The Functional Effects of Biotinylation of Anti-angiotensin-Converting Enzyme Monoclonal Antibody in Terms of Targeting in Vivo

Abstract

The effect of modification with biotin N-hydroxysuccinimide ester of mouse monoclonal antibody to angiotensin-converting enzyme, anti-ACE Mab 9B9, on its targeting to endothelial cells has been studied in vitro and in vivo. By in vitro assay, Mab 9B9 biotinylated at a biotin/IgG molar ratio in reaction mixture (B/IgG ratio) of 0.7-2.2 bound streptavidin monovalently and retained antigen-binding capacity. Mab 9B9 biotinylated at a B/IgG ratio of 20 and higher bound streptavidin polyvalently. Extensive biotinylation (B/IgG ratio of 60 and higher) led to dramatic reduction of Mab 9B9 Ag-binding capacity and to reduction of Mab 9B9 recognition by goat polyclonal antibody to mouse IgG. Radiolabeled Mab 9B9 biotinylated at a B/IgG ratio of 6 (b6-Mab 9B9) bound effectively to cultured vascular endothelium, with affinity characteristics similar to nonbiotinylated Mab 9B9. Endothelial cells internalized both Mab 9B9 and b6-Mab 9B9 to the same extent (60% internalization at 3 h incubation at 37°C). Degradation of cell surface-associated Mab 9B9 or b6-Mab 9B9 was very low (<1% as measured by TCA solubility of radiolabel). In contrast, degradation of internalized b6-Mab 9B9 was more profound than that of Mab 9B9 (20 ± 3% vs 6 ± 1%, P <0.01). After injection in rats, radiolabeled b6-Mab 9B9 had a biodistribution pattern similar to that of radiolabeled Mab 9B9. Both preparations effectively accumulated in the lung (15-20% of injected dose/g of tissue vs 2% of injected dose/g of blood). Extensive biotinylation led to both a reduction of specific pulmonary uptake of Mab 9B9 and an enhanced blood clearance of Mab 9B9. Streptavidin binding to b6-Mab 9B9 did not alter biodistribution or pulmonary targeting of biotinylated antibody. We conclude that extensive biotinylation induces complex alterations of the functional properties of Mab 9B9. In contrast, b6-Mab 9B9 may serve as an affinity carrier for targeting of biotinylated compounds to the pulmonary endothelium.