Improvement of antibody affinity by introduction of basic amino acid residues into the framework region

Abstract

Antibodies are widely used not only as therapeutic agents but also as research tools and diagnostic agents, and extensive efforts have been made to generate antibodies that have higher affinity. It was recently reported that introduction of charged residues into the framework region of an antibody improved its affinity; however, the underlying molecular mechanism has not been elucidated. In this study, we used kinetic and thermodynamic analyses of the antibody–antigen interaction to investigate the molecular mechanism by which an antibody with introduced charged residues recognizes its antigen with higher affinity. The introduction of basic amino acid residues resulted in improvement of the affinity whereas the introduction of acidic residues weakened the interaction. For two mutant antigen-binding fragments (Fabs) with improved affinity (named K5- and R5-mutants), the balance between the association rate constant kon and the dissociation rate constant koff was distinct despite each mutant having the same number of charged residues. Moreover, thermodynamic analysis of the interactions in the transition state revealed a difference between the K5- and R5-mutants in terms of enthalpic energy change following formation of the encounter complex with the antigen. These results suggest that the affinity of the K5- and R5-mutants is improved by distinct mechanisms. Although the mutations destabilize the Fab and necessitate further studies, our strategy is expected to become a versatile and simple means to improve the affinity of antibodies to their antigens.

Semi-automated single-molecule microscopy screening of fast-dissociating specific antibodies directly from hybridoma cultures

“…For such antigens, one solution is to append epitope tags, such as FLAG, S, and V5 tags. Antibody engineering might also be used to produce fast-dissociating antibodies, for example, through amino acid substitutions within the antigen recognition sites of antibodies to accelerate dissociation from epitopes ( Fukunaga et al, 2018 ; Hugo et al, 2003 ).…”

Natural Catalytic IgGs Hydrolyzing Histones in Schizophrenia: Are They the Link between Humoral Immunity and Inflammation?

“…Therefore, some antibodies in schizophrenia acquire catalytic activity and rather high rates of catalysis due to their lower affinity than those of tight binding autoantibodies. Binding affinity is determined by the number of bonds formed with the antigen, and accordingly is determined by the amino acid residues that form the CDR [ 60 ]. Therefore, it can be assumed that the antibodies of patients with catalytic activity have a specific CDR structure.…”

The Bioinformatic and In Vitro Studies of Clostridioides Difficile Aminopeptidase M24 Revealed the Immunoreactive KKGIK Peptide

“…In the case of an antibody-antigen binding, it is able to strengthen the connection through hydrogen bonds or electrostatic forces [66,67]. In 2018, Fukunaga et al showed that the introduction of lysine to the Fab region of an antibody improves its affinity to the given antigen [68]. In turn, Thommesen et al pointed out the key role of lysine in the human IgG3 for the complement activation [69].…”