title: Pentamerization of single-domain antibodies from phage libraries: a novel strategy for the rapid generation of high-avidity antibody reagents.
abstract: We describe a novel type of molecule in which single-domain antibodies (sdAbs) isolated from a nai;ve llama single domain antibody library are linked to an oligomerization domain to generate high-avidity, antigen-binding reagents. An sdAb is fused to the B-subunit of Escherichia coli verotoxin, or shiga-like toxin, which self-assembles to form a homopentamer and results in simultaneous sdAb pentamerization and introduction of avidity. Molecular modeling indicated that this fusion protein (PDB: 1OJF), termed pentabody, has structural flexibility for binding to surface-presented antigen. In the instance of an sdAb specific for a peptide antigen, pentamerization resulted in a dramatic increase in functional affinity for immobilized antigen. The pentabody was expressed in high yield in E.coli in a non-aggregated state, and exhibited excellent thermostability and protease resistance. This technology provides a relatively rapid means of generating novel antigen-binding molecules that bind strongly to immobilized antigen. It is expected that pentavalent sdAbs will have general applicability in proteomics, immunochemical staining, cancer diagnosis and other applications in which antigens are presented multivalently.
authors.affiliation: Institute for Biological Sciences, National Research Council of Canada, K1A 0R6, Ottawa, Ont., Canada.
authors.forename: Jianbing
authors.initials: J
authors.lastname: Zhang
authors.forename: Jamshid
authors.initials: J
authors.lastname: Tanha
authors.forename: Tomoko
authors.initials: T
authors.lastname: Hirama
authors.forename: Nam Huan
authors.initials: NH
authors.lastname: Khieu
authors.forename: Rebecca
authors.initials: R
authors.lastname: To
authors.forename: Hong
authors.initials: H
authors.lastname: Tong-Sevinc
authors.forename: Emily
authors.initials: E
authors.lastname: Stone
authors.forename: Jean Robert
authors.initials: JR
authors.lastname: Brisson
authors.forename: C Roger
authors.initials: CR
authors.lastname: MacKenzie
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000235:genetics*; D000915:Antibody Affinity; D001427:Bacterial Toxins / Q000235:genetics; D002161:Camelids, New World; D019281:Dimerization; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D007202:Indicators and Reagents; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D019151:Peptide Library; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology
chemical_list: D000906:Antibodies; D001427:Bacterial Toxins; D007202:Indicators and Reagents; D010446:Peptide Fragments; D019151:Peptide Library; D011993:Recombinant Fusion Proteins