title: Molecular imprint of enzyme active site by camel nanobodies: rapid and efficient approach to produce abzymes with alliinase activity.
abstract: Screening of inhibitory Ab1 antibodies is a critical step for producing catalytic antibodies in the anti-idiotypic approach. However, the incompatible surface of the active site of the enzyme and the antigen-binding site of heterotetrameric conventional antibodies become the limiting step. Because camelid-derived nanobodies possess the potential to preferentially bind to the active site of enzymes due to their small size and long CDR3, we have developed a novel approach to produce antibodies with alliinase activities by exploiting the molecular mimicry of camel nanobodies. By screening the camelid-derived variable region of the heavy chain cDNA phage display library with alliinase, we obtained an inhibitory nanobody VHHA4 that recognizes the active site. Further screening with VHHA4 from the same variable domain of the heavy chain of a heavy-chain antibody library led to a higher incidence of anti-idiotypic Ab2 abzymes with alliinase activities. One of the abzymes, VHHC10, showed the highest activity that can be inhibited by Ab1 VHHA4 and alliinase competitive inhibitor penicillamine and significantly suppressed the B16 tumor cell growth in the presence of alliin in vitro. The results highlight the feasibility of producing abzymes via anti-idiotypic nanobody approach.
authors.affiliation: Xinjiang Key Laboratory of Biological Resources and Genetic Engineering College of Life Science and Technology, Xinjiang University, Urumqi 830046, China. jwli67@sina.com
authors.forename: Jiang-Wei
authors.initials: JW
authors.lastname: Li
authors.forename: Lijie
authors.initials: L
authors.lastname: Xia
authors.forename: Youhong
authors.initials: Y
authors.lastname: Su
authors.forename: Hongchun
authors.initials: H
authors.lastname: Liu
authors.forename: Xueqing
authors.initials: X
authors.lastname: Xia
authors.forename: Qinxia
authors.initials: Q
authors.lastname: Lu
authors.forename: Chunjin
authors.initials: C
authors.lastname: Yang
authors.forename: Kalbinur
authors.initials: K
authors.lastname: Reheman
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry; D000888:Antibodies, Anti-Idiotypic / Q000737:chemistry*; D001665:Binding Sites; D002162:Camelus; D013437:Carbon-Sulfur Lyases / Q000737:chemistry; D002384:Catalysis; D020134:Catalytic Domain; D004791:Enzyme Inhibitors / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004798:Enzymes / Q000737:chemistry; D005260:Female; D007700:Kinetics; D008969:Molecular Sequence Data; D036103:Nanotechnology / Q000379:methods*; D009369:Neoplasms / Q000276:immunology / Q000378:metabolism; D010396:Penicillamine / Q000737:chemistry; D019151:Peptide Library
chemical_list: D000906:Antibodies; D000888:Antibodies, Anti-Idiotypic; D004791:Enzyme Inhibitors; D004798:Enzymes; D019151:Peptide Library; D013437:Carbon-Sulfur Lyases; C019243:alliin lyase; D010396:Penicillamine