WO2008079890A1

WO2008079890A1 - Hepatitis c virus (hcv) inhibitory monoclonal antibody and bindable ligand thereof

Info

Publication number: WO2008079890A1
Application number: PCT/US2007/088173
Authority: WO
Inventors: Kirsten Nagashima; Sofija Andjelic; William Olson
Original assignee: Progenics Pharmaceuticals (Nevada), Inc.
Priority date: 2006-12-20
Filing date: 2007-12-19
Publication date: 2008-07-03

Abstract

Monoclonal antibodies (MAbs), or a portion thereof, that inhibit HCV infection of susceptible cells are described. The MAbs of the invention inhibit infection by HCV of different genotypes, including genotypes 1 and 2 and combinations thereof. One MAb designated PA-30, having the foregoing characteristics, is produced by a hybridoma cell line, also called PA-30 (ATCC Accession No. PTA-7278). The PA-30 MAb binds a ligand expressed by human B cells, liver cells, and mitogen-stimulated T cells. The MAbs, including PA-30, or humanized or chimeric forms thereof, alone or together with other antiviral agents, can provide therapeutic and prophylactic reagents and drugs for use in methods of treating and preventing infection of cells susceptible to HCV infection, and in methods of blocking HCV entry into cells from or within a subject infected with HCV, or at risk of infection by HCV.

Description

HEPATITIS C VIRUS (HCV) INHIBITORY MONOCLONAL ANTIBODY AND

BINDABLE LIGAND THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS:

[0001] This application claims benefit of U.S. Provisional Patent Application No. 60/876,488, filed December 20, 2006, the entire contents of which are incorporated by reference herein.

[0002] This invention was made with support under United States Government Grant No. AI069675 from the National Institutes of Health. Accordingly, the United States Government has certain rights in the subject invention.

[0003] Throughout this application, certain publications are referenced. Full citations for these publications may be found immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention relates.

BACKGROUND OF THE INVENTION

[0004] First recognized in 1989, Hepatitis C virus (HCV) infects the liver and is responsible for the majority of cases of non-A, non-B hepatitis (Alter and Seef, 1993). Infections are typically chronic and lifelong; many infected individuals are healthy and unaffected for decades, while others develop chronic hepatitis or liver cirrhosis, the latter often leading to hepatocellular carcinoma (Fry and Flint, 1997; Lauer and Walker, 2001).

[0005] Although screening of the blood supply has drastically reduced new transmissions of the virus, there exists a large cohort of infected individuals who will require treatment in the coming decades. The World Health Organization estimates that 3% of the world's population, or some 170 million people, are currently infected with HCV, with another 3 to 4 million new infections occurring each year. Approximately 3.9 million Americans have been infected with HCV, making it the most common chronic blood-borne viral infection in the United States. Chronically infected persons are at risk of developing severe and potentially life-threatening liver disease, including cirrhosis and hepatocellular carcinoma, and HCV infection is the leading cause of liver transplantation in the U.S.

[0006] HCV infection and its clinical sequelae are the leading causes of liver transplantation in the Unites States. No vaccine is currently available, and the two licensed therapies, interferon- alpha and ribavirin, which are both non-specific anti-viral agents with incompletely understood mechanisms of action, are only modestly efficacious (McHutchison et al, 1998). Thus, while the best long-term response rates are obtained with a combination of interferon alpha-2b and ribavirin, only a minority of individuals treated with this combination achieves the desired result of no detectable serum HCV RNA six months after stopping treatment (McHutchison et al., 1998). Moreover, as an antiviral therapy, these drugs are expensive and exhibit severe, life- threatening toxicities, including neutropenia, hemolytic anemia and severe depression. Thus, there is an urgent need for the development of new therapeutic approaches and agents to combat HCV infection.

[0007] Co-infection with HIV-I and HCV is also common, particularly in intravenous drug abusers and hemophiliacs (Poles, M. A. and D. T. Dieterich, 2000). HIV-I infection increases HCV virus load, liver-related mortality and the risk of perinatal transmission of HCV and may accelerate the course of HCV disease (Dieterich, D, T., 2002). Similarly, HCV infection increases the frequency of complications in HIV-I -infected individuals, and co-infected individuals progress to AIDS or to death significantly faster than in patients infected with only HIV-I. (Id.)

