Anti-hepatitis c virus antibody and uses thereof

The present invention concerns human antibodies capable of specifically binding to conformation-dependent epitopes of Hepatitis C virus (HCV) glycoprotein E2 and various uses thereof.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including any manufacturer\'s specifications, instructions, etc.) are hereby incorporated herein by reference; however, there is no admission that any document cited is indeed prior art as to the present invention.

Hepatitis C virus (HCV) is the principal causative agent for non-A, non-B Hepatitis. The prevalence of HCV infection in the blood donor population has been estimated to range from 0.4 to 2% (Choo et al., 1989). Acute HCV infection leads, in more than 70% of the patients, to the development of chronic hepatitis that can evolve towards cirrhosis and hepatocellular carcinoma (Saito et al., 1990). HCV is an enveloped positive-stranded RNA virus which is classified in the Flaviviridae family (Francki et al., 1991, Miller et al., 1990). It contains a genome of about 9,500 nts encoding a polyprotein of 3010 to 3033 amino acids. Processing of the polyprotein by host and viral proteases results in the production of structural and nonstructural (NS) proteins (Rice et al., 1996). Structural proteins include a nucleocapsid and two putative virion envelope glycoproteins E1 and E2 (Miyamura et al., 1993). Non-structural proteins include NS2 to NS5 antigens.

In some individuals, acute infection successfully resolves indicating that HCV can be controlled by the host immune system. The mechanisms by which the host overcomes HCV infection remain unknown. Previous reports strongly suggest that humans and chimpanzees can generate virus-neutralizing antibodies (Choo et al., 1994, Farci et al., 1994, 1996, Shimizu et al., 1994). Successful in vivo protection of chimpanzees from primary infection by an homologous HCV isolate has been achieved following immunization with recombinant E1 and E2 proteins (Choo et al., 1994). In that study only those chimpanzees showing high anti-E2 antibody titers-were protected. While neutralizing antigenic domains were not identified, it was postulated that conformation of the immunogens was critical for the induction of neutralizing antibodies.

As there is to date no efficient in vitro replication system to grow the virus and develop neutralization assays, alternative assays to assess the biological function of anti-E1/E2 antibodies are actively searched for. Prevention of viral attachment onto presumed susceptible cells has been described in preliminary studies (Shimizu et al., 1994, Zibert et al., 1995). More recently, an “in vitro” neutralization of binding (NOB) assay has been developed that is exploiting the specific binding of a highly purified E2 protein onto susceptible target cells (Rosa et al., 1996). This assay allows the quantitative evaluation of NOB antibodies that are capable of neutralizing the binding of E2 onto such cells. Using this system, Rosa et al., have shown that only those chimpanzees immunized with E1 and E2 proteins that developed high anti-NOB titers were protected against challenged infection (Rosa et al., 1996), suggesting that NOB activity could be an indication for “in vivo” neutralization of viral infection. In HIV infection, a similar model has recently shown that affinity of antibody binding to envelope glycoprotein oligomers was a good predictor for virus neutralization (Fouts et al., 1997). Another way to assess the biological activity of anti-E1 and/or anti-E2 antibodies consists in testing the ability of such antibodies to recognize native structures believed to exist on the surface of virions. In vitro studies have shown that E1 and E2 interact to form non-covalently linked complexes (Deleersnyder et al., 1997, Ralston et al., 1993). Such complexes have been proposed to represent functional subunits of HCV virions (Deleersnyder et al., 1997, Dubuisson et al., 1994, Dubuisson and Rice, 1996, Ralston et al., 1993). Probing for the B-cell repertoire in viral infections is critical for the understanding of pathogenesis associated with these infections. Human monoclonal antibodies provide an alternative method to do so. Isolation and characterization of such antibodies have been reported in the case of HCV for only a limited number of viral antigens. These include the nucleocapsid, the NS3 and NS4 proteins (Akatsuka et al., 1993, Cerino et al., 1991, 1993, Chan et al., 1996, Mondelli et al., 1994) and more recently the glycoprotein E2 (Chan et al., 1996). In this latter case, authors used the phage display technology coupled with the use of synthetic peptides for the screening of the anti-E2 immune reactivity and were able to obtain specific IgG single-chain Fvs that recognized the E2 sequence. While a specific linear epitope sequence was identified, no biological activity for the anti-E2 antibody was described and the putative role of this antibody in the control or progression of infection remains undefined. Recently, WO97/40176 described immunoglobulin molecules obtained from a combinatorial library, which are capable of specifically binding with HCV E2 antigen. Although Fab-fragments of such immunoglobulins were demonstrated to have binding activity in a neutralization of binding assay recombinantly expressed Fab clones and corresponding whole IgG molecules were found to be negative in neutralizing the binding of the HCV E2 polypeptide.

