Single-domain antibodies (sdAbs)， the variable domains of camelid heavy chain-only antibodies， are generally thought to poorly recognize nonproteinaceous small molecules and carbohydrates in comparison with conventional antibodies. However， the structures of anti-methotrexate， anti-triclocarban and anti-cortisol sdAbs revealed unexpected contributions of the non-hypervariable "CDR4" loop， formed between β-strands D and E of framework region 3， in binding. Here， we investigated the potential role of CDR4 in sdAb binding to a hapten， 15-acetyl-deoxynivalenol (15-AcDON)， and to carbohydrates. We constructed and panned a phage-displayed library in which CDR4 of the 15-AcDON-specific sdAb， NAT-2... More
Single-domain antibodies (sdAbs)， the variable domains of camelid heavy chain-only antibodies， are generally thought to poorly recognize nonproteinaceous small molecules and carbohydrates in comparison with conventional antibodies. However， the structures of anti-methotrexate， anti-triclocarban and anti-cortisol sdAbs revealed unexpected contributions of the non-hypervariable "CDR4" loop， formed between β-strands D and E of framework region 3， in binding. Here， we investigated the potential role of CDR4 in sdAb binding to a hapten， 15-acetyl-deoxynivalenol (15-AcDON)， and to carbohydrates. We constructed and panned a phage-displayed library in which CDR4 of the 15-AcDON-specific sdAb， NAT-267， was extended and randomized. From this library， we identified one sdAb， MA-232， bearing a 14-residue insertion in CDR4 and showing improved binding to 15-AcDON by ELISA and surface plasmon resonance. On the basis of these results， we constructed a second set of phage-displayed libraries in which the CDR4 and other regions of three hapten- or carbohydrate-binding sdAbs were diversified. With the goal of identifying sdAbs with novel glycan-binding specificities， we panned the library against four tumor-associated carbohydrate antigens but were unable to enrich binding phages. Thus， we conclude that while CDR4 may play a role in binding of some rare hapten-specific sdAbs， diversifying this region through molecular engineering is probably not a general solution to sdAb carbohydrate recognition in the absence of a paired V domain.
