Disclosed is a hybridoma cell line which produces human antibodies capable of binding to the hepatitis C virus (HCV) E2 glycoprotein and capable of neutralizing HCV infection in vivo in an animal model, as well as antibodies produced by the cell line. Also disclosed are various uses of said antibodies in the prevention and treatment of HCV infection. Peripheral blood lymphocytes obtained from human donors having a high titer of anti HCV E2 antibodies are transformed in vitro by Epstein-Barr virus and then fused with heteromyeloma cells to generate hybridomas secreting human antibodies having a high affinity and specificity to HCV E2 glycoprotein.

FIELD OF THE INVENTION

The present invention concerns a hybridoma cell line producing human antibodies capable of binding to hepatitis C virus envelope glycoprotein, antibodies produced by the cell line and various uses thereof.

BACKGROUND OF THE INVENTION

Hepatitis C virus (HCV) infection is a major worldwide health problem. Approximately 170 million individuals worldwide are infected by HCV and chronically infected patients carry a high risk of developing cirrhosis and hepatocellular carcinoma (Cohen 1999 Science 285:26 30).

Interferon-.alpha. either alone or in combination with ribavirin is used for therapy of HCV showing efficacy in between 20% and 40% of patients respectively.

HCV is an enveloped virus the genetic information for which is encoded in a 9.5 kilo bases positive strand RNA genome. A highly conserved noncoding region of 341 base pairs is localized at the 5'-end of this viral genome, which is followed by a long open-reading frame coding for a polyprotein of approximately 3,010 amino acids. Two putative envelope glycoproteins, E1 (gp35) and E2 (gp72) have been identified with 5 or 6 and 11 N-linked glycosylation sites, respectively. A high level of genetic variability is associated with the envelope genes. This is highly accentuates at the 5'-end of the E2 gene, where two hypervariable regions termed HVR1 and HVR2, have been described (Kato et al., 1992 Bioch. Biophys. Res. Commun. 189:119 127).

Studies using HCV E1 E2 proteins expressed in mammalian cells showed that infected individuals have an antibody response to HCV E2 (Harada, et al., 1994 J. Gen. Virol. 76:1223 1231). Recent work proposes the existence of neutralizing antibodies in serum from HCV infected patients (Rosa et al., 1996 PNAS (USA) 93:1759 1763; Zibert et al., 1995 Virology 208:653 661; Zibert et al., 1997 J. Virol. 71: 4123 4127).

Investigators employed surrogate assays to provide insights into virus neutralization since the virus cannot be grown in vitro (Houghton. Hepatitis C viruses. In Fields B N, Knipe D M, Howley P M (eds) Virology. Lippincott-Raven, Philadelphia, pp1035 1053). One surrogate assay, the neutralization of binding (NOB) assay, evaluates the ability of a given antibody or serum to prevent the association of HCV E2 protein with a human T-cell line (Rosa et al., 1996 PNAS (USA) 93:1759 1763).

Habersetzer et al., 1998 Virology 249:3241 describes human monoclonal antibodies capable of inhibiting the interaction of HCV E2 with human cells in vitro. Burioni et al., 1998 Hepatology 28:810 814 report human recombinant Fabs for the HCV E2 protein similarly capable of inhibiting the interaction of HCV with human cells in vitro.

PCT patent application WO 200005266 discloses antibodies comprising at least one complementarity determining region (CDR) of the variable domain of a human antibody that specifically recognize a conformation-dependent epitope of HCV E2 and are capable of precipitating E1/E2 complexes.

PCT patent application WO 9740176 discloses a recombinant human antibody Fab portion capable of binding to HCV E2 obtained using a combinatorial antibody library. The relevance of such antibodies for therapy of HCV infection still needs to be demonstrated.

It is therefore of substantial interest to identify human monoclonal antibodies (Mabs) directed against the E2 glycoprotein that are capable of neutralizing HCV infection in vivo. Such antibodies may constitute a new alternative for the treatment of HCV infections.

Cardoso et al., J. Med. Virol. 55, 28 34 (1998) describes the isolation of human monoclonal antibodies capable of binding to hepatitis C virus envelope glycoproteins. One of the described antibodies (4F7) was further characterized and sequenced and is the subject matter of the present invention.

