Intracellular expression of a cloned antibody fragment interferes with hepatitis B virus surface antigen secretion.
We evaluated the potential of an intracellularly expressed antibody fragment to interfere with hepatitis B virus (HBV). Sequences coding for the immunoglobulin variable regions of the HBV surface antigen (HBsAg) specific monoclonal antibody 5C3 were isolated and characterized. A secretory pathway-targeted, 5C3 derived single chain Fv (sFv) fragment was expressed in HuH-7 hepatocellular carcinoma cells together with HBsAg. Quantification of extracellular HBsAg levels in the cell culture supernatant demonstrated that the presence of the 5C3 sFv equipped with a secretory pathway retention signal SEKDEL reduced extracellular HBsAg levels by a mean of 85%. Co-immunoprecipitation studies revealed that the 5C3 sFv targeted to the secretory pathway physically interacted with its target antigen, HBsAg. Confocal microscopy studies confirmed the intracellular expression and colocalization of the 5C3 sFv and HBsAg. We conclude that certain intracellularly expressed antibody fragments will substantially interfere with HBV antigen secretion from the cell.
Molecular Hepatology Laboratory, Massachusetts General Hospital Cancer Center and Harvard Medical School, Boston, Massachusetts, 02129, USA.
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zu Putlitz
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A
Skerra
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JR
Wands
D000595:Amino Acid Sequence; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular / Q000276:immunology*; D003001:Cloning, Molecular; D005455:Fluorescent Antibody Technique; D006514:Hepatitis B Surface Antigens / Q000276:immunology*; D006518:Hepatitis Viruses / Q000276:immunology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000276:immunology; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000276:immunology; D017422:Sequence Analysis, DNA; D014162:Transfection / Q000235:genetics; D014407:Tumor Cells, Cultured
D006514:Hepatitis B Surface Antigens; D007128:Immunoglobulin Fragments; D021382:Protein Sorting Signals; C062059:SEKDEL sequence