Pentamerization of Single-domain Antibodies from Phage Libraries: A Novel Strategy for the Rapid Generation of High-avidity Antibody Reagents

• Published in: Journal of molecular biology Vol. 335; no. 1; pp. 49 - 56
• Main Authors: Zhang, Jianbing, Tanha, Jamshid, Hirama, Tomoko, Khieu, Nam Huan, To, Rebecca, Tong-Sevinc, Hong, Stone, Emily, Brisson, Jean-Robert, Roger MacKenzie, C
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 02.01.2004
• Subjects: Animals; Antibodies - chemistry; Antibodies - genetics; Antibody Affinity; antibody engineering; Bacterial Toxins - genetics; Camelids, New World; Dimerization; Drug Stability; Escherichia coli; Escherichia coli - genetics; Indicators and Reagents; multivalency; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Library; phage display; Protein Denaturation; Protein Engineering - methods; Protein Structure, Tertiary; proteomics; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - immunology; single-domain antibodies; Fab, antigen-binding fragment; BSA, bovine serum albumin; proteomics; Gb 3, globotriaosylceramide; RMSF, root-mean-square fluctuations; VT1B, verotoxin 1 B subunit; PTH, parathyroid hormone; RU, response unit; multivalency; antibody engineering; MD, molecular dynamics; phage display; scFv, single chain variable fragment; sdAb, single-domain antibody; single-domain antibodies; SPR, surface plasmon resonance
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2003.09.034

We describe a novel type of molecule in which single-domain antibodies (sdAbs) isolated from a naı̈ve 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.