1 Department of Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Moorenstr. 5, 40225 Düsseldorf, Germany
2 Department of Molecular Biotechnology, University of Aachen, Germany
Correspondence
Tobias Heintges
heintges{at}uni-duesseldorf.de
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ABSTRACT |
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INTRODUCTION |
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HCV NS3 is a multifunctional enzyme with three known catalytic activities segregated into two distinct domains. The serine protease activity resides in the N-terminal one-third of the protein. The nucleoside triphosphatase and helicase activities are localized in the remaining C-terminal domain (Gwack et al., 1997; Kim et al., 1997
; Kolykhalov et al., 1994
; Preugschat et al., 1996
). HCV NS3 has been reported to be processed internally (Shoji et al., 1999
; Yang et al., 2000
). The crystal structure of NS3 helicase has been resolved (Cho et al., 1998
; Kim et al., 1998
; Yao et al., 1997
). Helicase activity is reported to be necessary for RNA unwinding during the replication of the viral genome (Gwack et al., 1997
; Wardell et al., 1999
). Therefore, the HCV NS3 helicase is a promising target for HCV-specific drug therapy. Recently, a dimer model for NS3 helicase was proposed based on crystal structure and mutational analysis of the NS3 helicase (Cho et al., 1998
; Khu et al., 2001
). This provides new opportunities for designing compounds that inhibit dimerization specifically. Such inhibitors will be specific for the enzyme, as the dimer structure of the HCV helicase is unique (Khu et al., 2001
).
Intracellular antibodies (intrabodies) with inhibitory properties represent a new class of neutralizing molecules with great potential for gene therapy. Single-chain antibody fragments (sFvs) have been shown to possess favourable properties as therapeutic agents. These recombinant antibodies consist of the variable domains of heavy (VH) and light (VL) chain connected by a polypeptide linker, generating a fully functional antigen-specific binding unit (Chen et al., 1994a; Marasco, 1997
). The variable domains are expressed as a single polypeptide chain, increasing the stability of sFv fragments in comparison to complete immunoglobulins, e.g. in the reducing environment of the cytosol (Schouten et al., 2002
). Recombinant antibody technology (phage display) permits the generation of high-affinity antibodies that are highly specific for the target protein (Gram et al., 1992
; Hoogenboom & Winter, 1992
; Rader & Barbas, 1997
; Winter et al., 1994
). Combinatorial libraries of immunoglobulin genes can be expressed on the surface of filamentous bacteriophages, thus colocalizing the antibody protein with its encoding DNA sequence within the phage (Marks et al., 1991
; McCafferty et al., 1990
; Pope et al., 1996
). Phages displaying antibodies with high affinity against the target molecule are highly enriched during several rounds of affinity selection and reamplification (panning). sFvs with inhibitory properties against the target protein can be expressed directly inside cells, creating an active molecule for protein-based gene therapy. The employment of human sFvs should minimize the immune response against the foreign protein (Chen et al., 1994a
; Marasco, 1997
; Rader & Barbas, 1997
). The clinical evaluation of two human monoclonal antibodies (mAbs) binding to hepatitis B virus demonstrated that intravenous administration of these recombinant antibodies was well tolerated (Galun et al., 2002
).
Recently, the isolation and characterization of a murine mAb that inhibits HCV RNA-dependent RNA polymerase was described (Moradpour et al., 2002). In the present study, we report the generation of human sFv antibody fragments that bind to the HCV NS3 helicase, another enzyme essential for the HCV replication cycle. We show efficient neutralization of the helicase unwinding activity upon binding of high-affinity sFv antibodies.
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METHODS |
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Human sFv library and affinity selection.
The cloning of a combinatorial sFv library from patients chronically infected with HCV has been described previously (Tessmann et al., 2002). Briefly, human VH and VL fragments derived from bone marrow aspirate were cloned into the phagemid vector pAK100 (Krebber et al., 1997
). This phage-display vector allows the expression of an sFv as a gene III fusion protein at the surface of filamentous phages. N-terminal FLAG and C-terminal c-Myc tag sequences were used for the immunodetection of the sFv antibody fragments.
