title: Bivalent antibody phage display mimics natural immunoglobulin.
abstract: We report the development of a system for displaying bivalent antibody fragments on M13 bacteriophage in a manner that effectively mimics the binding behavior of natural antibodies. In the "bivalent display" format, two copies of antigen binding sites are displayed on the coat of a single phage particle. Bivalent display was first achieved by the insertion of a dimerization domain, consisting of an IgG1 hinge region and a homodimerizing GCN4 leucine zipper, between a Fab and the C-terminal domain of the M13 gene-3 minor coat protein. In a phagemid-based display system, the resulting "Fab'-zip-phage" particles display bivalent Fabs that resemble natural IgGs. An important functional consequence of bivalent display is an avidity effect, which results in a greatly reduced off-rate for phage bound to immobilized antigen. The avidity effect improved the capture and retention of bivalent Fab'-zip-phage relative to monovalent Fab-phage both with antigen immobilized on plates and with cell surface antigen. To examine the requirements for bivalent display on phage, we systematically trimmed down the dimerization domain and found that a single cysteine was sufficient to confer the same avidity effect conferred by the complete dimerization domain. Bivalent antibody phage display should be useful for many applications. In particular, the technology should aid in the production of antibodies against difficult antigens, and also, in selections that require dimerization for activity.
authors.affiliation: Department of Protein Engineering, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
authors.forename: Chingwei V
authors.initials: CV
authors.lastname: Lee
authors.forename: Sachdev S
authors.initials: SS
authors.lastname: Sidhu
authors.forename: Germaine
authors.initials: G
authors.lastname: Fuh
mesh_terms: D000595:Amino Acid Sequence; D018033:Antibodies, Bispecific / Q000737:chemistry* / Q000235:genetics; D017104:Bacteriophage M13 / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000235:genetics; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics; D016350:Leucine Zippers; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D019151:Peptide Library
chemical_list: D018033:Antibodies, Bispecific; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D019151:Peptide Library