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Rapid identification of highly potent human anti-GPCR antagonist monoclonal antibodies
mAbs ( IF 5.6 ) Pub Date : 2020-04-28
Martin J. Scott, Amanda Jowett, Martin Orecchia, Peter Ertl, Larissa Ouro-Gnao, Julia Ticehurst, David Gower, John Yates, Katie Poulton, Carol Harris, Michael J. Mullin, Kathrine J. Smith, Alan P. Lewis, Nick Barton, Michael L. Washburn, Ruud de Wildt

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.



中文翻译:

快速鉴定高效人抗GPCR拮抗剂单克隆抗体

摘要

复杂的细胞靶标,例如G蛋白偶联受体(GPCR),离子通道和其他多跨膜蛋白,对治疗性抗体的发现提出了重大挑战,这主要是因为从细胞膜提取靶蛋白的稳定性很差。为了评估是否可以利用有限的一组膜结合抗原格式来鉴定针对此类靶标的功能抗体,我们选择了具有治疗意义的GPCR(CCR1),并使用基于体外酵母的抗体发现平台(Adimab TM值),以加快命中率。最初,我们使用抗体库的一个子集,以“探索”方法比较了两种过量表达人CCR1的生物素化抗原形式。使用链霉亲和素包被的珠子分离结合剂,表达为酵母上清液,并在适当的细胞系上使用高通量结合测定和流式细胞术进行筛选。然后使用完整的文库多样性选择最合适的抗原以分离目标结合物。该方法确定了总共183个具有不同重链序列的mAb。克隆的子集在原代细胞趋化分析中显示出高效能,IC 50值在低nM /高pM范围内。为了评估进一步提高亲和力的可行性,纯化了全长hCCR1蛋白,从高亲和力和低亲和力单克隆抗体构建了互补决定区多样化的文库,并通过荧光激活的细胞分选技术分离出了改良的结合物。对于较低亲和力的亲本mAb观察到显着的亲和力增强,而对于高亲和力的mAb则没有观察到。这些数据举例说明了一种方法,该方法可以使用全细胞作为抗原快速产生有效的人单克隆抗体,以挑战靶标,并在需要时定义鉴定亲和力成熟变体的途径。

更新日期:2020-04-28
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