Library growth, phage preparation and soluble antibody expression were carried out as described (Harrison et al., 1996). Phages were prepared by infecting the library stock with VCS M13 helper phage (Stratagene). A phage solution of 1012 to 1013 transducing units diluted in PBS containing 2 % BSA was incubated in microtitre plates coated with approximately 200 ng recombinant NS3 helicase for 2 h at room temperature. The microtitre plate was washed extensively (20 times) with PBS containing 0·1 % Tween-20 to remove nonspecifically bound phages. Subsequently, phages bound to NS3 helicase were eluted with 100 mM triethylamine and neutralized immediately with equal amounts of 1 M Tris/HCl, pH 7·4. Eluted phages were used for reinfection of the supressor E. coli strain XL-1 Blue for phage propagation and for subsequent rounds of selection. The panning procedure was repeated three times. Soluble expression and screening were carried out using the E. coli nonsupressor strain HB2151. Antibody purification and ELISA were described previously (Tessmann et al., 2002
).
Sequencing and V gene alignment.
Custom sequencing was carried out by MWG Biotech. The alignment of the nucleotide sequences of rearranged V genes to their closest germline V, D and J segments was performed using V base DNA Plot (http://www.mrc-cpe.cam.ac.uk/) and IgBlast (http://www.ncbi.nlm.nih.gov/igblast/). The determination of immunoglobulin families was performed according to Kabat et al. (1991) and Martin (1996)
. Multiple sequence alignment was performed using CLUSTALX (Higgins et al., 1996
).
Determination of equilibrium dissociation constants.
Biomolecular interactions were measured by surface plasmon resonance (SPR) using BIACORE 2000 (BioSensor) in HBS-EP buffer [150 mM NaCl, 3·4 mM EDTA, 0·05 % (v/v) surfactant P20, 10 mM HEPES, pH 7·4]. The purified NS3 helicase protein was immobilized on a CM5-rg sensor chip using the Amine Coupling kit (BioSensor). Sample protein (sFv antibodies) was diluted in Hanks' balanced salt buffer and injected at different concentrations at a flow-rate of 30 µl min-1. The surface was regenerated with 10 µl 30 mM HCl between sample injections. On- and off-rates were determined using the BIAevaluation 3.0 software (BioSensor), assuming a single-step monovalent interaction.
NS3 helicase-mediated DNA-unwinding assay.
The helicase activity assay was developed in ELISA format. The substrate was composed of two annealed complementary DNA strands: the 5'-biotin-labelled template strand (5'-GGTTTAAAAAATAGGAGGGACAACGTCGTGACTGGGAAAACTCCCCGGGTACCGAGCTCG-3') and the 5'-digoxigenin-labelled release strand (5'-GTTTTCCCAGTCACGACGTTGT-3') (MWG Biotech). The annealing of two oligonucleotides at a molar ratio of 1 : 3 (template : release) was carried out in 10 mM Tris/HCl, 25 mM NaCl and 1 mM EDTA, pH 7·6, by heating to 100 °C for 10 min followed by cooling slowly to room temperature. High-binding, polystyrene half-area microtitre plates (Costar) were coated with 10 µg streptavidine ml-1 (Fluka) and blocked with 2 % BSA in PBS. The helicase substrate (concentration of the template strand, 0·4 pmol per well, 100 µl) was transferred to the preblocked plate and incubated for 1 h at room temperature. After extensive washing with buffer M (25 mM MOPS, 2 mM DTT, 3 mM MgCl2), HCV helicase was added in buffer M supplemented with 2·5 mM ATP and 20 pmol per well of the capture oligonucleotide 5'-ACAACGTCGTGACTGGGAAAAC-3', which is complementary to the release strand (helicase reaction buffer). After incubation at 37 °C for 1 h, the plate was washed six times with PBS. Release strand not unwound by helicase was detected with alkaline phosphatase conjugated to an anti-digoxigenin Fab-fragment from sheep (Roche), followed by p-nitrophenylphosphate substrate. Absorbance was measured at 405 nm after 30 min of incubation at room temperature.
Calculation of the fraction of bound helicase.
The fraction of complexed helicase was calculated assuming a one-step monovalent interaction that is described by the following law of mass action:
where [helicase] is the equilibrium concentration of free helicase, [sFv] is the equilibrium concentration of free sFv, [helicase : : sFv] is the equilibrium concentration of helicasesFv complex and KD is the equilibrium dissociation constant.
