title: Characterization of scFv-Ig constructs generated from the anti-CD20 mAb 1F5 using linker peptides of varying lengths.
abstract: The heavy (VH) and light (VL) chain variable regions of the murine anti-human CD20 mAb 1F5 were cloned, and four single-chain Ab (scFv) molecules were constructed using linker peptides of variable lengths to join the VH and VL domains. Three constructs were engineered using linker peptides of 15, 10, and 5 aa residues consisting of (GGGGS)3, (GGGGS)2, and (GGGGS)1 sequences, respectively, whereas the fourth was prepared by joining the VH and VL domains directly. Each construct was fused to a derivative of human IgG1 (hinge plus CH2 plus CH3) to facilitate purification using staphylococcal protein A. The aggregation and CD20 binding properties of these four 1F5 scFv-Ig derivatives produced were investigated. Both size-exclusion HPLC column analysis and Western blots of proteins subjected to nonreducing SDS-PAGE suggested that all four 1F5 scFv-Ig were monomeric with m.w. of approximately 55 kDa. The CD20 binding properties of the four 1F5 scFv-Ig were studied by ELISA and flow cytometry. The 1F5 scFv-Ig with the 5-aa linker (GS1) demonstrated significantly superior binding to CD20-expressing target cells, compared with the other scFv-Ig constructs. Scatchard analysis of the radiolabeled monovalent GS1 scFv-Ig revealed a binding avidity of 1.35 x 108 M-1 compared with an avidity of 7.56 x 108 M-1 for the native bivalent 1F5 Ab. These findings suggest that the GS1 scFv-Ig with a short linker peptide of approximately 5 aa is the best of the engineered constructs for future studies.
authors.affiliation: Department of Biological Structure, University of Washington, Seattle 98195, USA.
authors.forename: D
authors.initials: D
authors.lastname: Shan
authors.forename: O W
authors.initials: OW
authors.lastname: Press
authors.forename: T T
authors.initials: TT
authors.lastname: Tsu
authors.forename: M S
authors.initials: MS
authors.lastname: Hayden
authors.forename: J A
authors.initials: JA
authors.lastname: Ledbetter
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D018951:Antigens, CD20 / Q000276:immunology*; D017209:Apoptosis / Q000276:immunology; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology; D001483:Base Sequence; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers / Q000096:biosynthesis / Q000138:chemical synthesis; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis* / Q000276:immunology*; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D018951:Antigens, CD20; D017931:DNA Primers; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D010455:Peptides