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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 k_on and the dissociation rate constant k_off 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.

SUBMITTER: Fukunaga A

PROVIDER: S-EPMC6068084 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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

Fukunaga Atsushi A Maeta Shingo S Reema Bajaj B Nakakido Makoto M Tsumoto Kouhei K

Biochemistry and biophysics reports 20180714

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 ...[more]

PMID: 30073208

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Project description:HIV-1 remains a global health concern and to date, nearly 38 million people are living with HIV. The complexity of HIV-1 pathogenesis and its subsequent prevalence is influenced by several factors including the HIV-1 subtype. HIV-1 subtype variation extends to sequence variation in the amino acids of the HIV-1 viral proteins. Of particular interest is the transactivation of transcription (Tat) protein due to its key function in viral transcription. The Tat protein predominantly functions by binding to the transactivation response (TAR) RNA element to activate HIV-1 transcriptional elongation. Subtype-specific Tat protein sequence variation influences Tat-TAR binding affinity. Despite several studies investigating Tat-TAR binding, it is not clear which regions of the Tat protein and/or individual Tat amino acid residues may contribute to TAR binding affinity. We, therefore, conducted a scoping review on studies investigating Tat-TAR binding. We aimed to synthesize the published data to determine (1) the regions of the Tat protein that may be involved in TAR binding, (2) key Tat amino acids involved in TAR binding and (3) if Tat subtype-specific variation influences TAR binding. A total of thirteen studies met our inclusion criteria and the key findings were that (1) both N-terminal and C-terminal amino acids outside the basic domain (47-59) may be important in increasing Tat-TAR binding affinity, (2) substitution of the amino acids Lysine and Arginine (47-59) resulted in a reduction in binding affinity to TAR, and (3) none of the included studies have investigated Tat subtype-specific substitutions and therefore no commentary could be made regarding which subtype may have a higher Tat-TAR binding affinity. Future studies investigating Tat-TAR binding should therefore use full-length Tat proteins and compare subtype-specific variations. Studies of such a nature may help explain why we see differential pathogenesis and prevalence when comparing HIV-1 subtypes.

Dataset Information

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

Publications

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

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