Where KD was derived from the rate constants determined by SPR:
The fraction of helicase that is complexed by sFv:
Where the unknown equilibrium concentrations are replaced by the known total concentrations (c0) of sFv and helicase:
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RESULTS |
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Generation of human antibodies by phage display and affinity measurements
The human sFv library, derived from bone marrow aspirate of chronically infected patients and cloned into a phage-display vector (Tessmann et al., 2002), was used for affinity selection. Previously, we described isolation of high-affinity sFv antibodies binding to denatured NS3 helicase (Tessmann et al., 2002
). In the present study, we carried out affinity selection (panning) against an enzymatically active NS3 helicase domain expressed in insect cells. The rationale behind this strategy was that the use of a biologically functional enzyme as a template for panning would allow us to select antibodies against conformational epitopes not represented in the denatured protein. After three rounds of affinity selection, a number of clones which bound strongly to the target protein was isolated. Some clones were shown to possess identical DNA sequences, thus encoding the same antibody fragment. Distinct clones, designated sFvs 6, 20, 41, 44, 42, 52 and 59, were chosen for further characterization. The subgrouping of the variable domains and identification of the closest germline V, D and J segment counterparts (Table 1
) was performed by alignment using the V Base database (Cambridge, UK) and immunoglobulin germline V genes (NCBI). The deduced protein sequences of the selected antibodies are shown in Fig. 3
. An unusual and interesting feature of antibody fragment sFv 6 has been identified. The CDR-H1 and CDR-H3 regions of this clone each contain a single cysteine. Cysteines are not found in the germline genes of the VH exons and only paired cysteines are found in the exons for D2 elements. The closest D segment identified for sFv6 is D3-22/D3-10, in which the cysteines are missing. Thus, both cysteines must have been acquired during affinity maturation. Interestingly, the two cysteines are in close vicinity, allowing the formation of a disulfide bond connecting CDR-H1 and CDR-H3. Alternatively, free cysteines may be a requirement for binding of sFv 6 to helicase.
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Inhibition of NS3 helicase by recombinant antibodies
To assess the inhibitory potential of selected sFvs by DNA-unwinding assay, we used both antibody and helicase proteins purified by Ni-NTA (Fig. 1B). The sFv antibodies were preincubated with the enzyme in reaction buffer, the helicase reaction was performed and the fraction of unwound substrate was determined. During initial screening, we identified seven recombinant antibodies that inhibited helicase-mediated DNA unwinding at nanomolar concentrations, with efficacies ranging from 20 % to complete abrogation of helicase activity (Fig. 5
). sFv 1-2, selected against denatured NS3 protein, was able to neutralize the helicase reaction, but all other inhibitory antibodies were obtained from selection against enzymatically active helicase (sFvs 59, 42, 6, 20, 44 and 41). The dose-response curves of enzyme inhibition at varying concentrations of the sFv proteins and at constant concentration of the enzyme (18 nM) are shown in Fig. 6
(A). The inhibitory activities of sFvs 42 and 59 are in good agreement with the theoretically calculated amounts of helicase bound to antibody, based on the equilibrium dissociation constants determined by SPR analysis (4·4 and 4·9 nM, respectively; Fig. 6B
). These two antibodies are equally effective and at concentration of 65 nM inhibit NS3 helicase to 100 %. sFvs 6 and 20 had higher equilibrium dissociation constants (18 and 8·6 nM) and dissociation rate constants (1·6x10-3 s-1 and 9·4x10-4 s-1) and, consequently, were less effective in inhibiting NS3 helicase, with efficacies of up to 60 %. A higher deviation of the inhibition from the theoretically calculated amount of bound helicase may be due to the faster dissociation kinetics during the inhibition assay, or this may also indicate a more complex mode of enzyme inhibition (e.g. noncompetitive), but elucidation of the mechanisms of helicase inhibition requires further investigation. sFvs 41 and 44 (possessing the same light chain and a similar heavy chain), which could be tested only at lower concentrations (up to 30 nM) because of poor expression in E. coli, still showed inhibitory effects.
