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Rapid identification of highly potent human anti-GPCR antagonist monoclonal antibodies.

Abstract：

:Complex cellular targets such as G protein-coupled receptors (GPCRs), ion channels, and other multi-transmembrane proteins represent a significant challenge for therapeutic antibody discovery, primarily because of poor stability of the target protein upon extraction from cell membranes. To assess whether a limited set of membrane-bound antigen formats could be exploited to identify functional antibodies directed against such targets, we selected a GPCR of therapeutic relevance (CCR1) and identified target binders using an in vitro yeast-based antibody discovery platform (AdimabTM) to expedite hit identification. Initially, we compared two different biotinylated antigen formats overexpressing human CCR1 in a 'scouting' approach using a subset of the antibody library. Binders were isolated using streptavidin-coated beads, expressed as yeast supernatants, and screened using a high-throughput binding assay and flow cytometry on appropriate cell lines. The most suitable antigen was then selected to isolate target binders using the full library diversity. This approach identified a combined total of 183 mAbs with diverse heavy chain sequences. A subset of clones exhibited high potencies in primary cell chemotaxis assays, with IC50 values in the low nM/high pM range. To assess the feasibility of any further affinity enhancement, full-length hCCR1 protein was purified, complementary-determining region diversified libraries were constructed from a high and lower affinity mAb, and improved binders were isolated by fluorescence-activated cell sorting selections. A significant affinity enhancement was observed for the lower affinity parental mAb, but not the high affinity mAb. These data exemplify a methodology to generate potent human mAbs for challenging targets rapidly using whole cells as antigen and define a route to the identification of affinity-matured variants if required.

journal_name

MAbs

journal_title

mAbs

authors

Scott MJ,Jowett A,Orecchia M,Ertl P,Ouro-Gnao L,Ticehurst J,Gower D,Yates J,Poulton K,Harris C,Mullin MJ,Smith KJ,Lewis AP,Barton N,Washburn ML,de Wildt R

doi

10.1080/19420862.2020.1755069

subject

Has Abstract

pub_date

2020-01-01 00:00:00

pages

1755069

issue

eissn

1942-0862

issn

1942-0870

journal_volume

pub_type

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Rapid identification of highly potent human anti-GPCR antagonist monoclonal antibodies.