THE FUNCTIONAL-EFFECTS OF BIOTINYLATION OF ANTI-ANGIOTENSIN-CONVERTING ENZYME MONOCLONAL-ANTIBODY IN TERMS OF TARGETING IN-VIVO

The effect of modification with biotin N-hydroxysuccinimide ester of m ouse monoclonal antibody to angiotensin-converting enzyme, anti-ACE Ma b 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 strepta vidin monovalently and retained antigen-binding capacity. Mab 9B9 biot inylated at a B/IgG ratio of 20 and higher bound streptavidin polyvale ntly. Extensive biotinylation (B/IgG ratio of 60 and higher) led to dr amatic reduction of Mab 9B9 Ag-binding capacity and to reduction of Ma b 9B9 recognition by goat polyclonal antibody to mouse IgG. Radiolabel ed Mab 9B9 biotinylated at a B/IgG ratio of 6 (b(6)-Mab 9B9) bound eff ectively to cultured vascular endothelium, with affinity characteristi cs similar to nonbiotinylated Mab 9B9. Endothelial cells internalized both Mab 9B9 and bs-Mab 9B9 to the same extent (60% internalization at 3 h incubation at 37 degrees C). Degradation of cell surface-associat ed Mab 9B9 or b(6)-Mab 9B9 was very low (<1% as measured by TCA solubi lity of radiolabel). In contrast, degradation of internalized b(6)-Mab 9B9 was more profound than that of Mab 9B9 (20 +/- 3% vs 6 +/- 1%, P <0.01). After injection in rats, radiolabeled b(6)-Mab 9B9 had a biodi stribution pattern similar to that of radiolabeled Mab 9B9. Both prepa rations 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 a n enhanced blood clearance of Mab 9B9. Streptavidin binding to bs-Mab 9B9 did not alter biodistribution or pulmonary targeting of biotinylat ed antibody. We conclude that extensive biotinylation induces complex alterations of the functional properties of Mab 9B9. In contrast, b(6) -Mab 9B9 may serve as an affinity carrier for targeting of biotinylate d compounds to the pulmonary endothelium. (C) 1995 Academic Press, Inc .