Complete inhibition of hepatitis C virus helicase activity by human antibody fragments cloned by phage display

Monday, March 31,2003

18

imately 17 months later. Liver histology was scored independently by two pathologists. Post-injection reactions were similar to placebo (alum). At week 48, ALT had decreased significantly in El patients (mean -17%; 95%CI: -25%, 9%), but not in the placebo group. At week 69, improved (>2 points) or stabilised total Ishak scores were seen in 19 patients (79%), diminished perisinusoidal fibrosis in 10 patients (42%) and negativation/marked reduction in E2 immunostaining in 11 patients (46%). Twenty-one patients (88%) developed a significant El-specific T-cell response. The increase in anti-El antibody levels correlated with the decrease in total Ishak score, the relative decrease in Ishak fibrosis scores, and ALT (all p<=O.Ol). Seven patients with the strongest increase in antibody levels showed a mean change of -0.9 (95%CI: -1.5, -0.2) forMetavirfibrosis and of -37% (95%CI: -49%, -25%) for ALT. Serum HCV-RNA remained unchanged. El therapeutic vaccination is well tolerated and could halt progression of liver fibrosis.

30 nucleotides of the core coding sequence was PCR-amplified before treatment (baseline) and at the end of treatment (EOT). Direct sequencing was performed before therapy in 24 patients, at EOT in 12, and extensive quasispecies analysis was performed in 8 patients at both time points (30 clones per time point). ISIS induced a virological response (more than 1 log HCV RNA decrease) in 3 patients, all receiving 2 mg/kg. The lack of virological response to ISIS 14803 was not due to primary HCV resistance resulting from mutations in the drug target site or neighboring regions. Four weeks of ISIS 14803 did not select variants bearing mutations in the drug target site, suggesting no secondary HCV resistance to ISIS 14803. In most patients, there was no significant evolution of the HCV IRES during therapy, pointing to strong conservatory constraints on HCV IRES genetic evolution in spite of ISIS 14803 administration.

I

50

COMPLETE ACTIVITY

INHIBITION

OF HEPATITIS C VIRUS HELICASE

BY HUMAN ANTIBODY

FRAGMENTS

CLONED

BY

PHAGE DISPLAY 0. Artsaenko,

K. Tessmann,

of Gastroenterology,

A. Erhardt, D. Haussinger,

Hepatology

Heinrich-Heine-University,

and Infectious

Dusseldor$

Dept

T. Heintges.

Diseases,

Germany

Hepatitis C Virus helicase is essential for viral replication. It is therefore a promising target for HCV-therapy. Specific inhibition of the NS3 helicase activity by intracellularly expressed antibodies is expected to terminate HCV replication in infected cells. The aim of the present study is the generation of human antibody fragments neutralising viral NS3 helicase activity. Methods: Phage-display technology has been employed for the isolation of single-chain variable domain antibody fragments against HCV helicase. Human immunoglobulin genes were cloned into a phagemid and displayed on the surface of filamentous bacteriophages. Affinity selection was carried out against enzymatically-active helicase domain expressed in a baculovirus system. Results A large library of human immunoglobulin genes was cloned from bone marrow aspirate of patients infected with HCV. Single-chain antibody fragments binding to NS3 protein with high affinity (KD 10.’ to 10m9 10 M) were selected, sequenced and characterized in detail. To evaluate inhibitory properties of the selected antibodies, a helicase-mediated DNAunwinding assay was developed in ELISA-format. Recombinant antibodies inhibiting HCV-helicase at nM concentrations with efficacies ranging from 30% to a complete abrogation of enzymatic activity were identified. Conclusions In conclusion, we generated recombinant human high-affinity antibody fragments against hcv NS3 protein. Complete inhibition of NS3 helicase activity, essential for HCV life cycle, has been demonstrated. These antibody fragments may allow a novel gene therapeutic strategy by intracellular immunization and provide new insight into rational drug design of small molecule inhibitors for essential HCV proteins.

I

49

HCV RESISTANCE

TO ISIS 14803, AN ANTISENSE

OLIGONUCLEOTIDE

INHIBITOR

REGION

ON ANTIVIRAL

SEQUENCE

OF HCV. EFFECT

OF TARGET

I 51

EFFICACY

France;

2Scripps

Clinic, La Jolla, California, Car&bad,

California,

WITH CHRONIC COMBINATION ANALYSIS

USA; ‘ISIS

USA

ISIS 14803 is a 20.base phosphorothioate antisense oligodeoxynucleotide that inhibits HCV replication and protein expression in cell culture and mouse models. Its target sequence is located within the 5’ noncoding region, in a conserved region of the internal ribosome entry segment (IRES) spanning nt 330-349. Our aims were to determine whether treatment failm-e is related to primary or secondary HCV resistance to this specific HCV replication inhibitor. 28 patients with chronic hepatitis C included in a doseescalation trial were treated with ISIS 14803 tiw for 4 weeks intravenously. A 5’ noncoding region fragment spanningthe full IRES including the first

OF PEGINTERFERON

PLUS RIBAVIRIN

M. Soler’, J.G. McHutchison2, T.J. Kwoh3, EA. Dorr3, J.-M. Pawlotsky ‘. ‘Hopi& Hem-i Mondos Universite Paris XII, Creteil, Pharmaceuticals,

EFFICACY

FOR 24 WEEKS

ALFA-2A (40KD) (PEGASYS) IN GENOTYPE

HEPATITIS C FOLLOWED THERAPY

1 PATIENTS

BY MONO OR

FOR 22 WEEKS:

OF AN OPEN, MULTICENTER,

PRELIMINARY RANDOMIZED

TRIAL

J.F! Bronowicki’,

D. 0uzan2, T. Asselah3, H. Desmorat4, J.P. Zarski’, J. Foucher6, M. Bourliere7, C. Renou’, A. Tran’, l? Melin9, C. Hezode”, M. Chevallier”, M. Bouvier”, J.M. Pawlotsky lo, I. Lonjon-Domanec’2. ‘Hasp Brabois,

‘Hasp

Beaujon,

Nancy; 2Amaud

Tzanck Institute, St Laurent Du Var;

Clichy; 4Clin Du Part, Toulouse;

6Hosp Haul Leveque,

Pessac;

2, Nice; ‘Hasp Saint, Dizier; “Hasp Meyrieun;

‘Univ Hasp, Grenoble;

7Hosp Saint Joseph, Hew-i Mondos

Marseille;

‘Hasp Acad

Creteil; “Lab

12Roche, Neuilly France

Recent clinical studies have shown that patients infected with HCV genotype 1 derive significant benefit from combination therapy with Peginter-