Development of a novel mammalian display system for selection of antibodies against membrane proteins |
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Authors: | Nathan Robertson Nancy Lopez-Anton Shalom A. Gurjar Hena Khalique Zainab Khalaf Siobhan Clerkin Vaughan R. Leydon Richard Parker-Manuel Alexander Raeside Tom Payne Tim D. Jones Len Seymour Ryan Cawood |
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Affiliation: | 1.OXGENE, Medawar Centre, Oxford, United Kingdom;2.Anticancer Viruses and Cancer Vaccines Group, Department of Oncology, University of Oxford, Oxford, United Kingdom |
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Abstract: | Reliable, specific polyclonal and monoclonal antibodies are important tools in research and medicine. However, the discovery of antibodies against their targets in their native forms is difficult. Here, we present a novel method for discovery of antibodies against membrane proteins in their native configuration in mammalian cells. The method involves the co-expression of an antibody library in a population of mammalian cells that express the target polypeptide within a natural membrane environment on the cell surface. Cells that secrete a single-chain fragment variable (scFv) that binds to the target membrane protein thereby become self-labeled, enabling enrichment and isolation by magnetic sorting and FRET-based flow sorting. Library sizes of up to 109 variants can be screened, thus allowing campaigns of naïve scFv libraries to be selected against membrane protein antigens in a Chinese hamster ovary cell system. We validate this method by screening a synthetic naïve human scFv library against Chinese hamster ovary cells expressing the oncogenic target epithelial cell adhesion molecule and identify a panel of three novel binders to this membrane protein, one with a dissociation constant (KD) as low as 0.8 nm. We further demonstrate that the identified antibodies have utility for killing epithelial cell adhesion molecule–positive cells when used as a targeting domain on chimeric antigen receptor T cells. Thus, we provide a new tool for identifying novel antibodies that act against membrane proteins, which could catalyze the discovery of new candidates for antibody-based therapies. |
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Keywords: | mammalian display epithelial cell adhesion molecule (EpCAM) chimeric antigen receptor T cells (CAR-T) therapeutic antibody discovery antibody antibody engineering membrane protein immunotherapy |
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