Carbohydrate-directed conjugation of cobra venom factor to antibody by selective derivatization of the terminal galactose residues.
Cobra venom factor (CVF) can cause cell death by complement-mediated bystander cell lysis. Several studies have investigated CVF for application in cancer therapy by conjugating CVF to antibodies against tumor cell surface-specific antigens via the side-chain amino acid residues. In most cases, the activity of CVF was markedly impaired, presumably by modification of the factor B binding domain due to random derivatization. Since CVF is a glycoprotein and its oligosaccharide chains are distal to the factor B binding domain, coupling of CVF to antibodies through its oligosaccharide chains is expected to yield immunoconjugates with retention of CVF activity and elimination of the immunoreactivity of the terminal alpha-galactosyl residues. In this study, we investigated the carbohydrate site-directed conjugation of CVF to a monoclonal IgG specific to a cell-surface antigen of human ovarian cancer cells. The terminal galactosyl residues of CVF were selectively modified at C-6 by treatment with galactose oxidase, and the generated aldehyde groups were derivatized in situ with hydrazides containing either protected thiol or maleimide functional groups. The CVF derivatives were allowed to react with thiol groups introduced to the antibody by derivatization with 2-iminothiolane to yield carbohydrate site-directed CVF-antibody conjugates. In both cases, 30-40% of the antibody cross-linked to CVF to yield predominantly monovalent CVF-antibody conjugates. The purified immunoconjugates retained 70-75% of CVF activity and significant level of antigen-binding capacity. This is the first study to exploit the oligosaccharide chains of CVF for the preparation of active immunoconjugates.
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Department of Biochemistry and Molecular Biology, 3900 Reservoir Road, Georgetown University Medical Center, Washington, DC 20007, USA.
Gowda
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D000818:Animals; D000912:Antibodies, Neoplasm / Q000737:chemistry*; D000951:Antigens, Neoplasm / Q000276:immunology; D002241:Carbohydrates / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D003169:Complement Inactivator Proteins / Q000737:chemistry; D003432:Cross-Linking Reagents; D004546:Elapid Venoms / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005690:Galactose / Q000737:chemistry; D006168:Guinea Pigs; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry; D015394:Molecular Structure; D010051:Ovarian Neoplasms / Q000276:immunology; D014407:Tumor Cells, Cultured
D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D002241:Carbohydrates; D003169:Complement Inactivator Proteins; D003432:Cross-Linking Reagents; D004546:Elapid Venoms; D018796:Immunoconjugates; C016310:cobra venom factor; D005690:Galactose