Characterization of adalimumab Fab variants with the CH1 domain replaced by the Cα1 domain of IgA

IF 2.2 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Biochemistry and Biophysics Reports Pub Date : 2025-09-19 DOI:10.1016/j.bbrep.2025.102269

Rara Sugimoto , Hitomi Nakamura , Masato Kiyoshi , Akiko Ishii-Watabe , Naoko Oda-Ueda , Takatoshi Ohkuri

{"title":"Characterization of adalimumab Fab variants with the CH1 domain replaced by the Cα1 domain of IgA","authors":"Rara Sugimoto , Hitomi Nakamura , Masato Kiyoshi , Akiko Ishii-Watabe , Naoko Oda-Ueda , Takatoshi Ohkuri","doi":"10.1016/j.bbrep.2025.102269","DOIUrl":null,"url":null,"abstract":"<div><div>Antibody constant domains (C domains) contribute to structural stability. However, studies focusing on Fab fragments with heterologous constant domains are limited. Here, we engineered an IgA-type Fab (Fab_CH1IgA) by replacing the CH1 domain of an IgG1-type adalimumab Fab with the corresponding Cα1 domain of IgA1. Fab_CH1IgA was expressed in CHO cells at approximately 50 mg L<sup>−1</sup> and purified to homogeneity. DSC showed comparable thermal stability, with <em>T</em><sub>m</sub> = 74.8 °C for Fab_CH1IgA and 75.2 °C for the IgG1-type Fab. SPR analysis showed similar antigen-binding kinetics, with <em>K</em><sub>D</sub> = 2.23 n M for Fab_CH1IgA and 1.77 nM for the IgG1-type Fab. Structural analysis identified Pro124 and Tyr211 in the C domain as part of the hydrophobic core-bridging variable and constant domains. Substitution of these residues with their IgG-type counterparts reduced thermal stability, underscoring the critical contribution of V–C domain interactions. Although the sequence identity between the IgG1 and IgA1 constant domains was not particularly high, the CH1 domain in adalimumab Fab could be replaced by the IgA Cα1 domain without markedly compromising stability or activity. These findings highlight cooperative packing at the V–C interface, offering important insights into Fab engineering to enhance function while preserving biophysical integrity, and supporting the design of IgG–IgA chimeric antibodies and novel chimeric Fab formats.</div></div>","PeriodicalId":8771,"journal":{"name":"Biochemistry and Biophysics Reports","volume":"44 ","pages":"Article 102269"},"PeriodicalIF":2.2000,"publicationDate":"2025-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biochemistry and Biophysics Reports","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2405580825003565","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}

引用次数: 0

Abstract

Antibody constant domains (C domains) contribute to structural stability. However, studies focusing on Fab fragments with heterologous constant domains are limited. Here, we engineered an IgA-type Fab (Fab_CH1IgA) by replacing the CH1 domain of an IgG1-type adalimumab Fab with the corresponding Cα1 domain of IgA1. Fab_CH1IgA was expressed in CHO cells at approximately 50 mg L⁻¹ and purified to homogeneity. DSC showed comparable thermal stability, with T_m = 74.8 °C for Fab_CH1IgA and 75.2 °C for the IgG1-type Fab. SPR analysis showed similar antigen-binding kinetics, with K_D = 2.23 n M for Fab_CH1IgA and 1.77 nM for the IgG1-type Fab. Structural analysis identified Pro124 and Tyr211 in the C domain as part of the hydrophobic core-bridging variable and constant domains. Substitution of these residues with their IgG-type counterparts reduced thermal stability, underscoring the critical contribution of V–C domain interactions. Although the sequence identity between the IgG1 and IgA1 constant domains was not particularly high, the CH1 domain in adalimumab Fab could be replaced by the IgA Cα1 domain without markedly compromising stability or activity. These findings highlight cooperative packing at the V–C interface, offering important insights into Fab engineering to enhance function while preserving biophysical integrity, and supporting the design of IgG–IgA chimeric antibodies and novel chimeric Fab formats.

查看原文本刊更多论文

CH1结构域被IgA的Cα1结构域取代的阿达木单抗Fab变异体的特征

抗体恒定结构域（C结构域）有助于结构的稳定性。然而，对具有异源恒定结构域的Fab片段的研究还很有限。在这里，我们通过用IgA1的Cα1结构域替换igg1型阿达木单抗Fab的CH1结构域来设计一个iga型Fab （Fab_CH1IgA）。Fab_CH1IgA在CHO细胞中以约50 mg L−1的浓度表达并纯化至均匀性。DSC显示了相当的热稳定性，Fab_CH1IgA的Tm = 74.8°C， igg1型Fab的Tm = 75.2°C。SPR分析显示了相似的抗原结合动力学，Fab_CH1IgA的KD = 2.23 nM， igg1型Fab的KD = 1.77 nM。结构分析发现C结构域的Pro124和Tyr211是疏水核心桥接可变和恒定结构域的一部分。将这些残基替换为igg类型的对应物降低了热稳定性，强调了V-C结构域相互作用的关键贡献。虽然IgG1和IgA1恒定结构域之间的序列一致性不是特别高，但在阿达木单抗Fab中，CH1结构域可以被IgA c - α1结构域取代，而不会显著影响稳定性或活性。这些发现强调了V-C界面的协同包装，为Fab工程提供了重要的见解，以增强功能，同时保持生物物理完整性，并支持IgG-IgA嵌合抗体和新型嵌合Fab格式的设计。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

来源期刊

Biochemistry and Biophysics Reports Biochemistry, Genetics and Molecular Biology-Biophysics

CiteScore

4.60

自引率

0.00%

发文量

191

审稿时长

59 days

期刊介绍： Open access, online only, peer-reviewed international journal in the Life Sciences, established in 2014 Biochemistry and Biophysics Reports (BB Reports) publishes original research in all aspects of Biochemistry, Biophysics and related areas like Molecular and Cell Biology. BB Reports welcomes solid though more preliminary, descriptive and small scale results if they have the potential to stimulate and/or contribute to future research, leading to new insights or hypothesis. Primary criteria for acceptance is that the work is original, scientifically and technically sound and provides valuable knowledge to life sciences research. We strongly believe all results deserve to be published and documented for the advancement of science. BB Reports specifically appreciates receiving reports on: Negative results, Replication studies, Reanalysis of previous datasets.