SUMMARY OF THE INVENTION

In accordance with the present invention, a hybridoma cell line is provided which secretes human antibodies capable of binding to the hepatitis C virus envelope glycoprotein E2 and capable of neutralizing HCV infection in vivo in an animal model. In accordance with the invention, peripheral blood mononuclear cells (PBMC) were obtained from human individuals having anti HCV E1/E2 antibodies. PBMC from the human donor may be obtained either by whole blood donation or by leukophoresis. The human PBMC are then transformed in vitro by Epstein-Barr virus (EBV) (Simoneit at al. 1994 Hybridoma 13:9 13). After transformation the resulting anti HCV producing lymphoblastoid cells are fused in vitro preferably with a human-mouse fusion partner such as a heteromyeloma by techniques well known in the art (e.g. Kohler & Milstein, Nature, 256:495 497, 1975). The generated hybridoma cell lines are either cultured in vitro in a suitable medium wherein the desired monoclonal antibody is recovered from the supernatant or, alternatively the hybridoma cell lines may be injected intraperitoneally into mice and the antibodies harvested from the malignant ascitis or serum of these mice. The supernatants of the hybridoma cell lines are screened by any of the methods known in the art such as enzyme linked immunosorbent assay (ELISA) or radioimmunoassy (RIA) for presence of anti HCV E1/E2 antibodies using HCV E1/E2 as a substrate for antibody binding. The human monoclonal anti HCV E1/E2 antibodies thus produced are further examined in a small animal model of HCV infection for their ability to neutralize the virus or reduce the viral load. Virus neutralization or the reduction in viral load may be measured, for example, by RT-PCR analysis of HCV RNA in the animal's sera or by the number of HCV positive mice.

In accordance with the preferred embodiment of the present invention, a hybridoma cell line which was deposited on May 17, 2000, at the European Collection of Cell Cultures (ECACC, CAMR, Salisbury, Wiltshire, SP4 0JG, UK) under the Accession No. 00051714 is provided. Anti HCV B2 human monoclonal antibodies secreted by the above hybridoma cell line designated herein as "HCV-AB 68" as well as fragments thereof retaining the antigen binding characteristics of the antibodies are also provided. Such fragments may be, for example, Fab or F(ab).sub.2 fragments obtained by digestion of the whole antibody with various enzymes as known and described extensively in the art. The antigen binding characteristics of an antibody are determined by using standard assays such as RIA, ELISA or FACS (Fluorescence activated cell sorter) analysis.

The antibodies of the invention have a relatively high affinity to HCV E2 being in the range of about 10.sup.-9 M and 10.sup.-11 M as determined by a BIAcore 2000 instrument (Pharmacia Biosensor).

The antigen bound by the antibodies defined above also constitutes an aspect of the invention.

Further aspects of the present invention are various prophylactic and therapeutic uses of the HCV-AB 68 monoclonal antibodies. In accordance with this aspect of the invention, pharmaceutical compositions comprising the HCV-AB 68 antibodies may be used for the treatment of chronic hepatitis C patients by administering to such patients a therapeutically effective amount of the antibodies or fragments thereof capable of binding to HCV E2. A therapeutically effective amount being an amount effective in alleviating the symptoms of the HCV infection or reducing the number of circulating viral particles in an individual. Such pharmaceutical compositions may also be used, for example, for passive immunization of newborn babies born to HCV positive mothers, and for passive immunization of liver transplantation patients to prevent possible recurrent HCV infections in such patients. A further aspect of the invention is a pharmaceutical composition comprising a therapeutically effective amount of the antibodies of the invention combined with at least one other anti viral agent as an additional active ingredient. Such agents may include but are not limited to interferons, anti HCV monoclonal antibodies, anti HCV polyclonal antibodies, RNA polymerase inhibitors, protease inhibitors, IRES inhibitors, helicase inhibitors, immunomodulators, antisense compounds and ribozymes.

Claim 1 of 11 Claims

1. A human monoclonal antibody or fragment, capable of binding to HCV envelope glycoproteins and capable of neutralizing HCV infection in vivo, being selected from the group consisting of: (a) a human monoclonal antibody HCV-AB 68, which is secreted by the hybridoma cell line deposited in the European Collection of Cell Cultures (ECACC) under Accession No. 00051714, or a fragment thereof which retains the antigen binding characteristics of HCV-AB68; and (b) a human monoclonal antibody or fragment thereof comprising at least a heavy chain variable region whose amino acid sequence is depicted in FIG. 4 (SEQ ID NO: 4) and a light chain variable region whose amino acid sequence is depicted in FIG. 4 (SEQ ID NO: 3).

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