Cloning and characterization of a single-chain fragment of monoclonal antibody to ACE suitable for lung endothelial targeting.
Monoclonal antibody (mAb) 9B9 to angiotensin-converting enzyme (ACE) demonstrates selective accumulation in lung tissue of the rat, hamster, cat, monkey and human after systemic injection. It has also been demonstrated that mAb 9B9 is the useful tool for targeting therapeutic agents or genes to lung endothelium. In this study, we describe the generation and characterization of a single-chain derivative (scFv) of mAb9B9 (scFv 9B9). In vitro, scFv9B9 retains the ability of the parental antibody to recognize human and rat ACE when expressed both on the surface of phage and as a soluble protein in prokaryotic and eukaryotic expression systems. The ability of scFv 9B9 presented by phage or the soluble protein labeled with I(125) to recognize ACE in the pulmonary circulation was also confirmed in an in vivo rat model. Sequence analysis revealed a putative glycosylation site in close proximity to the complementarity determining region 2 (CDR2) of the scFv 9B9 heavy chain. Mutation of Asn68 to Gln in the heavy chain of scFv 9B9 eliminated the glycosylation site and significantly improved the binding affinity of scFv 9B9 to human ACE as determined by cell ELISA and Western Blot. Moreover, Asn68Gln scFv 9B9 showed a greater rate of secretion at 30°C than wild type scFv 9B9, but had a decreased thermal stability at 37°C. The development of a stable and functional single-chain format of mAb 9B9 which specifically recognizes human and rat ACE represents a novel antibody-based reagent suitable for targeted delivery of drugs/genes to the pulmonary circulation.
Balyasnikova
Irina V
IV
Department of Anesthesiology, University of Illinois at Chicago, Chicago, IL 60637, USA. ibalyasn@surgery.bsd.uchicago.edu
Berestetskaya
Julia V
JV
Visintine
David J
DJ
Nesterovitch
Andrew B
AB
Adamian
Larisa
L
Danilov
Sergei M
SM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000918:Antibody Specificity; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D003001:Cloning, Molecular; D022801:Complementarity Determining Regions; D006224:Cricetinae; D003412:Cricetulus; D004337:Drug Carriers; D042783:Endothelial Cells / Q000201:enzymology* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D006031:Glycosylation; D006801:Humans; D006825:Hybridomas; D008168:Lung / Q000098:blood supply*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D007703:Peptidyl-Dipeptidase A / Q000276:immunology*; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D051381:Rats; D057127:Single-Chain Antibodies / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013045:Species Specificity; D013329:Structure-Activity Relationship; D013696:Temperature; D014162:Transfection
D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D004337:Drug Carriers; D057127:Single-Chain Antibodies; D007703:Peptidyl-Dipeptidase A