Generation of full-length functional antibody against preS2 of hepatitis B virus in hepatic cells in vitro from bicistrons mediated by gutless adenovirus.
BACKGROUND
Monoclonal antibodies (mAbs) have been developed as effective therapeutics for a wide variety of diseases. Delivery of mAbs by gene transfer provides an option for overcoming the difficulties in mAb production and manufacturing processes. However, for the polymeric structure of full-length mAbs, it is important to design an optimal gene transfer system for mAb generation.


METHODS
Gutless adenovirus and liver-specific promoter transthyretin (TTR) were combined to deliver bicistronic mAb genes in human hepatic cell lines. In order to optimize the bicistrons for mAb generation, four bicistrons were designed and compared, and the most efficient one was selected. ELISA and Western blot were conducted to evaluate mAb products in the supernatants.


RESULTS
Our data showed that all of four gutless adenoviruses elicited liver-specific mAb production in HepG2 and Hep3B hepatic cell lines. It was observed that the L2AH bicistron construct (comprising an immunoglobulin light-chain cDNA situated 5' of a heavy-chain cDNA, with a foot-and-mouth disease virus 2A cleavage site in the middle, subcloned into the helper-dependent adenovirus plasmid pGL) could induce the highest level expression of mAb (about 5.0 microg/mL in Hep3B) among these four constructs. Importantly, the mAb products by gene transfer methods retained specific antigen-binding activity.


CONCLUSIONS
Our studies gave further evidence that it was feasible to produce active full-length mAb in human hepatic cell lines in vitro by a special gene delivery system. Moreover, we developed an optimized bicistron gene transfer system for future gene therapy research, which may also be of use in industrial mAb production.
Yin
Yuexiang
Y
Xinyuan Institute of Medicine and Biotechnology, School of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, Zhejiang, China.
Lin
Fang
F
Zhuang
Qiang
Q
Liu
Li
L
Qian
Cheng
C
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000502:physiology; D045744:Cell Line, Tumor; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics / Q000276:immunology; D005822:Genetic Vectors / Q000008:administration & dosage / Q000096:biosynthesis / Q000276:immunology; D006367:HeLa Cells; D006508:Hepatitis Antibodies / Q000096:biosynthesis* / Q000502:physiology; D006514:Hepatitis B Surface Antigens / Q000276:immunology*; D006515:Hepatitis B virus / Q000276:immunology*; D022781:Hepatocytes / Q000276:immunology* / Q000378:metabolism* / Q000821:virology; D006801:Humans; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000276:immunology*
D000911:Antibodies, Monoclonal; D006508:Hepatitis Antibodies; D006514:Hepatitis B Surface Antigens; D011498:Protein Precursors; C050807:presurface protein 2, hepatitis B surface antigen