title: Molecular modeling and affinity determination of scFv antibody: proper linker peptide enhances its activity.
abstract: One of existing strategies to engineer active antibody is to link V(H) and V(L) domains via a linker peptide. How the composition, length, and conformation of the linker affect antibody activity, however, remains poorly understood. In this study, a dual approach that coordinates molecule modeling, biological measurements, and affinity evaluation was developed to quantify the binding activity of a novel stable miniaturized anti-CD20 antibody or single-chain fragment variable (scFv) with a linker peptide. Upon computer-guided homology modeling, distance geometry analysis, and molecular superimposition and optimization, three new linker peptides PT1, PT2, and PT3 with respective 7, 10, and 15 residues were proposed and three engineered antibodies were then constructed by linking the cloned V(H) and V(L) domains and fusing to a derivative of human IgG1. The binding stability and activity of scFv-Fc chimera to CD20 antigen was quantified using a micropipette adhesion frequency assay and a Scatchard analysis. Our data indicated that the binding affinity was similar for the chimera with PT2 or PT3 and approximately 24-fold higher than that for the chimera with PT1, supporting theoretical predictions in molecular modeling. These results further the understanding in the impact of linker peptide on antibody structure and activity.
authors.affiliation: Institute of Basic Medical Sciences, P.O. Box 130 (3), Taiping Road, Beijing 100850, P.R. China.
authors.forename: Xin
authors.initials: X
authors.lastname: Gu
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Jia
authors.forename: Jiannan
authors.initials: J
authors.lastname: Feng
authors.forename: Beifen
authors.initials: B
authors.lastname: Shen
authors.forename: Ying
authors.initials: Y
authors.lastname: Huang
authors.forename: Shusheng
authors.initials: S
authors.lastname: Geng
authors.forename: Yingxun
authors.initials: Y
authors.lastname: Sun
authors.forename: Yugang
authors.initials: Y
authors.lastname: Wang
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Mian
authors.initials: M
authors.lastname: Long
mesh_terms: D001665:Binding Sites; D008956:Models, Chemical; D018448:Models, Immunological; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry* / Q000276:immunology*; D011485:Protein Binding; D011487:Protein Conformation; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000276:immunology* / Q000648:ultrastructure
chemical_list: D010455:Peptides; D057127:Single-Chain Antibodies