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DISCUSSION |
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The antibodyantigen interaction studied by SPR and evaluated by fitting the results to mathematical models revealed different kinetics parameters of the selected clones, with association rate constants between 8·7x104 and 8·4x105 M-1 s-1 and dissociation rate constants between 7·1x10-4 and 1·6x10-3 s-1, resulting in apparent equilibrium constants ranging from 2·5x10-9 to 4·4x10-8. In some cases, we observed deviations from the simple monovalent one-step binding model, which can be caused by the presence of dimeric or multimeric structures. Moreover, it has been observed that only a small fraction of about 15 % of the immobilized helicase was recognized by different antibodies. This might be an indication that immobilization of the helicase led to partial, and for sFv 52, to complete, inactivation or masking of a binding epitope. It has been reported that NS3 helicase may exist as a homodimer in vivo (Khu et al., 2001). Because of the coupling procedure used, the immobilization of the enzyme is undirected and any of the exposed lysine residues can be modified. This may affect the capability of dimerization. Alternatively, immobilization may arrest helicase conformationally and might affect binding of antibodies that require helicase to undergo a conformational change either before or upon binding. Taken together, kinetics analysis indicates differences in antibodyantigen interaction modes among the cloned sFvs and future studies will aim to resolve the underlying mechanisms of binding and inhibition of helicase activity. The affinity data obtained by SPR measurements are in good agreement with the individual inhibition rates of the sFvs, which were determined with the established enzymatic activity assay. This assay allows the reproducible quantification of helicase unwinding activity and provides a rapid screening method for inhibitory compounds. We report efficient inhibition of HCV HS3 helicase in vitro at nanomolar concentrations of the sFvs. To date, no intracellular system for monitoring the helicase activity of HCV in vivo has been described. An option for the investigation of the inhibitory properties of antibodies expressed intracellularly could be a new HCV replicon system, which has been developed recently for the assessment of functions of HCV nonstructural proteins (Lohmann et al., 1999
). A similar HCV replicon was constructed by Pflugheber et al. (2002)
to examine the role of the HCV NS5 protein in disrupting a host antiviral pathway. Intracellular immunization with inhibitory antibodies in a cell line harbouring a HCV replicon may be a next step for the target evaluation. This could be used to assess the efficiency of inhibition of virus replication via intracellular inactivation of one of the viral enzymes, e.g. the helicase, and may add to our knowledge of designing improved HCV inhibitory drugs.
Inhibitory effects of sFv fragments used as intrabodies have been shown with various intracellular target proteins. Various intrabodies were shown to be effective against human immunodeficiency virus replication and infection (Marasco et al., 1999; Mhashilkar et al.; 1999
; Poznansky et al., 1998
; Kitamura et al., 1999
; BouHamdan et al., 1999
; Goncalves et al., 2002
). Other intrabodies were used successfully against various oncogenes (Jean et al., 2000
; Deshane et al., 1995
; Chen et al., 2002
).
To achieve antiviral effects against HCV, the cDNA encoding inhibitory antibodies have to be transduced into hepatocytes (reviewed by Wu et al., 2002). Successful transduction has been demonstrated with, for example, retrovirus and lentivirus vectors (BouHamdan et al., 1999
; Chen et al., 1994b
; Marasco et al., 1999
; Richardson et al., 1998
). To date the first clinical studies involving patients with ovarian cancer are ongoing using adenovirus vectors to express anti-erbB-2 intrabodies (Alvarez et al., 2000
; Deshane et al., 1995
). Using the appropriate gene transfer vectors, intracellular immunization with intrabodies could be a promising approach for treatment of chronic HCV infection.
Once sFv fragments have been isolated, the cloning and expression of complete human IgG, IgA or IgM antibodies is possible (Boel et al., 2000). Production of recombinant antibodies in a heterologous system, such as transgenic yeasts or plants, is simple and cheap in comparison to the isolation of antibodies from the sera of HCV-infected patients. Moreover, there is no risk of infection with HCV or other human pathogens with the use of recombinant high-affinity antibodies for diagnostic and therapeutic purposes.
Further experiments will examine the molecular mechanism of the antibody-mediated enzyme inhibition. This information may provide a structural basis for the molecular mimicry of antibody recognition loops and may aid in the rational design of small molecule inhibitors of essential viral proteins.
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ACKNOWLEDGEMENTS |
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Received 17 April 2003;
accepted 28 May 2003.