[0008] Hepatocytes are the primary target cells for HCV infection, and infection results in the progressive loss of liver function. HCV RNA, protein and virus-like particles have been visualized in liver biopsies of HCV-positive individuals and also have been correlated with liver disease (Boisvert, J. et al., 2001; Pal, S. et al., 2006). Recent studies have demonstrated robust replication of cloned HCV isolates in human hepatoma cells but not other cell lines in vitro (Lindenbach, B. D. et al., 2005; Wakita, T. et al., 2005; Zhong, J., P. et al., 2005). Other cell types also may be susceptible to HCV infection (Lerat, H. et al., 1998; Navas, M. C, et al., 2002) and may contribute to the extrahepatic manifestations of HCV infection (Agnello, V. and F. G. De Rosa, 2004).

[0009] Tt has been reported that HCV can replicate in peripheral blood mononuclear cells (PBMC) from chronically infected HCV+ patients (Moldvay, J. et al., 1994; Lerat, H. et al., 1998, Laskus, T. et al., 2000; Bare, P. et al., 2005). It has also been shown that HCV can infect lymphoid cells, preferentially B lymphocytes, monocytes and macrophages (Caussin- Schwemling, C. et al., 2001; Boisvert, J. et al., 2001 ; Sung et al., 2003). However, in these types of cells, HCV replication typically occurs at very low levels and the number of viral particles that are released are low and thus may not influence plasma viral loads (Rodriguez- Iήigo, E. et al., 2000; Boisvert, J. et al.. 2001 ; Bare, P. et al., 2005). Nonetheless, PBMC, B lymphocytes and monocytes/macrophages may serve as extra-hepatic reservoirs that may account for virus occurrence and persistence, particularly in immunosuppressed individuals (Laskus, T. et al., 2000; Bare, P. et al., 2005).

[0010] HCV genomes exhibit considerable sequence diversity and have been classified into six major genotypes (exhibiting <70% sequence identity), which are further divided into subtypes (exhibiting >70% identity), (Manns, M. P., et al., 2001 ; Zein, N. N., 2000). Genotypes 1 and 2 of HCV constitute 80-100% of all HCV infections in North America, Europe and Japan. Of these, genotype Ib is a more aggressive strain that is associated with more severe liver disease and reduced response to existing therapies (Zein, N. N., 2000). Genotype 3 constitutes approximately one-third of infections in Southeast Asia and Australia. Genotype 4 accounts for nearly all infections in Egypt and was transmitted in large part by re-use of needles during a campaign to treat schistosomes (blood flukes), (Frank, C, et al., 2000). Genotypes 5 and 6 are most commonly found in Southern Africa and Southeast Asia (Zein, N. N., 2000).

[0011] The HCV genome is a 9.6 kb positive-sense, single-stranded RNA molecule that replicates exclusively in the cytoplasm of infected cells (Rice, 1996). The genomic RNA encodes a .about.3000 amino acid polyprotein that is processed to generate at least ten proteins termed C, El, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B (Grakoui et al., 1993; Rice, 1996; Lauer and Walker, 2001). The C protein constitutes the nucleocapsid; El and E2 are transmembrane envelope glycoproteins; p7 is a membrane spanning protein of unknown function; and the various non-structural (NS) proteins have replication functions (Bartenschlager and Lohmann, 2000; Op De Beeck et al., 2001).

[0012] The envelope glycoproteins of HCV are thought to play a crucial role in viral infectivity through their direct effect on various processes, including the packaging of virions, the attachment of virions to target cells, fusion with and entry into these cells, and the budding of viruses from cell membranes before another round of cell infection can be initiated. Li particular, virus entry into susceptible target cells is mediated by the HCV envelope glycoproteins El and E2. HCV entry into host cells requires attachment of the viral particle to the cell surface, followed by fusion of the viral envelope with the cellular membrane.