The present invention relates to novel antibodies comprising at least one complementarity determining region (CDR) of the variable domain of a human antibody which is capable of specifically recognizing a conformation dependent epitope of HCV glycoprotein E2. Furthermore, the present invention relates to antigens recognized by said antibodies. In addition, the present invention relates to a polynucleotide encoding the above-described antibody or antigen, vectors comprising said polynucleotide as well as cells comprising the afore-mentioned polynucleotide or vector. A further aspect of the invention is a method for preparing antibodies capable of recognizing conformation dependent epitopes of HCV glycoprotein E2 and that are capable of neutralizing the binding of the E2 protein onto susceptible cells. The present invention further involves pharmaceutical and diagnostic compositions comprising the afore-mentioned antibodies, antigens, polynucleotides, vectors or cells as well as the use of the afore-described compounds in various therapeutic and diagnostic applications.

Accordingly, the technical problem of the invention is to provide means and methods for the treatment and prevention of HCV infection in humans.

The solution to this technical problem is achieved by providing the embodiments characterized in the claims, namely antibodies are provided that 1) recognize conformation-dependent determinant(s), 2) were capable of recognizing antigens derived from different HCV genotypes and 3) were able to precipitate noncovalently associated E1E2 complexes believed to exist on the surface of virion particles; and 4) are capable of neutralizing the binding of E2 protein onto susceptible cells suggesting the potential of the antibodies for in vivo neutralization. Such antibodies are particularly useful for the development of therapeutic or preventive strategies to fight infection by a highly mutable agent such as HCV.

Accordingly, the invention relates to an antibody comprising at least one (preferably two, more preferably three, four or five, and most preferably six) complementarity determining region (CDR) of the V_H and/or V_L region of a human antibody comprising the amino acid sequence encoded by the DNA sequence depicted in FIG. 5 (V_L) (SEQ ID NO: 1) and FIG. 6 (V_H) (SEQ ID NO: 3) that specifically recognizes a conformation-dependent epitope of Hepatitis C Virus glycoprotein E2 and is capable of precipitating covalently or non-covalently associated E2/E1 complexes. Alternatively, and/or in addition the antibody of the invention comprises at least 1, 2 or 3 CDR(s) of the V_L region of a human immunoglobulin chain comprising the amino acid sequence of SEQ ID NO: 6 and encoded by the DNA sequence depicted in SEQ ID NO: 5 which represents an allelic variant of the V_L encoding DNA sequence of SEQ ID NO: 1 (FIG. 5).

The person skilled in the art knew that each variable domain (the heavy chain V_H and light chain V_L) of an antibody comprises three hypervariable regions, sometimes called complementarity determining regions or “CDRs” flanked by four relatively conserved framework regions or “FRs”. The CDRs contained in the variable regions of the antibody of the invention can be determined, e.g., according to Kabat, Sequences of Proteins of Immunological Interest (U.S. Department of Health and Human Services, third edition, 1983, fourth edition, 1987, fifth edition 1990). The person skilled in the art will readily appreciate that the variable domain of the antibody having the above-described variable domain can be used for the construction of other polypeptides or antibodies of desired specificity and biological function. Thus, the present invention also encompasses polypeptides and antibodies comprising at least one CDR of the above-described variable domain and which advantageously has substantially the same or similar binding properties as the antibody described in the appended examples. The person skilled in the art will readily appreciate that using the variable domains or CDRs described above antibodies can be constructed according to methods known in the art, e.g., as described in EP-A1 0 451 216 and EP-A1 0 549 581.

