A strategy for phage display selection of functional domain-exchanged immunoglobulin scaffolds with high affinity for glycan targets

Monoclonal antibodies are essential reagents for deciphering gene or protein function and have been a fruitful source of therapeutic and diagnostic agents. However, the use of anticarbohydrate antibodies to target glycans for these purposes has been less successful. Glycans contain less hydrophobic functionality than do proteins or nucleic acids, thus individual glycan–antibody interactions are relatively weak. Information encoded by glycans often involves subtle variations of branched oligosaccharides that cannot be detected with conventional antibodies. Here we describe a new phage display selection strategy for identification of high-affinity and specific glycan antibodies. We designed and characterized a phage clone that functionally displays the unique architectural scaffold of 2G12, an antibody that targets oligomannoses on the HIV-1 glycoprotein gp120. The two heavy chain variable domains of 2G12 exchange positions to create an extended recognition surface containing four oligomannose binding sites per IgG molecule. We designed and characterized a phage clone in which this domain exchange architecture was recapitulated as an antigen binding fragment dimer [(Fab)2]on the phage surface by protein engineering. The functional display of the 2G12 (Fab)2 fragment was validated by Western blot and phage enzyme-linked immunosorbent assay. Furthermore, we demonstrate that this 2G12 (Fab)2 display system is amenable to selection of functional clones using a mock selection. These results provide proof-of-concept that the privileged 2G12 domain-exchanged scaffold can be used for design of novel antibody libraries that are biased toward glycan recognition.

► Anti-glycan antibodies with high affinity and specificity are difficult to obtain.
► Synthetic antibody technology circumvents many limitations of other antibody isolation methods.
► 2G12 is an unusual domain-exchanged scaffold that binds oligomannose on HIV-1 gp41.
► We designed and characterized a phage clone that displays 2G12 on its surface.
► This clone can be used for synthetic antibody library development against glycans.