[0013] Liver tropism maps to the HCV envelope glycoproteins El and E2 (Bartosch, B., et al., 2003; Bertolini, L. et al., 1993; Cormier, E. G. et al., 2004; Hsu, M. et ai., 2003; Zhang, J. et al., 2004). El is homologous to Class II fusion proteins of flaviviruses and alphaviruses (Garry, R. F. and S. Dash, 2003). E2 is a receptor-binding subunit with affinity for both CD81, which serves as an entry co-receptor for HCV (Cormier, E. G. et al., 2004) and scavenger receptor class B type 1 (SR-Bl) (Scarselli, E. et al., 2002), another molecule implicated in HCV entry (Bartosch, B. et al., 2003; Lavillette, D. et al., 2005; Voisset, C. et al., 2005). El and E2 are released from the HCV polyprotein by signal peptidase and associate into E1E2 heterodimers, which mediate fusion (Op De Bceck, A. et a]., 2004; Voisset, C. and J. Dubuisson, 2004).

[0014J In mammalian cell-based expression systems, the molecular weight of mature, full length El is about.35 kDa and that of E2 is about.72 kDa (Grakoui et al., 1993; Matsuura et al., 1994; Spaete et al., 1992). El and E2 are present as a non-covalently associated heterodimer, hereinafter referred to as E1E2, on the virus surface and undergo extensive posttranslational modification by N- linked glycosylation (Lauer and Walker, 2001).

[0015] Synthesis of the HCV structural proteins, C-El-E2-p7, in the cell is followed by translocation into the endoplasmic reticulum (ER), which is accompanied by cleavage of internal signal sequences by ER-resident signal peptidases (Bartenschlager and Lohmann, 2000; Op De Beeck et al., 2001; Reed and Rice, 2000). It is assumed that HCV buds into the ER and matures by passage through cytoplasmic vesicles (Pettersson, 1991). Studies of the subcellular localization of HCV envelope glycoproteins and particles in cells transfected or infected in vitro suggest vesicle-based morphogenesis of HCV (Dash et al., 1997; Egger et al., 2002; Greive et al., 2002; Iocovacci et al., 1997; Pietschmann et al., 2002; Serafino et al., 1997; Shimizu et al., 1996). However, HCV-like particles have been detected in the cytoplasm of hepatocytes from infected patients, which suggests budding at the plasma membrane (DeVos et al., 2002), although the budding and maturation process of HCV have not yet been fully delineated.

[0016] Two common experimental models of viral entry are cell-cell membrane fusion between receptor- and envelope glycoprotein-expressing cells, and entry of "reporter" viruses pseudotyped with heterologous envelope glycoproteins. Both systems rely on cell surface- associated expression of functional envelope glycoproteins. However, achieving expression of El and E2 on the surface of cells has proven to be elusive.

[0017] Serum- and plasma-associated HCV antibodies have been reported to prevent infection in humans. For example, prospective clinical trials and retrospective studies have demonstrated that neutralizing antibodies naturally present in polyclonal immune globulin preparations protect patients exposed to HCV via transfusions, dialysis, sexual contact and horizontal transmission (al Khaja, N. et al., 1991; Borgia, G., 2004; Conrad, M. E. and S. M. Lemon, 1987; Knodell, R. G. et al., 1976; Knodeil, R. G. et al., 1977; Kuhns, W. J. et al., 1976; Piazza, M. et al., 1998; Sanchez-Quijano, A. et al., 1988; Seeff, L. B. et al., 1977; Simon, N., 1984; Sugg, U. et al., 1985). Immune globulin isolated from plasma containing hepatitis B virus (HBV) cross- protected against HCV re-infection following liver transplant. Because there is a high rate of co- infection with HBV and HCV, the cross-protection was attributed to HCV neutralizing antibodies in the preparations. In support of this view, when the source plasma was screened for HCV antibodies and HCV-positive units were excluded during manufacturing, cross-protection was significantly diminished, and rates of HCV re-infection increased (Feray, C. et al., 1998).

