Cloning and molecular characterization of human high affinity antibody fragments against Hepatitis C virus NS3 helicase

Tessmann, Kathi; Erhardt, Andreas; Häussinger, Dieter; Heintges, Tobias

doi:10.1016/s0166-0934(02)00017-4

Cited by 13 publications

(18 citation statements)

References 48 publications

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“…Data are expressed as the means of triplicates±SEM. sFvs 10-1, 10-2, 9-1, 1-1, 1-2 and 7-4 were selected against commercially available recombinant NS3 helicase produced in E. coli (Mikrogen; Tessmann et al, 2002). sFvs 59, 42, 6, 20, 52, 41 and 44 were selected against baculovirus-expressed helicase in the present study.…”

Section: Discussionmentioning

confidence: 99%

“…Previously, we described isolation of highaffinity 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.…”

Section: Generation Of Human Antibodies By Phage Display and Affinitymentioning

confidence: 97%

“…The human sFv library, derived from bone marrow aspirate of chronically infected patients and cloned into a phagedisplay vector (Tessmann et al, 2002), was used for affinity selection. Previously, we described isolation of highaffinity sFv antibodies binding to denatured NS3 helicase ( Tessmann et al, 2002).…”

Section: Generation Of Human Antibodies By Phage Display and Affinitymentioning

confidence: 99%

“…The cloning of a combinatorial sFv library from patients chronically infected with HCV has been described previously (Tessmann et al, 2002). Briefly, human V H and V L fragments derived from bone marrow aspirate were cloned into the phagemid vector pAK100 (Krebber et al, 1997).…”

mentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

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Abrogation of hepatitis C virus NS3 helicase enzymatic activity by recombinant human antibodies

Artsaenko

Tessmann

Sack³

et al. 2003

Journal of General Virology

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The hepatitis C virus (HCV) NS3 protein possesses both protease and helicase activities and is essential for virus replication and maturation. Specific inhibition of NS3 enzymatic activity can be achieved by antibody binding. Transduction of hepatocytes with encoding cDNA leading to intracellular expression of antibody fragments is expected to terminate HCV replication in infected cells. The objective of the present study was the generation of human antibody fragments that neutralize the viral NS3 helicase activity for gene therapeutic applications and drug design. A human immunoglobulin phage-display library cloned from bone marrow aspirate of patients infected with HCV was used for affinity selection against HCV NS3 helicase. Antibody fragments with high affinity to HCV helicase were isolated. To evaluate the inhibitory potential of isolated single-chain antibody fragments, a helicase-mediated, DNA-unwinding enzymatic assay was developed in ELISA format. Recombinant protein comprising the full-length HCV NS3 helicase domain was expressed in the baculovirus expression system. Recombinant antibodies that inhibit the HCV helicase at nanomolar concentrations, with efficacies ranging from 20 % to complete abrogation of enzymatic unwinding activity, were identified. These antibody fragments may be useful for novel gene therapeutic strategies that employ intracellular immunization and may provide new insights into the design of small molecule inhibitors of essential HCV proteins.

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Section: Discussionmentioning

confidence: 99%

Section: Generation Of Human Antibodies By Phage Display and Affinitymentioning

confidence: 97%

Section: Generation Of Human Antibodies By Phage Display and Affinitymentioning

confidence: 99%

mentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

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Abrogation of hepatitis C virus NS3 helicase enzymatic activity by recombinant human antibodies

Artsaenko

Tessmann

Sack³

et al. 2003

Journal of General Virology

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Specific targeting of hepatitis C virus core protein by an intracellular single-chain antibody of human origin

Karthe

Tessmann

et al. 2008

Hepatology

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The hepatitis C virus (HCV) core protein is essential for viral genome encapsidation and plays an important role in steatosis, immune evasion, and hepatocellular carcinoma. It may thus represent a promising therapeutic target to interfere with the HCV life-cycle and related pathogenesis. In this study, we used phage display to generate single-chain variable domain antibody fragments (scFv) to the core protein from bone marrow plasma cells of patients with chronic hepatitis C. An antibody with high-affinity binding (scFv42C) was thus identified, and the binding site was mapped to the PLXG motif (residues 84-87) of the core protein conserved among different genotypes. Whereas scFv42C displayed diffuse cytoplasmic fluorescence when expressed alone in the Huh7 human hepatoma cell line, cotransfection with the core gene shifted its subcellular distribution into that of core protein. The intracellular association of scFv42C with its target core protein was independently demonstrated by the fluorescence resonance energy transfer technique. Interestingly, expression of the single-chain antibody reduced core protein levels intracellularly, particularly in the context of full HCV replication. Moreover, cell proliferation as induced by the core protein could be reversed by scFv4C coexpression. Therefore, scFv42C may represent a novel anti-HCV agent, which acts by sequestering core protein and attenuating core protein-mediated pathogenesis. (HEPATOLOGY 2008;48:702-712.)

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HCV‐hepatocellular carcinoma: New findings and hope for effective treatment

Dash

Haque

Joshi

et al. 2005

Microscopy Res & Technique

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We present here a comprehensive review of the current literature plus our own findings about in vivo and in vitro analysis of hepatitis C virus (HCV) infection, viral pathogenesis, mechanisms of interferon action, interferon resistance, and development of new therapeutics. Chronic HCV infection is a major risk factor for the development of human hepatocellular carcinoma. Standard therapy for chronic HCV infection is the combination of interferon alpha and ribavirin. A significant number of chronic HCV patients who cannot get rid of the virus infection by interferon therapy experience long-term inflammation of the liver and scarring of liver tissue. Patients who develop cirrhosis usually have increased risk of developing liver cancer. The molecular details of why some patients do not respond to standard interferon therapy are not known. Availability of HCV cell culture model has increased our understanding on the antiviral action of interferon alpha and mechanisms of interferon resistance. Interferons alpha, beta, and gamma each inhibit replication of HCV, and the antiviral action of interferon is targeted to the highly conserved 5'UTR used by the virus to translate protein by internal ribosome entry site mechanism. Studies from different laboratories including ours suggest that HCV replication in selected clones of cells can escape interferon action. Both viral and host factors appear to be involved in the mechanisms of interferon resistance against HCV. Since interferon therapy is not effective in all chronic hepatitis C patients, alternative therapeutic strategies are needed to treat chronic hepatitis C patients not responding to interferon therapy. We also reviewed the recent development of new alternative therapeutic strategies for chronic hepatitis C, which may be available in clinical use within the next decade. There is hope that these new agents along with interferon will prevent the occurrence of hepatocellular carcinoma due to chronic persistent hepatitis C virus infection. This review is not inclusive of all important scientific publications due to space limitation.

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Cloning and molecular characterization of human high affinity antibody fragments against Hepatitis C virus NS3 helicase

Cited by 13 publications

References 48 publications

Abrogation of hepatitis C virus NS3 helicase enzymatic activity by recombinant human antibodies

Abrogation of hepatitis C virus NS3 helicase enzymatic activity by recombinant human antibodies

Specific targeting of hepatitis C virus core protein by an intracellular single-chain antibody of human origin

HCV‐hepatocellular carcinoma: New findings and hope for effective treatment

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