Role of the non-hypervariable FR3 D-E loop in single-domain antibody recognition of haptens and carbohydrates

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_L domain.