In vitro affinity maturation of antibody against membrane-bound GPCR molecules
Open accessAbstract
G protein-coupled receptors (GPCRs), also known as seven-transmembrane domain receptors, are among the most important targets against which many small molecule drugs have been developed. However, only two antibody drugs targeting GPCRs have been approved for clinical use although many antibody drugs against non-GPCR protein targets have been successfully developed for various disease indications. One of the challenges for developing anti-GPCR drugs is the high difficulty to perform affinity maturation due to their insolubility in aqueous solutions. To address this issue, CHO cell display libraries of single-chain variable fragments (scFvs) and full-length antibodies were maturated directly against vesicle probes prepared from CHO cells displaying the endothelin A receptor (ETaR) GPCR. The probe in the vesicle form ensures the physiological conformation and functional activity of the protein and avoids issues with membrane protein insolubility. The size of the vesicle had a clear effect on protein-ligand interaction; we used small-sized vesicles with low expression levels of GPCRs for the affinity maturation. Four rounds of affinity maturation combining vesicles as probes with the CHO cell display platform improved affinity by 13.58-fold for scFvs and 5.05-fold for full-length antibodies. We expect that this method will not only be used for the affinity maturation of antibodies against GPCRs but will also be used to mature antibodies for other types of proteins where the conformation/activity of which depends on the proper membrane environment.
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In vitro affinity maturation methods, such as random mutagenesis with display technologies, improve antibody binding against target proteins but are time consuming and labor intensive.
“In vitro affinity maturation methods, such as random mutagenesis with display technologies, has been shown to improve antibody binding against target proteins, but such approaches are time consuming and labor intensive (27–32).”
From Deep learning guided optimization of human antibody against SARS-CoV-2 variants with broad neutralization (2022) · cites this paper as [32]
Conventional in vitro affinity maturation usually takes a long time with poor efficiency due to its small library content, incompatible optimization for multiple goals, and other technical obstacles.
“The conventional in vitro affinity maturation usually takes a long time with poor efficiency due to its small library content, incompatible optimization for multiple goals, and other technical obstacles (32, 49).”
From Deep learning guided optimization of human antibody against SARS-CoV-2 variants with broad neutralization (2022) · cites this paper as [32]
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- Authors
- Wang, J., An, L., Zhao, Y., Zhang, C., Li, S., Ye, C., Jing, S., & Hang, H.
- Affiliations
- Chinese Academy of Sciences (CN)
- Corresponding author
- Jing, Shuqian
- Hang, Haiying
- Venue
- Applied Microbiology and Biotechnology
- Published
- 2019-07-29
- Volume
- 103
- Issue
- 18
- Pages
- 7703-7717
- ISSN
- 0175-7598 (ISSN-L)
- 1432-0614
- PMC
- PMC6719327
- OpenAlex
- W2965717316
- Cited by
- 11
- References
- 65
- Citations by year
- 2026: 1 · 2025: 1 · 2024: 3 · 2022: 3 · 2021: 1 · 2020: 2
- FWCI
- 0.99
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Locations
- Applied Microbiology and Biotechnology (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Funders
- Development of Ministry of Science and Technology (2014CB910402)
- National Natural Science Foundation of China (No. 31870923)
- National Key R&D Program of China (2017YFF0205404)
- Beijing Science and Technology Project (Z181100003818023)
- Topics
- Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Receptor Mechanisms and Signaling
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
- Keywords
- affinity maturation, G protein-coupled receptors, membrane proteins, antibody drug development, scFv, ETA receptor, protein-ligand interactions, membrane protein conformation
- MeSH terms
- Animals, Antibody Affinity, Cricetulus, Ligands, Molecular Conformation, CHO Cells, Receptor, Endothelin A, Single-Chain Antibodies
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