Abstract
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.
Highlights
In recent decades, antibodies have become more and more important in therapeutics, as evidenced by an increasing number of FDA-approved monoclonal antibodies (Schrama et al 2006)
We intended to establish an antibody affinity maturation platform using CHO cells for displaying antibody and vesicles prepared from CHO cells expressing the G protein-coupled receptors (GPCRs) endothelin A receptor (ETaR) as probes
We established a platform to mature the affinity of an antibody against the natively conformed GPCR (ETaR) displayed on the vesicles prepared from cells expressing the GPCR
Summary
Antibodies have become more and more important in therapeutics, as evidenced by an increasing number of FDA-approved monoclonal antibodies (Schrama et al 2006). GPCRs served as one class of the most important therapeutic targets, and approximately 34% of all currently marketed drugs are targeted to GPCRs (Hauser et al 2017; Hauser et al 2018; Hutchings et al 2017; Raskandersen et al 2011; Raskandersen et al 2014; Santos et al 2017). In spite of their importance in pathogeny and treatment, only two antibody drugs targeting GPCRs have been approved for clinical use. The development of GPCR-targeting antibody drugs has been beset by difficulties in the preparation of native and functional form of antigens as well as the lack of a suitable antibodymaturation platform for selectivity (Jo and Jung 2016)
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