Renaut Laurence, Monnet Céline, Dubreuil Olivier, Zaki Ouafa, Crozet Fabien, Bouayadi Khalil, Kharrat Hakim, Mondon Philippe
MILLEGEN SA, Immeuble BIOSTEP-Bâtiment A, Labege, France.
Methods Mol Biol. 2012;907:451-61. doi: 10.1007/978-1-61779-974-7_26.
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.
随着越来越多的治疗性抗体被开发出来，需要有强大的方法来有效地提高抗体候选物的亲和力和/或特异性。在此，我们描述了我们强大的平台，该平台结合了单链抗体片段（scFv）亲和力成熟和IgG高通量筛选。在利用我们的随机诱变技术（MutaGen™）创造多样性后，scFv文库通过引入β-内酰胺酶基因的融合系统进行全面筛选，以基于氨苄青霉素抗性选择读框内且无终止密码子的变体。由此构建的高质量scFv文库随后利用噬菌体展示技术在体外进行靶标筛选。与标准程序不同的是，我们不是将有限数量的亲和力成熟的scFv作为IgG分子来生产，而是开发了一种克隆系统，将整个选定的scFv库直接转移到一个IgG表达载体中，从而允许在哺乳动物细胞中快速进行IgG小规模生产（96孔）。我们的整合流程使我们能够生成高质量的scFv文库并测试众多IgG变体，增加了选择最佳治疗性抗体候选物的机会。
