Potent enzyme inhibitors derived from dromedary heavy-chain antibodies.
Evidence is provided that dromedary heavy-chain antibodies, in vivo-matured in the absence of light chains, are a unique source of inhibitory antibodies. After immunization of a dromedary with bovine erythrocyte carbonic anhydrase and porcine pancreatic alpha-amylase, it was demonstrated that a considerable amount of heavy-chain antibodies, acting as true competitive inhibitors, circulate in the bloodstream. In contrast, the conventional antibodies apparently do not interact with the enzyme's active site. Next we illustrated that peripheral blood lymphocytes are suitable for one-step cloning of the variable domain fragments in a phage-display vector. By bio-panning, several antigen-specific single-domain fragments are readily isolated for both enzymes. In addition we show that among those isolated fragments active site binders are well represented. When produced as recombinant protein in Escherichia coli, these active site binders appear to be potent enzyme inhibitors when tested in chromogenic assays. The low complexity of the antigen-binding site of these single-domain antibodies composed of only three loops could be valuable for designing smaller synthetic inhibitors.
Lauwereys
M
M
Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Paardenstraat 65, 1640-Sint-Genesius Rode, Belgium. mlauwere@vub.ac.be
Arbabi Ghahroudi
M
M
Desmyter
A
A
Kinne
J
J
Hölzer
W
W
De Genst
E
E
Wyns
L
L
Muyldermans
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity; D000918:Antibody Specificity; D002162:Camelus; D002257:Carbonic Anhydrase Inhibitors; D002256:Carbonic Anhydrases / Q000276:immunology / Q000378:metabolism; D002417:Cattle; D004791:Enzyme Inhibitors / Q000276:immunology* / Q000302:isolation & purification; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000145:classification / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000145:classification / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment; D013552:Swine; D000516:alpha-Amylases / Q000037:antagonists & inhibitors* / Q000276:immunology
D002257:Carbonic Anhydrase Inhibitors; D004791:Enzyme Inhibitors; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D000516:alpha-Amylases; D002256:Carbonic Anhydrases