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. CONCLUSION: 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.

Transfer of suppressor of cytokine signaling 3 by an oncolytic adenovirus induces potential antitumor activities in hepatocellular carcinoma.

UNLABELLED: The constitutive activation of signal transducer and activator of transcription 3 (STAT3) participates in carcinogenesis through up-regulation of genes encoding apoptosis inhibitors and cell cycle regulators, such as Bcl-xL, cyclins D1 and D2, and c-myc. Suppressor of cytokine signaling 3 (SOCS3) is one of the negative regulators of cytokine signaling and is frequently silenced in diverse cancers. In this study, we explored whether restoration of SOCS3 by oncolytic adenoviral vectors could inhibit the constitutive activation of the Janus kinase/STAT pathway and suppress tumor growth. Our data showed that SOCS3 was down-expressed in all liver tumor cell lines. The incorporation of SOCS3 or SOCS3 fused with cell-penetrating peptides (cpp-SOCS3) did not alter adenoviral replication selectively in liver tumor cells. The infection of cells with adenovirus CN305 (AdCN305)-SOCS3 and AdCN305-cpp-SOCS3 resulted in dramatic cytotoxicity in liver tumor cells. However, no cytotoxic effect was observed in normal cells infected with these vectors. Infection of liver tumor cells with AdCN305-SOCS3 and AdCN305-cpp-SOCS3 resulted in nearly complete inhibition of STAT3 phosphorylation and down-regulation of cyclin D1 and Bcl-xL. Treatment of the established tumor by AdCN305-SOCS3 and AdCN305-cpp-SOCS3 caused significant suppression of tumor growth. The suppression of tumor growth was due to the inhibition of STAT3 phosphorylation and induction of tumor cell death. CONCLUSION: This study suggests that transfer of SOCS3 by an oncolytic adenovirus represents a potent approach for cancer therapy.