The term “conformation-dependent epitope of Hepatitis C Virus glycoprotein E2” denotes the non-linear nature of the epitope recognized by the antibody of the invention. This means that the antigen\'s determinants of the epitope are provided by the three-dimensional structure of the HCV glycoprotein E2 rather than by the amino acid sequence as such.

The term “capable of precipitating covalently or non-covalently associated E2/E1 complexes” refers to the ability of the antibody of the invention to precipitate E1 and E2 noncovalently associated complexes which are believed to exist on the virion particle.

The term “capable of neutralizing the binding of E2 protein onto susceptible cells” describes the ability of candidate antibodies to neutralize the binding of highly purified E2 (neutralizing of binding or NOB) onto cells susceptible to HCV infection; see also Example 4. Advantageously, the antibody of the invention has an NOB activity at a concentration of about 1 μg/ml, preferably at a concentration of about 0.1 μg/ml and most preferably at a concentration of about 0.03 μg/ml.

In accordance with the present invention a screening assay that specifically allows the detection of anti-E2 antibodies capable of recognizing E2 directly expressed in cells without the requirement of antigen purification was chosen to identify and purify antibodies directed at conformation-dependent determinants. The assay was also based on expression of a genotype 1a derived antigen thus allowing for the characterization of cross-reactive anti-E2 antibodies and epitopes. Using this approach, two clones have been obtained producing anti-E2 antibodies from two HCV chronically infected patients. The first clone (clone 503) was obtained from one patient (patient 1) infected by a genotype 4 isolate while the second clone (clone 108) was derived from a second patient (patient 2) infected by a genotype 1b isolate. It could be shown that the HMabs displayed in addition a good reactivity against a genotype 1b antigen suggesting that the determinant(s) targeted by these antibodies are conserved among at least two of the main prevalent viral subtypes found in the world (subtypes 1a and 1b). In view of the above, it can be reasonably expected that the antibody of the present invention is also capable of reacting with antigens of other genotypes such as 2, 3a, 4, 5 and/or 6. The binding activity of an antibody of the invention concerning these genotypes can be easily tested in accordance with the methods as described in the examples.

The results obtained in accordance with the present invention indicate that the determinants recognized by the HMabs of the invention are targeted at conformation-dependent domains of E2 since linear determinants using different screening approaches, including peptide-scanning, Western-blots and immunofluorescence analysis using expressed truncated domains of the protein could not be identified; see Example 2. On the other hand, immunoprecipitation studies performed under reducing or non-reducing conditions indicated that the HMabs recognized a conformation-dependent-determinant. Under non-reducing conditions, these antibodies precipitated covalently as well as noncovalently associated E1E2 complexes; see Example 3.2. The latter are thought to be functional subunits incorporated in the virion particle (Deleersnyder et al., 1997). The present data, in particular obtained from kinetic analysis of epitope formation strongly indicate that the two HMabs recognize domains of the E2 protein that appear to be folded early. Such domains would stay accessible as the protein further matures, until it adopts its final conformation characteristic of the form of E2 susceptible to be present on the surface of virions. The kinetic analysis, together with the NOB data (i.e. antibody 503, displaying NOB activity) also suggest that the two antibodies recognize different determinants; see Example 4. Alternatively, that affinity of the antibodies for the E2 protein differs.

