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In vitro affinity maturation of antibody against membrane-bound GPCR molecules
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...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Springer Berlin Heidelberg
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719327/ https://www.ncbi.nlm.nih.gov/pubmed/31359103 http://dx.doi.org/10.1007/s00253-019-10030-x |
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author | Wang, Jie An, Lili Zhao, Yun Zhang, Cheng Li, Shengnan Ye, Chen Jing, Shuqian Hang, Haiying |
author_facet | Wang, Jie An, Lili Zhao, Yun Zhang, Cheng Li, Shengnan Ye, Chen Jing, Shuqian Hang, Haiying |
author_sort | Wang, Jie |
collection | PubMed |
description | 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s00253-019-10030-x) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-6719327 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Springer Berlin Heidelberg |
record_format | MEDLINE/PubMed |
spelling | pubmed-67193272019-09-19 In vitro affinity maturation of antibody against membrane-bound GPCR molecules Wang, Jie An, Lili Zhao, Yun Zhang, Cheng Li, Shengnan Ye, Chen Jing, Shuqian Hang, Haiying Appl Microbiol Biotechnol Methods and Protocols 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s00253-019-10030-x) contains supplementary material, which is available to authorized users. Springer Berlin Heidelberg 2019-07-29 2019 /pmc/articles/PMC6719327/ /pubmed/31359103 http://dx.doi.org/10.1007/s00253-019-10030-x Text en © The Author(s) 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. |
spellingShingle | Methods and Protocols Wang, Jie An, Lili Zhao, Yun Zhang, Cheng Li, Shengnan Ye, Chen Jing, Shuqian Hang, Haiying In vitro affinity maturation of antibody against membrane-bound GPCR molecules |
title | In vitro affinity maturation of antibody against membrane-bound GPCR molecules |
title_full | In vitro affinity maturation of antibody against membrane-bound GPCR molecules |
title_fullStr | In vitro affinity maturation of antibody against membrane-bound GPCR molecules |
title_full_unstemmed | In vitro affinity maturation of antibody against membrane-bound GPCR molecules |
title_short | In vitro affinity maturation of antibody against membrane-bound GPCR molecules |
title_sort | in vitro affinity maturation of antibody against membrane-bound gpcr molecules |
topic | Methods and Protocols |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719327/ https://www.ncbi.nlm.nih.gov/pubmed/31359103 http://dx.doi.org/10.1007/s00253-019-10030-x |
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