Construction and characterization of an intracellular single-chain human antibody to hepatitis C virus non-structural 3 protein
Introduction
Hepatitis C virus (HCV) is an enveloped virus with a plus-strand RNA genome encoding a polyprotein of approximately 3010 amino acids [1]. This polyprotein is cleaved co- and post-translationally into mature viral proteins by host cell signal peptidases and two viral enzymes designated the non-structural 2/3 (NS2/3) proteinase and the NS3/4A proteinase complex. The NS3 component of these proteinases is a 67 kDa multifunctional enzyme with three known catalytic activities segregated into two somewhat independent domains. The essential machinery of a serine-type protease is localized in the N-terminal one-third of the protein and nucleoside triphosphatase (NTPase) and helicase activities, presumably involved in the unwinding of the viral genome, reside in the remaining C-terminal region. The enzymatic functions of NS3 are essential for the virus replication cycle [2]. Therefore, NS3 presents an important target for antiviral therapy.
It is our hypothesis that binding of an antibody to the NS3 protein will block one or more functions of NS3. We previously generated a human monoclonal antibody, CM3.B6 (IgG1 kappa), that specifically recognizes an immunodominant conformational B-cell epitope (amino acids 1363–1454) within the helicase domain of HCV NS3 [3]. Recombinant Fab fragments generated from cells expressing CM3.B6 recognized the cognate antigen expressed in mammalian cells [4]. To explore the possibility that this antibody may have therapeutic potential, we constructed and characterized a single-chain form of this antibody (scFvNS3) for intracellular expression. To achieve high levels of scFvNS3 expression in hepatocytes, we employed recombinant adenovirus as gene vehicle. Most significantly, we demonstrate the potential of this scFv to inhibit HCV replication.
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Cell culture
HepG2 and Hep3269 (HepG2 cells stably transfected with HCV complementary DNA (cDNA) representing the C-terminal half of NS2 to the N-terminal one-third of NS5 and constitutively expressing NS3) [5] cells were cultured in Eagle's minimal essential medium (MEM) (Invitrogen, Carlsbad, CA) supplemented with 1 mM sodium pyruvate, 10 μM non-essential amino acids, 50 U/ml penicillin/streptomycin, and 10% fetal bovine serum. Hep3269 cells were grown on plates coated with collagen I (Becton Dickinson,
Antibody mediated inhibition of NS3 helicase activity
Given the highly specific binding of CM3.B6 to an epitope within the helicase domain of NS3, we investigated whether binding of this antibody could inhibit NS3 helicase activity. Increasing amounts of CM3.B6 or an irrelevant human monoclonal antibody (IgG1) were added to recombinant full-length NS3 under optimal conditions for NS3 helicase activity as determined previously [6]. CM3.B6 inhibited NS3 helicase up to 50% in a dose dependent manner; whereas, the irrelevant antibody inhibited
Discussion
Selective gene ablation with intracellular scFv targeted to specific cellular compartments represents a novel approach to a gene therapy for viral infection [23], [24], [25], [26], [27], [28], [29]. Several recent studies report the construction and characterization of scFv's to HCV NS3 [30], [31], RNA-dependent RNA polymerase [20], core [32] or envelope protein [33]; however to date, none have demonstrated a biological consequence of scFv binding to HCV proteins. Here, we report the
Acknowledgements
We thank Dr Paola Gallinari for providing recombinant NS3, Dr T. Miyamura for providing Hep3269 cells and Chiron, Inc. for supplying rabbit polyclonal anti-NS3. This study was supported in part by NIH grants R01 CA68245 and R01 CA74242 to DTC; NIH grant R01 CA54576 to MAG and SD.
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2005, Molecular TherapyCitation Excerpt :Various delivery vectors have been developed for genetic transfer of antibodies, including baculovirus, vaccinia virus, rhabdovirus, and adeno-associated virus vectors [40–45]. Adenovirus gene transfer vectors have been used to deliver single-chain antibodies for testing in vivo systemic single-chain antibody levels, for in vitro viral load reduction, and for cancer applications [46–57]. An Ad vector engineered to express scFv (a single-chain antibody encoding the heavy- and light-chain variable domains connected by a linker) directed against B cell lymphoma idiotypes expressed enough scFv to elicit protective immunity in >40% of mice challenged with a lethal dose of a lymphoma cell line [49] and delivery of an Ad vector expressing anti-erbB2 scFv was used effectively as an anti-tumor agent for ovarian and extraovarian cancers [47].
Inhibition of hepatitis C virus nonstructural protein, helicase activity, and viral replication by a recombinant human antibody clone
2004, American Journal of PathologyCitation Excerpt :There are some reports of using recombinant antibody fragment to inhibit enzyme activities of NS3 protein.47 In most of these studies, including ours, single chain antibodies have been used to inhibit either protease or helicase activity of NS3 protein and polymerase activity of NS5B protein.13,47,48 However, the single chain variant of this mouse monoclonal antibody was found to lose the ability to bind helicase as compared to the parent antibody.
Intrabodies as therapeutic agents
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