title: Affinity maturation of a CDR3-grafted VHH using in silico analysis and surface plasmon resonance.
abstract: A requirement for advancing antibody-based medicine is the development of proteins that can bind with high affinity to a specific epitope related to a critical protein activity site. As a part of generating such proteins, we have succeeded in creating a binding protein without changing epitope by complementarity-determining region 3 (CDR3) grafting (Inoue et al., Affinity transfer to a human protein by CDR3 grafting of camelid VHH. Protein Sci. 20, 1971-1981). However, the affinity of the target-binding protein was low. In this manuscript, the affinity maturation of a target-binding protein was examined using CDR3-grafted camelid single domain antibody (VHH) as a model protein. Several amino acids in the CDR1 and CDR2 regions of VHH were mutated to tyrosines and/or serines and screened for affinity-matured proteins by using in silico analysis. The mutation of two amino acids in the CDR2 region to arginine and/or aspartic acid increased the affinity by decreasing the dissociation rate. The affinity of designed mutant increased by ∼20-fold over that of the original protein. In the present study, candidate mutants were narrowed down using in silico screening and computational modelling, thus avoiding much in vitro analytical effort. Therefore, the method used in this study is expected to be one of the useful for promoting affinity maturation of antibodies.
authors.affiliation: Kyowa Hakko Kirin Co., Ltd., 3-6-6 Asahi-machi, Machida, Tokyo 194-8533; and Department of Bioinformatics, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan.
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Inoue
authors.forename: Akiko
authors.initials: A
authors.lastname: Suganami
authors.forename: Isao
authors.initials: I
authors.lastname: Ishida
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Tamura
authors.forename: Yoshitake
authors.initials: Y
authors.lastname: Maeda
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002161:Camelids, New World; D003198:Computer Simulation; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D061905:Single-Domain Antibodies / Q000737:chemistry* / Q000235:genetics; D020349:Surface Plasmon Resonance
chemical_list: D011994:Recombinant Proteins; D061905:Single-Domain Antibodies
keywords: VHH; affinity maturation; in silico analysis; peptide grafting; scaffold