[0018] Additionally, prior to the introduction of methods for screening and removing plasma containing HCV antibodies, there were no documented cases of HCV transmission associated with FDA-approved intravenous immune globulin preparations (IVIG). However, an outbreak of HCV transmission did occur shortly after the introduction of HCV screening in what became known as the "Gammagard incident" (1994 MMWR Morb. Mortal. WkIy. Rep.; Bresee, J. S. et al., 1996; Flora, K. et al., 1996; Gomperts, E. D., 1996; Healey, C. J. et al., 1996). In this incident, approximately 80 cases of HCV infection were attributed to the first two lots of screened Gammagard, while no cases of infection were associated with the prior six lots of unscreened product. Gammagard is currently subjected during manufacturing to procedures that inactivate HCV.

[0019] Approximately 15% of infected patients clear HCV via immune mechanisms. Although the correlates of clearance remain poorly understood, several studies have implicated humoral immune responses. Resolution of infection has been associated with the development of antibodies that block E2 binding to CD81 (Ishii, K., et al., 1998) or that bind the hypervariable region 1 (HVRl) of E2 (Allander, T. et al., 1997; Isaguliants, M. G. et al., 2002; Zibert, A. et al., 1997), which is important for HCV entry (Callens, N. et al., 2005). In a large, well-defined cohort of patients with acute HCV infection, outcome was associated with the extent of sequence evolution in E2. Self-limiting infection was associated with limited evolution in HRVl, whereas progression was associated with significant genetic evolution over the same time period (Farci, P. et al., 2000). The sequence changes were temporally associated with seroconversion and are consistent with selective pressure by the host humoral immune system. Collectively, these studies demonstrate that HCV antibodies can play an important role in preventing and/or clearing virus in a number of clinical settings and provide clinical support for antibody-based therapies of HCV infection.

[0020] Research in the area of HCV receptor biology is still ongoing. For example, it has been reported that CD81 functions as a post-attachment entry co-receptor for HCV and that other hepatocyte-specifϊc factors act in concert with CD81 to mediate HCV binding and entry (Cormier, E. G., 2004). More recently, the tight junction protein, claudin 1 has been reported to be associated with the susceptibility of human hepatoma cell lines to replicating HCV (HCVcc) and to retroviral particles pseudotyped with HCV E1E2 (HCVpp), (MJ. Evans et al., Nature Letters, 2007). In addition, L-SIGN (liver/lymph node-specific intercellular adhesion molecule- 3-grabbing non-integrin; CD209L) has been shown to be a liver-specific capture receptor for HCV (Gardner, J. P. et al., 2003). Patient-derived HCV virions were found to bind specifically to L-SIGN, and this interaction was mapped to HCV E2 Qd.). However, L-SIGN does not function as a traditional entry receptor, but rather binds HCV and mediates trans-infection of adjacent liver cells (Cormier, E. G., 2004). Accordingly, it has been proposed that L-SlGN present on liver sinusoidal endothelial cells may bind blood-borne HCV, concentrate virus in the liver, and mediate trans-infection of neighboring hepatocytes to facilitate the establishment and maintenance of HCV infection in vivo (Cormier, E. G., 2004; Gardner, J. P. et al., 2003). Scarselli et al. identified human scavenger receptor class B type 1 (SR-Bl) as an HCV receptor, by cross-linking soluble HCV E2 to HeρG2 cells (Scarselli, E. et al., 2002). SR-Bl, an 82kD protein having nine N-linked glycosylation sites, is a member of the CD36 superfamily and is highly expressed on hepatocytes. Other members of the superfamily do not bind sE2 (Id.).

[0021] Although recent experiments have suggested that claudin 1 may be involved in a late step in viral entry, after the virus has bound to cells and interacted with the CD81 co-receptor (MJ. Evans et al., Nature Letters, 2007), it is highly likely that additional, as-yet-unidentified receptors and cofactors are necessary for HCV to infect cells. This is supported by the fact that some human cell lines containing known HCV co-receptors still do not become infected. HCV also does not enter some human cells that express all of the HCV entry-associated factors. Moreover, the virus is not able to infect mouse cells that have been engineered to express the human receptors. In view of the several types of cells that exhibit susceptibility to HCV infection and the inability of known co-receptors to render various cell types susceptible to HCV infection, an understanding and elucidation of receptor and co-receptor molecules involved with infection of cells by HCV is not yet complete.

