Function-based high-throughput screening for antibody antagonists and agonists against G protein-coupled receptors

Huanhuan Ren,Jian Li,Jianqing Xu,Liaoyuan A Hu,Yingli Ma,Philip Tagari,Mei-Yun Zhang,Ning Zhang

doi:10.1038/s42003-020-0867-7

Huanhuan Ren, Jian Li + Show 6 more

Open Access

https://doi.org/10.1038/s42003-020-0867-7

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Abstract

Hybridoma and phage display are two powerful technologies for isolating target-specific monoclonal antibodies based on the binding. However, for complex membrane proteins, such as G protein-coupled receptors (GPCRs), binding-based screening rarely results in functional antibodies. Here we describe a function-based high-throughput screening method for quickly identifying antibody antagonists and agonists against GPCRs by combining glycosylphosphatidylinositol-anchored antibody cell display with β-arrestin recruitment-based cell sorting and screening. This method links antibody genotype with phenotype and is applicable to all GPCR targets. We validated this method by identifying a panel of antibody antagonists and an antibody agonist to the human apelin receptor from an immune antibody repertoire. In contrast, we obtained only neutral binders and antibody antagonists from the same repertoire by phage display, suggesting that the new approach described here is more efficient than traditional methods in isolating functional antibodies. This new method may create a new paradigm in antibody drug discovery.

Highlights

Hybridoma and phage display are two powerful technologies for isolating target-specific monoclonal antibodies based on the binding
Ten G protein-coupled receptors (GPCRs)-targeting monoclonal antibody (mAb) are currently in clinical trials[19], among which, GMA102 is a humanized GLP-1R-binding mAb fused to GLP-1 peptide, and the rest are antibody antagonists or neutral binders conjugated to isotopes
JN126 and JN241 are APJ single domain antibody (sdAb) previously isolated from the APJ immune library by phage display, while JN126P13 is an antibody-ligand fusion with apelin 13 covalently linked to the C-terminus of the neutral binding sdAb JN126

Summary

Introduction

Hybridoma and phage display are two powerful technologies for isolating target-specific monoclonal antibodies based on the binding. We obtained only neutral binders and antibody antagonists from the same repertoire by phage display, suggesting that the new approach described here is more efficient than traditional methods in isolating functional antibodies This new method may create a new paradigm in antibody drug discovery. The conformation flexibility and low immunogenicity and antigenicity of GPCRs pose challenges to generating target-specific antibody binders, let alone functional antibodies against GPCRs. If the immunization was successful, hybridoma screening or phage-displayed library panning and screening may lead to the identification of neutral binders and, sometimes, antibody antagonists[15,16], but rarely antibody agonists. Ten GPCR-targeting mAbs are currently in clinical trials[19], among which, GMA102 is a humanized GLP-1R-binding mAb fused to GLP-1 peptide, and the rest are antibody antagonists or neutral binders conjugated to isotopes None of these antibodies are antibody agonists. Developing a function-based antibody high-throughput screening method would be of great value for identifying mAbs with desired biofunction to complex membrane protein targets

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