title: Immunotargeting of antioxidant enzyme to the pulmonary endothelium.
abstract: Oxidative injury to the pulmonary endothelium has pathological significance for a spectrum of diseases. Administration of antioxidant enzymes, superoxide dismutase (SOD) and catalase (Cat), has been proposed as a method to protect endothelium. However, neither these enzymes nor their derivatives possess specific affinity to endothelium and do not accumulate in the lung. Previously we have described a monoclonal antibody to angiotensin-converting enzyme (ACE) that accumulates selectively in the lung after systemic injection in rats, hamsters, cats, monkeys, and humans. In the present work we describe a system for selective intrapulmonary delivery of CuZn-SOD and Cat conjugated with biotinylated anti-ACE antibody mAb 9B9 (b-mAb 9B9) by a streptavidin (SA)-biotin bridge. Both enzymes biotinylated with biotin ester at biotin/enzyme ratio 20 retain enzymatic activity and bind SA without loss of activity. We have constructed tri-molecular heteropolymer complexes consisting of b-mAb 9B9, SA, and biotinylated SOD or biotinylated Cat and have studied biodistribution and pulmonary uptake of these complexes in the rat after i.v. injection. Biodistribution of biotinylated enzymes was similar to that of nonmodified enzymes. Binding of SA markedly prolonged lifetime of biotinylated enzymes in the circulation. In contrast to enzymes conjugated with nonspecific IgG, other enzyme derivatives, and nonmodified enzymes, biotinylated enzymes conjugated with b-mAb 9B9 accumulated specifically in the rat lung (9% of injected SOD/g of lung tissue and 7.5% of injected Cat/g of lung tissue). Pulmonary uptake of nonmodified enzymes or derivatives with nonspecific IgG did not exceed 0.5% of injected dose/g. Both SOD and Cat conjugated with b-mAb 9B9 were retained in the rat lung for at least several hours. Trichloracetic acid-precipitable radiolabeled Cat was associated with microsomal and plasma membrane fractions of the lung tissue homogenate. Thus, modification of antioxidant enzymes with biotin and SA-mediated conjugation with b-mAb 9B9 prolongs the circulation of enzymes resulting in selective accumulation in the lung and intracellular delivery of enzymes to the pulmonary endothelium. These results provide the background for an approach to provide protection of pulmonary endothelium against oxidative insults.
authors.affiliation: Insitute for Environmental Medicine, University of Pennsylvania Medical Center, Philadelphia, 19104, USA.
authors.forename: V R
authors.initials: VR
authors.lastname: Muzykantov
authors.forename: E N
authors.initials: EN
authors.lastname: Atochina
authors.forename: H
authors.initials: H
authors.lastname: Ischiropoulos
authors.forename: S M
authors.initials: SM
authors.lastname: Danilov
authors.forename: A B
authors.initials: AB
authors.lastname: Fisher
mesh_terms: D000818:Animals; D002374:Catalase / Q000493:pharmacokinetics*; D002417:Cattle; D006224:Cricetinae; D004727:Endothelium / Q000378:metabolism; D004912:Erythrocytes / Q000201:enzymology; D000882:Haplorhini; D006801:Humans; D008099:Liver / Q000201:enzymology; D008168:Lung / Q000378:metabolism*; D008297:Male; D008657:Metabolic Clearance Rate; D007703:Peptidyl-Dipeptidase A / Q000493:pharmacokinetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D013482:Superoxide Dismutase / Q000493:pharmacokinetics*; D014018:Tissue Distribution
chemical_list: D002374:Catalase; D013482:Superoxide Dismutase; D007703:Peptidyl-Dipeptidase A