[0022] Because the mechanism(s) and target molecule(s) that allow HCV infection of susceptible cells are currently under investigation, the generation and production of agents that are able to reduce or prevent the ability of HCV to infect susceptible cells, as well as extra- hepatic cells and tissues, are important goals to achieve in the ongoing work to reduce and eradicate HCV and infection by HCV. In view of the clear need for safe, effective and targeted anti-HCV therapies, a variety of new therapeutic and prophylactic agents, e.g., anti-HCV antibodies, HCV inhibitory antibodies and the like, will be extremely beneficial as drug candidates for treating and preventing HCV infection. Such agents and/or their target ligands may also be useful as therapeutics and as diagnostic tools for the screening of patient samples.

SUMMARY OF THE INVENTION

[0023] The present invention provides novel monoclonal antibodies (MAbs) that inhibit infection of susceptible cells by Hepatitis C Virus (HCV) of diverse genotypes. The monoclonal antibodies of the present invention block or inhibit entry of HCV into susceptible target cells. ^'throughout this application, the terms "block'^* HCV infection and "inhibit" HCV infection are used interchangeably. [0024] The present invention provides a monoclonal antibody (mAb) or a portion thereof that blocks infection of HCV of different genotypes and binds a ligand present on human B lymphocytes, B cell lines, and on at least one other HCV susceptible cell, including human liver cells. In one embodiment, the monoclonal antibody of the present invention binds human B cells in a dose-dependent manner. In another embodiment, the monoclonal antibody of the invention binds Ramos cells and Daudi cells.

[0025] In an embodiment, the present invention provides a monoclonal antibody (MAb) or portion thereof that binds cell populations comprising human peripheral blood mononuclear cells (PBMCs) including monocytes, primary human T cells and B cells, for example, human primary B cells. In an embodiment, the present invention provides a monoclonal antibody (MAb) or portion thereof that binds human liver cells, including human liver cell lines.

[0026] The invention provides a monoclonal antibody or a portion thereof which inhibits entry of Hepatitis C Virus (HCV) into cells susceptible to infection by HCV, binds human B lymphocytes, binds human liver cells, and binds CD4+ and/or CD8+ T lymphocytes following stimulation with mitogen. In an embodiment, the human B lymphocytes are selected from the group consisting of peripheral blood mononuclear cells having a CD4^~CD8XD19⁺CD20⁺ phenotype, Ramos cells and Daudi cells. In an embodiment, the liver cells are Hep3b cells, HepG2 cells, or primary human hepatocytes. In an embodiment, the mitogen-stimulated T lymphocytes are CD4+ T lymphocytes. In an embodiment, the mitogen-stimulated T lymphocytes are CD8+ T lymphocytes. In an embodiment, the monoclonal antibody or portion thereof inhibits the entry of HCV of genotype 1, genotype 2, or a combination thereof into susceptible cells. In an embodiment, the monoclonal antibody or portion thereof inhibits the entry of HCV of genotypes Ia, Ib, 2b and/or la/2b into susceptible cells. In an embodiment, the monoclonal antibody or portion thereof inhibits entry of HCV of genotype Ia into susceptible cells. In an embodiment, the monoclonal antibody or portion thereof inhibits entry of HCV of genotype Ib into susceptible cells. In an embodiment, the monoclonal antibody or portion thereof inhibits entry of HCV of genotype 2b into susceptible cells. In an embodiment, the monoclonal antibody or portion thereof inhibits entry of HCV of genotype la/2b into susceptible cells. In an embodiment, the monoclonal antibody or portion thereof inhibits entry of HCV into cells susceptible to HCV infection as determined by a median IC50 value of 5 μg/mL or less, or of 2 μg/mL or less.

