Intracellular expression of a cloned antibody fragment interferes with hepatitis B virus surface antigen secretion

Jasper Zu Putlitz; Arne Skerra; Jack R. Wands

doi:10.1006/bbrc.1999.0149

Intracellular expression of a cloned antibody fragment interferes with hepatitis B virus surface antigen secretion

Jasper Zu Putlitz, Arne Skerra, Jack R. Wands

Research output: Contribution to journal › Article › peer-review

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Abstract

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 co-localization of the 5C3 sFv and HBsAg. We conclude that certain intracellularly expressed antibody fragments will substantially interfere with HBV antigen secretion from the cell.

Original language	English
Pages (from-to)	785-791
Number of pages	7
Journal	Biochemical and Biophysical Research Communications
Volume	255
Issue number	3
DOIs	https://doi.org/10.1006/bbrc.1999.0149
State	Published - 24 Feb 1999
Externally published	Yes

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title = "Intracellular expression of a cloned antibody fragment interferes with hepatitis B virus surface antigen secretion",

abstract = "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 co-localization of the 5C3 sFv and HBsAg. We conclude that certain intracellularly expressed antibody fragments will substantially interfere with HBV antigen secretion from the cell.",

author = "Putlitz, {Jasper Zu} and Arne Skerra and Wands, {Jack R.}",

note = "Funding Information: Supported by Grants CA-35711 and AA-02169 from the National Institutes of Health. J.z.P. is on leave from the Department of Internal Medicine II, University of Freiburg, Germany and is supported by the Stipendienprogramm “Infektionsforschung” of the German Cancer Research Center, Heidelberg, Germany. We would like to thank Tobias Heintges for the kind gift of a HCV core antigen specific sFv expression construct, Rolf I. Carlson for assistance with hybridoma work, Darrell Peterson for the kind gift of MAb H166 and Stefan Wieland for a HBsAg expression construct. We would like to gratefully acknowledge Yimin Ge, Cutaneous Biology Research Center, Massachusetts General Hospital, for help with confocal microscopy and analysis of images.",

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N2 - 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 co-localization of the 5C3 sFv and HBsAg. We conclude that certain intracellularly expressed antibody fragments will substantially interfere with HBV antigen secretion from the cell.

AB - 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 co-localization of the 5C3 sFv and HBsAg. We conclude that certain intracellularly expressed antibody fragments will substantially interfere with HBV antigen secretion from the cell.

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Intracellular expression of a cloned antibody fragment interferes with hepatitis B virus surface antigen secretion

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