Abstract
As a growing number of therapeutic antibodies are developed, robust methods to efficiently improve the affinity and/or specificity of antibody candidates are needed. Here we describe our powerful platform that combines scFv affinity maturation and IgG high-throughput screening. After creating diversity with our random mutagenesis technology (MutaGen™), the scFv libraries are fully cleaned using a fusion system introducing the beta-lactamase gene to select in-frame and stop codon free variants on the basis of ampicillin resistance. The high-quality scFv libraries thereby constructed are then selected on the target in vitro using phage display technology. Contrary to standard procedures, instead of producing a limited number of affinity matured scFv as IgG molecules, we developed a cloning system to directly transfer the entire pool of selected scFv into an IgG expression vector permitting rapid IgG small-scale production (96 wells) in mammalian cells. Our integrated process allows us to generate high-quality scFv libraries and test numerous IgG variants, increasing the chances to select the best therapeutic antibody candidate.
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Renaut, L. et al. (2012). Affinity Maturation of Antibodies: Optimized Methods to Generate High-Quality ScFv Libraries and Isolate IgG Candidates by High-Throughput Screening. In: Chames, P. (eds) Antibody Engineering. Methods in Molecular Biology, vol 907. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-974-7_26
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DOI: https://doi.org/10.1007/978-1-61779-974-7_26
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-973-0
Online ISBN: 978-1-61779-974-7
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