[0027] The present invention provides a monoclonal antibody (MAb) as described above that blocks infection of susceptible cells by HCV of different genotypes. Tn an embodiment, the monoclonal antibody of the invention blocks infection by HCV of genotypes 1 and 2, including subtypes thereof, e.g., Ia, Ib, 2a, 2b, la/2b, and other combinations thereof.

[0028J The invention provides an HCV inhibitory monoclonal antibody or portion thereof that (i) inhibits entry of Hepatitis C Virus (HCV) into cells susceptible to infection by HCV; (ii) binds a ligand of approximately 45-65 kDa expressed by human B lymphocytes; and (iii) binds human liver cells. In an embodiment, the ligand is highly expressed by human B lymphocytes, e.g., peripheral blood mononuclear cells having a CD4^~CD8^~CD19⁺CD20⁺ phenotype, Ramos cells and Daudi cells. In a particular embodiment, the ligand is highly expressed by Ramos cells. In an embodiment, the monoclonal antibody or portion thereof binds human liver cells, e.g., Hep3b cells, HepG2 cells, or primary human hepatocytes. In an embodiment, the monoclonal antibody or portion thereof binds CD4+ and CD8+ T lymphocytes following exposure to cell stimulation or activation factors. In an embodiment, the monoclonal antibody or portion thereof binds CD4+ and CD8+ T lymphocytes following stimulation with mitogen, e.g. phytohemagglutinin (PHA). In an embodiment, HCV is of genotype 1, genotype 2, or a combination thereof. In an embodiment, the monoclonal antibody or portion thereof inhibits HCV infection at a median IC50 value of 5 μg/mL or less. In an embodiment, the monoclonal antibody or portion thereof inhibits HCV infection at a median IC50 value of 2 μg/mL or less.

[0029] The invention provides an HCV inhibitory monoclonal antibody or portion thereof that (i) inhibits entry of Hepatitis C Virus (HCV) into cells susceptible to infection by HCV; (ii) binds a ligand of approximately 45-60 kDa expressed by human B lymphocytes; and (iii) binds human liver cells. In an embodiment, the ligand is highly expressed by human B lymphocytes, e.g., peripheral blood mononuclear cells having a CD4^~CD8^~CD19⁺CD20⁺ phenotype, Ramos cells and Daudi cells. In a particular embodiment, the ligand is highly expressed by Ramos cells. In an embodiment, the monoclonal antibody or portion thereof binds human liver cells, e.g., Hep3b cells, HepG2 cells, or primary human hepatocytes. Tn an embodiment, the monoclonal antibody or portion thereof binds CD4+ and CD8+ T lymphocytes following exposure to cell stimulation or activation factors. In an embodiment, the monoclonal antibody or portion thereof binds CD4+ and CD8+ T lymphocytes following stimulation with mitogen, e.g. phytohemagglutinin (PHA). In an embodiment, HCV is of genotype 1, genotype 2, or a combination thereof. In an embodiment, the monoclonal antibody or portion thereof inhibits HCV infection at a median IC50 value of 5 μg/mL or less. In an embodiment, the monoclonal antibody or portion thereof inhibits HCV infection at a median IC50 value of 2 μg/mL or less.

