Leucine zipper dimerized bivalent and bispecific scFv antibodies from a semi-synthetic antibody phage display library.
This report describes the construction of leucine zipper-based dimerization cassettes for the conversion of recombinant monomeric scFv antibody fragments to bivalent and bispecific dimers. A truncated murine IgG3 hinge region and a Fos or Jun leucine zipper were cloned into four scFv fragments previously isolated from a synthetic antibody phage display library. Cysteine residues flanking the zipper region were introduced to covalently link dimerized scFv fragments. The secreted fusion proteins were shown to spontaneously and efficiently form stable Fos-Fos or Jun-Jun homodimers in the Escherichia coli periplasm at levels comparable to their monovalent counterparts. The bivalent (scFv)2 fragments performed well in enzyme-linked immunosorbent assay, flowcytometric, and immunohistochemical analysis. Fos and Jun homodimer (scFv)2 antibodies with different specificities could be reduced, reshuffled, and reoxidized to form preparations of functional bispecific (scFv)2 Fos-Jun heterodimers. These Fos and Jun fusion protein cassettes provide a universal basis for the construction of dimeric scFv antibodies with enhanced avidity or dual specificity.
de Kruif
J
J
Department of Immunology, Utrecht University, The Netherlands.
Logtenberg
T
T
D000595:Amino Acid Sequence; D000818:Animals; D018033:Antibodies, Bispecific / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D000918:Antibody Specificity; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity; D017931:DNA Primers; D016208:Databases, Factual; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D007074:Immunoglobulin G; D007150:Immunohistochemistry; D016350:Leucine Zippers / Q000276:immunology*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016760:Proto-Oncogene Proteins c-fos / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology; D016755:Proto-Oncogene Proteins c-jun / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification
D018033:Antibodies, Bispecific; D017931:DNA Primers; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D046911:Macromolecular Substances; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D011994:Recombinant Proteins