The most encouraging result obtained in accordance with the present invention was the demonstration that one of the HMabs displayed strong NOB activity. These observations together with Rosa\'s et al. (Rosa et al., 1996), indicate that the determinant(s) recognized by NOB antibodies are likely directed at conformation-dependent domains of E2, domains that appear to be conserved between different genotypes. Such domains seem to be distinct from the hypervariable region 1 (HVR) that has been shown to contain neutralization epitopes. In a recent study, Zibert et al. (Zibert et al., 1997) have been able to correlate early appearance of antibodies directed at a non-conformational structure found in the HVR with acute self-limited infection. Results from the study suggests the critical existence and role of antibodies directed at a linear determinant of E2 in the control of HCV infection, observations that are in agreement with a study originally performed in the chimpanzee model by Farci et al. (Farci et al., 1996). Authors in this latter study generated a hyperimmune serum directed at a peptide from the HVR, serum that contained antibodies capable to neutralize the infectivity of a well characterized inoculum in vitro. A similar experiment was also performed by Shimizu et al. (Shimizu et al., 1996). Thus, all of these studies strongly suggest that neutralization of HCV would mostly be type-specific involving the participation of variable, non-conserved epitopes. Nonetheless, recent observations have begun to suggest the existence of other neutralization determinants, cross reactive and not directed at the HVR. In the vaccination study by Choo et al., induced neutralizing antibodies were not directed at the HVR of E2 but apparently at other determinants carried by the antigen (Choo et al., 1994). Abrignani has recently observed a correlation between spontaneous resolution of chronic infection and appearance of high anti-NOB antibody titers (Abrignani 1997). In patients described in the Examples hereinafter, high or measurable neutralization of binding of E2 was not restricted to sera from patients infected with genotype 1a isolates, thus suggesting the existence of cross-reactive epitopes such as those described in the present application. As it was difficult to find a direct correlation between NOB titers of a purified MAb and titers found in patients sera (both patients in our study had similar NOB serum titers >1:1000), it was surprising that antibody 503 has an NOB activity detectable at very low concentration (0.03 μg/ml) providing for a potent activity; see Example 4.

The HMAbs produced in accordance with the present invention are expected to be useful tools to study further the biogenesis, folding and assembly of HCV glycoproteins as well as for characterization of the virion structure and a putative cell-surface receptor. As the antibody of the invention exemplified by Ab 503 represents the first HMAb described to date as having NOB activity, this antibody is particularly useful for passive immunization studies. Antibody infusion studies have demonstrated, in the case of lentiviruses, a beneficial role of administered neutralizing antibodies in the control and even the prevention of infection in different animal models (Conley et al., 1996, Emini et al., 1992, Putkonen et al., 1991).

In a preferred embodiment of the invention, said antibody is a monoclonal antibody, a polyclonal antibody, a single chain antibody, humanized antibody, or fragment thereof that specifically binds said HCV E2 glycoprotein also including bispecific antibody, synthetic antibody, antibody fragment, such as Fab, Fv or scFv fragments etc., or a chemically modified derivative of any of these. Monoclonal antibodies can be prepared, for example, by the techniques as originally described in Köhler and Milstein, Nature 256 (1975), 495, and Galfre, Meth. Enzymol. 73 (1981), 3, which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals with modifications developed by the art. Furthermore, antibodies or fragments thereof to the aforementioned epitopes can be obtained by using methods which are described, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual”, CSH Press, Cold Spring Harbor, 1988. When derivatives of said antibodies are obtained by the phage display technique, surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies which bind to an epitope of the conformation-dependent HCV glycoprotein E2 epitope (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). The production of chimeric antibodies is described, for example, in WO89/09622. Methods for the production of humanized antibodies are described in, e.g., EP-A1 0 239 400 and WO90/07861. A further source of antibodies to be utilized in accordance with the present invention are so-called xenogenic antibodies. The general principle for the production of xenogenic antibodies such as human antibodies in mice is described in, e.g., WO 91/10741, WO 94/02602, WO 96/34096 and WO 96/33735. As discussed above, the antibody of the invention may exist in a variety of forms besides complete antibodies; including, for example, Fv, Fab and F(ab)₂, as well as in single chains; see e.g. WO88/09344. In case of bispecific antibodies where one specificity is directed to an HCV E2 glycoprotein epitope and the other preferably to a T cell antigen such as CD3, it is advantageous if the binding site recognizing the viral epitope has a high affinity in order to capture the virus or target cells which have been infected with HCV and can be destroyed with high efficiency. On the other hand, the binding affinity of the binding site recognizing, e.g., a T cell should be in the order of those of the natural T cell receptor/ligand interaction or of that usually found for the interaction of the T-cell costimulatory molecules with their receptor.

The antibodies of the present invention or their corresponding immunoglobulin chain(s) can be further modified using conventional techniques known in the art, for example, by using amino acid deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) known in the art either alone or in combination. Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are well known to the person skilled in the art; see, e.g., Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y.