[0030] The present invention provides a novel monoclonal antibody designated PA-30 that is produced by a hybridoma cell line, also designated PA-30, which was deposited at the American Type Culture Collection under the terms of the Budapest Treaty. The PA-30 monoclonal antibody is designated ATCC Accession No. PTA-7278. In an embodiment, the PA-30 monoclonal antibody or portion thereof inhibits entry of Hepatitis C Virus (HCV) into cells susceptible to infection by HCV, binds human B lymphocytes, binds human liver cells, and binds CD4+ and/or CD8+ T lymphocytes following stimulation with mitogen. In an embodiment, the human B lymphocytes are selected from the group consisting of peripheral blood mononuclear cells having a CD4^~CD8^~CD19 CD20⁺ phenotype, Ramos cells and Daudi cells. In an embodiment, the liver cells are Hep3b cells, HepG2 cells, or primary human hepatocytes. In an embodiment, the mitogen-stimulated T lymphocytes are CD4+ T lymphocytes. In an embodiment, the mitogen-stimulated T lymphocytes are CD8+ T lymphocytes. In an embodiment, the monoclonal antibody or portion thereof inhibits the entry of HCV of genotype 1, genotype 2, or a combination thereof into susceptible cells. In an embodiment, the monoclonal antibody or portion thereof inhibits the entry of HCV of genotypes Ia, Ib, 2b and/or la/2b into susceptible cells. In an embodiment, the monoclonal antibody or portion thereof inhibits entry of HCV of genotype Ia into susceptible cells. In an embodiment, the monoclonal antibody or portion thereof inhibits entry of HCV of genotype Ib into susceptible cells. In an embodiment, the monoclonal antibody or portion thereof inhibits entry of HCV of genotype 2b into susceptible cells. In an embodiment, the monoclonal antibody or portion thereof inhibits entry of HCV of genotype la/2b into susceptible cells. In an embodiment, the monoclonal antibody or portion thereof inhibits entry of HCV into cells susceptible to HCV infection as determined by a median IC50 value of 5 μg/mL or less, or of 2 μg/mL or less, hi an embodiment, the PA-30 monoclonal antibody binds a ligand of approximately 45-65 kDa expressed by human B cells. In an embodiment, the PA-30 monoclonal antibody binds a ligand of approximately 60 kDa expressed by human B cells. In an embodiment, the approximately 60 kDa ligand comprises a component of a cell membrane-associated protein complex. Such cell membrane proteins may be associated prior to HCV infection of a susceptible cell. Alternatively, the proteins may form a complex at the time of or at a time subsequent to virus attachment and/or infection of the cell, In an embodiment, the PA-30 monoclonal antibody or portion thereof binds liver cells and stimulated T lymphocytes, e.g., mitogen-stimulated T lymphocytes.

[0031 ] The present invention provides a monoclonal antibody PA-30 produced by a hybridoma cell line designated PA-30 (ATCC Accession No. PTA-7278), or a portion of antibody PA-30, that inhibits HCV infection of susceptible cells. [0032] The present invention provides a monoclonal antibody PA-30 produced by a hybridoma cell line designated PA-30 (ATCC Accession No. PTA-7278), or a portion of antibody PA-30, that inhibits HCV infection of cells infected by HCV pseudoparticles (HCVpp) of different genotypes.

[0033] The present invention provides a monoclonal antibody PA-30 produced by a hybridoma cell line designated PA-30 (ATCC Accession No. PTA-7278), or a portion of antibody PA-30, that binds human B cells and liver cells.

[0034] The present invention provides a monoclonal antibody PA-30 produced by a hybridoma cell line designated PA-30 (ATCC Accession No. PTA-7278), or a portion of antibody PA-30, that binds a protein (ligand) present on and/or expressed by human B cells and liver cells. In an embodiment, the protein (ligand) is highly expressed on the surface of the human B cells. Throughout this specification, the terms protein, ligand, or PA-30 ligand, bindable by the PA-30 monoclonal antibody, are used interchangeably.

[0035] The present invention provides a monoclonal antibody PA-30 produced by a hybridoma cell line designated PA-30 (ATCC Accession No. PTA-7278), or a portion of antibody PA-30, that binds a protein of approximately 60 kDa that is present on human B cells.

[0036] The present invention provides a hybridoma cell line designated PA-30 (ATCC Accession No. PTA-7278), which produces a monoclonal antibody designated PA-30.

[0037] The present invention further provides a cell which expresses a monoclonal antibody designated PA-30 (ATCC Accession No. PTA-7278). The invention provides a monoclonal antibody PA-30 produced by a hybridoma cell line designated PA-30 (ATCC Accession No.

PTA-7278).

