The functional effects of biotinylation of anti-angiotensin-converting enzyme monoclonal antibody in terms of targeting in vivo

Anal Biochem. 1995 Apr 10;226(2):279-87. doi: 10.1006/abio.1995.1226.

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 degrees 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).(ABSTRACT TRUNCATED AT 250 WORDS)

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Antibodies, Monoclonal / immunology
  • Antibodies, Monoclonal / metabolism*
  • Antibodies, Monoclonal / pharmacokinetics
  • Antigen-Antibody Reactions
  • Bacterial Proteins / metabolism
  • Biotin / analogs & derivatives*
  • Biotin / metabolism
  • Cells, Cultured
  • Endothelium, Vascular / metabolism
  • Humans
  • Lung / metabolism*
  • Metabolic Clearance Rate
  • Mice
  • Peptidyl-Dipeptidase A / immunology*
  • Peroxidase / metabolism
  • Protein Binding
  • Protein Denaturation
  • Rats
  • Rats, Sprague-Dawley
  • Streptavidin
  • Succinimides / metabolism*
  • Tissue Distribution

Substances

  • Antibodies, Monoclonal
  • Bacterial Proteins
  • Succinimides
  • biotinyl N-hydroxysuccinimide ester
  • Biotin
  • Streptavidin
  • Peroxidase
  • Peptidyl-Dipeptidase A