De Novo Selection of High-Affinity Antibodies from Synthetic Fab Libraries Displayed on Phage as pIX Fusion Proteins

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• 作者：Lei Shi ; John C. Wheeler ; Raymond W. Sweet ; Jin Lu ; Jinquan Luo ; Mark Tornetta ; Brian Whitaker ; Ramach ; ra Reddy ; Raymond Brittingham ; Lina Borozdina ; Qiang Chen ; Bernard Amegadzie ; David M. Knight ; Ju
• 关键词：immunoglobulin ; Fab libraries ; phage display ; affinity maturation ; therapeutic antibodies
• 刊名：Journal of Molecular Biology
• 出版年：2010
• 出版时间：26 March 2010
• 年：2010
• 卷：397
• 期：2
• 页码：385-396
• 全文大小：998 K

文摘

Filamentous phage was the first display platform employed to isolate antibodies in vitro and is still the most broadly used. The success of phage display is due to its robustness, ease of use, and comprehensive technology development, as well as a broad range of selection methods developed during the last two decades. We report here the first combinatorial synthetic Fab libraries displayed on pIX, a fusion partner different from the widely used pIII. The libraries were constructed on four V_L and three V_H domains encoded by IGV and IGJ germ-line genes frequently used in human antibodies, which were diversified to mirror the variability observed in the germ-line genes and antibodies isolated from natural sources. Two sets of libraries were built, one with diversity focused on V_H by keeping V_L in the germ-line gene configuration and the other with diversity in both V domains. After selection on a diverse panel of proteins, numerous specific Fabs with affinities ranging from 0.2 nM to 20 nM were isolated. V_H diversity was sufficient for isolating Fabs to most antigens, whereas variability in V_L was required for isolation of antibodies to some targets. After the application of an integrated maturation process consisting of reshuffling V_L diversity, the affinity of selected antibodies was improved up to 100-fold to the low picomolar range, suitable for in vivo studies. The results demonstrate the feasibility of displaying complex Fab libraries as pIX fusion proteins for antibody discovery and optimization and lay the foundation for studies on the structure–function relationships of antibodies.