Pentamerization of single-domain antibodies from phage libraries: a novel strategy for the rapid generation of high-avidity antibody reagents.
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.
Zhang
Jianbing
J
Institute for Biological Sciences, National Research Council of Canada, K1A 0R6, Ottawa, Ont., Canada.
Tanha
Jamshid
J
Hirama
Tomoko
T
Khieu
Nam Huan
NH
To
Rebecca
R
Tong-Sevinc
Hong
H
Stone
Emily
E
Brisson
Jean Robert
JR
MacKenzie
C Roger
CR
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
D000906:Antibodies; D001427:Bacterial Toxins; D007202:Indicators and Reagents; D010446:Peptide Fragments; D019151:Peptide Library; D011993:Recombinant Fusion Proteins