。1993年，在骆驼血清中发现了不含轻链且仅由重链组成的功能性抗体3。

The antibodies, referred to as heavy-chain antibodies (HCAbs), are unique because they lack the entire light chain and the first heavy chain constant region. The variable antigen-binding domain of a HCAb, referred to as the variable domain of the heavy-chain antibody (VHH), is generally functional as a single domain despite its small size of only 15 kDa4,5.

被称为重链抗体（HCAbs）的抗体是独特的，因为它们缺乏整个轻链和第一个重链恒定区。HCAb的可变抗原结合结构域，称为重链抗体（VHH）的可变结构域，尽管其尺寸仅为15 kDa4,5，但通常作为单个结构域起作用。

While conventional antibodies tend to form a flat surface or a groove as a paratope, the convex paratopes of VHHs have a smaller antigen-binding interface. Thus, VHHs tend to bind to concave-shaped epitopes, such as enzyme catalytic sites6,7,8. VHHs are easily produced by bacterial expression systems due to their small size and single domain, and their production cost is much lower than that of immunoglobulin G.

虽然常规抗体倾向于形成平坦的表面或凹槽作为互补位，但VHH的凸互补位具有较小的抗原结合界面。因此，VHH倾向于结合凹形表位，例如酶催化位点6,7,8。VHH由于其小尺寸和单结构域而易于由细菌表达系统产生，并且其生产成本远低于免疫球蛋白G。

In addition, the small size of VHHs leads to rapid extravasation followed by deep-tissue penetration and rapid blood clearance. Based on these characteristics, VHHs are an attractive alternative to antigen-binding fragments from conventional antibodies (e.g., Fabs and scFvs) in biotechnolog.

此外，VHH的小尺寸导致快速外渗，然后是深层组织渗透和快速血液清除。基于这些特征，VHH是生物技术中常规抗体（例如Fabs和scFv）的抗原结合片段的有吸引力的替代品。

Data availability

数据可用性

All the structural data used in this study are available in Protein Data Bank (https://www.rcsb.org/) and all the data generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.

这项研究中使用的所有结构数据都可以在蛋白质数据库中找到(https://www.rcsb.org/)在本研究期间生成和/或分析的所有数据均可在合理要求下从通讯作者处获得。

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Download referencesAcknowledgementsThis work was funded in part by the Japan Society for the Promotion of Science (Grant Nos. 21H05090 to M.N.; 19H05766 and 20H02531 to K.T.), the JST-Mirai Program (Grant No. JP MJMI21G6 to M.N.), the JST-CREST Program (Grant No. JP MJCR20H8 to K.T.), and the Japan Agency for Medical Research and Development (Grant Nos.

下载参考文献致谢这项工作部分由日本科学促进会（M.N.批准号21H05090；K.T.批准号19H05766和20H02531），JST Mirai计划（M.N.批准号JP MJMI21G6），JST-CREST计划（K.T.批准号JP MJCR20H8）和日本医学研究与发展机构（批准号：。

JP 18ak0101100 and 19am0401010to M.N., 20mk0101170 to K.T.).Author informationAuthor notesThese authors contributed equally: Makoto Nakakido and Seisho Kinoshita.Authors and AffiliationsDepartment of Bioengineering, School of Engineering, The University of Tokyo, Tokyo, JapanMakoto Nakakido, Seisho Kinoshita & Kouhei TsumotoDepartment of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Tokyo, JapanMakoto Nakakido & Kouhei TsumotoLaboratory of Medical Proteomics, The Institute of Medical Science, The University of Tokyo, Tokyo, JapanKouhei TsumotoAuthorsMakoto NakakidoView author publicationsYou can also search for this author in.

JP 18ak0101100和19AM0401010至M.N.，20mk0101170至K.T.）。作者信息作者注意到这些作者做出了同样的贡献：Makoto Nakakido和Seisho Kinoshita。作者和附属机构东京大学工程学院生物工程系，东京，东京，东京，东京大学，东京，东京大学，东京，东京，东京大学，东京，东京大学，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京，东京。

PubMed Google ScholarSeisho KinoshitaView author publicationsYou can also search for this author in

PubMed Google ScholarSeisho KinoshitaView作者出版物您也可以在

PubMed Google ScholarKouhei TsumotoView author publicationsYou can also search for this author in

PubMed Google ScholarKouhei TsumotoView作者出版物您也可以在

PubMed Google ScholarContributionsM.N. and K.T. conceptualized and administrated the research. S.K. conducted experiments and analyzed data. M.N. wrote the main manuscript and S.K. prepared figures. All authors reviewed the manuscript.Corresponding authorsCorrespondence to

PubMed谷歌学术贡献。N、 K.T.对这项研究进行了概念化和管理。S、 K.进行了实验并分析了数据。M、 N.撰写了主要手稿，S.K.准备了数字。所有作者都审阅了手稿。通讯作者通讯

Makoto Nakakido or Kouhei Tsumoto.Ethics declarations

Makoto Nakakido或Kouhei Tsumoto。道德宣言

Competing interests

相互竞争的利益

We declare that we filed a patent regarding the synthetic libraries described in this paper. The applicant is our institute, The University of Tokyo, and inventors are; Makoto Nakakido, Seisho Kinoshita, and Kouhei Tsumoto. Application number is JP2024-095995 and status of application is pending.

我们声明，我们已就本文所述的合成文库申请了专利。申请人是我们的研究所东京大学，发明人是；Makoto Nakakido，Seisho Kinoshita和Kouhei Tsumoto。申请号为JP2024-095995，申请状态待定。

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Reprints and permissionsAbout this articleCite this articleNakakido, M., Kinoshita, S. & Tsumoto, K. Development of novel humanized VHH synthetic libraries based on physicochemical analyses.

转载和许可本文引用本文Nakakido，M.，Kinoshita，S。＆Tsumoto，K。基于物理化学分析开发新型人源化VHH合成文库。

Sci Rep 14, 19533 (2024). https://doi.org/10.1038/s41598-024-70513-4Download citationReceived: 14 February 2024Accepted: 19 August 2024Published: 22 August 2024DOI: https://doi.org/10.1038/s41598-024-70513-4Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard.

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