Affinity Maturation for Antibody Engineering: The Critical Role of Residues on CDR Loops of Antibodies in Antigen Binding.

During the course of affinity maturation, antibodies exhibit enhanced antigen-binding affinities by altering the amino acids in their variable regions. Understanding the structural basis of these antibodies can be beneficial for antibody engineering. We determined the crystal structures of single-chain Fv (scFv) antibodies against (4-hydroxy-3-nitrophenyl)acetyl, C6 and E11, which had undergone affinity maturation. Compared with germline-type antibodies, the affinity-matured antibodies with somatic hypermutation from Lys58 to Arg58 of the heavy chain located in the complementarity-determining region 2 (CDR2) seemed to be critical for increasing the antigen-binding affinity. E11 possessed a disulfide bond at the base of CDR3 in the heavy chain, which contributed to a further increase in its antigen-binding affinity compared with that of C6. In this study, we generated several mutant scFvs of C6 and E11 and analyzed their antigen-binding thermodynamics using isothermal titration calorimetry. The results indicated that the CDR conformations could adjust antigen-binding not only at the mutated sites but also at the surrounding residues. The analysis of folding thermodynamics showed that the stability of the affinity-matured antibodies was lower than that of the germline-type antibodies and remarkably increased upon strong antigen binding. The results also indicated that the structural dynamics of the affinity-matured antibodies were greater than those of the germline-type antibodies and decreased upon antigen binding.