Abstract
Phage-displayed synthetic antibody (Ab) repertoires have become a major source of affinity reagents for basic and clinical research. Specific Abs identified from such libraries are often screened as fragments antigen binding (Fabs) produced in bacteria, and those with desired biochemical characteristics are reformatted for production as full-length immunoglobulin G (IgG) in mammalian cells. The conversion of Fabs to IgGs is a cumbersome and often rate-limiting step in the development of Abs. Moreover, biochemical properties required for lead IgG development are not always shared by the Fabs, and these issues are not uncovered until a significant effort has been spent on Abs that ultimately will not be useful. Thus, there is a need for simple and rapid techniques to convert phage-displayed Fabs to IgGs at an early stage of the Ab screening process. We report the generation of a highly diverse phage-displayed synthetic single-chain Fab (scFab) library, in which the light and heavy chains were tethered with an optimized linker. Following selection, pools of scFabs were converted to single-chain IgGs (scIgGs) en masse, enabling facile screening of hundreds of phage-derived scIgGs. We show that this approach can be used to rapidly screen for and select scIgGs that target cell-surface receptors, and scIgGs behave the same as conventional IgGs.