[0038] The present invention provides an antibody, either polyclonal or monoclonal, that binds the same ligand or epitope thereof as is bound by monoclonal antibody PA-30 (ATCC Accession No. PTA-7278), or a portion thereof. In one embodiment the antibody is monoclonal. In one embodiment, the antibody is polyclonal. In another embodiment, the antibody or portion thereof binds the same approximately 60 kDa B cell ligand as is bound by monoclonal antibody PA-30 (ATCC Accession No. PTA-7278). In another embodiment, the antibody or portion thereof competes with monoclonal antibody PA-30 for binding the approximately 60 kDa B cell ligand, or an epitope thereof.

[0039] In accordance with the present invention there is further provided a protein having an approximate molecular weight of about 60 kDa, which is present on human B cells and which is bound by monoclonal antibody PA-30 (ATCC Accession No. PTA-7278) or a bindable portion thereof.

[0040] This invention further provides a method of producing a monoclonal antibody that is an inhibitor of HCV entry into susceptible cells. The method involves immunizing a subject, e.g., a recipient animal, such as a rodent, e.g., mouse or rat, with cells that express functional HCV receptor and/or co-receptor molecules. In an embodiment, the cells used as immunogen are replication-competent for HCV and are permissive for HCV entry and infection. In an embodiment, the cells used as immunogen may be molecularly engineered to express a receptor or co-receptor molecule, for example, CD81, that renders the cell susceptible to HCV entry. In an embodiment, the invention provides a method of producing a monoclonal antibody which binds cells susceptible to HCV infection and inhibits entry of HCV into the HCV susceptible cells, which involves immunizing a subject with cells which (i) express functional HCV receptor and/or co-receptor molecules; (ii) are replication-competent for HCV and (iii) are permissive for HCV entry and infection; producing hybridoma cells comprising immune B cells from the immunized subject of (a); and assaying antibody generated by the hybridoma cells for the antibody's ability to bind HCV susceptible cells and inhibit entry of HCV into the HCV susceptible cells.

[0041 ] The present invention provides a method of inhibiting HCV infection of a cell susceptible to HCV infection using a monoclonal antibody of the invention, which is capable of inhibiting HCV infection of susceptible cells, for example, by blocking HCV entry into the cells, as described herein. It is to be understood that the described methods may encompass the use of one monoclonal antibody, for example, the PA-30 monoclonal antibody; a combination of one or more of the monoclonal antibodies, including PA-30; or an HCV inhibiting portion of the one or more monoclonal antibodies, e.g., the PA-30 monoclonal antibody or a portion thereof. In an embodiment, the HCV infection inhibiting monoclonal antibody or portion thereof is humanized or chimeric. In an embodiment, the HCV infection inhibiting monoclonal antibody or portion thereof is humanized or chimeric PA-30.

[0042] Accordingly, the present invention provides a method of inhibiting HCV infection of a cell susceptible to HCV infection which comprises contacting susceptible cells that are or may be in the presence of HCV virions or particles with a monoclonal antibody of the invention or a portion thereof that blocks HCV entry into susceptible cells, in an amount and under conditions such that the monoclonal antibody or portion thereof inhibits HCV virion or particle entry into the susceptible cells. In an embodiment, the HCV entry inhibiting monoclonal antibody or portion thereof is humanized or chimeric. In an embodiment, the HCV entry inhibiting monoclonal antibody or portion thereof is PA-30. In an embodiment, the HCV entry inhibiting monoclonal antibody or portion thereof is humanized or chimeric PA-30.

[0043] The present invention further provides a method of preventing, alleviating, or reducing HCV infection of cells susceptible to HCV infection which comprises contacting susceptible cells that may be exposed to HCV virions or particles with a monoclonal antibody of the invention or a portion thereof that blocks or inhibits HCV entry into susceptible cells, in an amount and under conditions such that the monoclonal antibody or portion thereof inhibits HCV virion or particle entry into the susceptible cells so as to prevent, alleviate, or reduce HCV infection of the cells. In an embodiment, the HCV entry inhibiting monoclonal antibody or portion thereof is humanized or chimeric. In an embodiment, the HCV entry inhibiting monoclonal antibody or portion thereof is PA-30. In an embodiment, the HCV entry inhibiting monoclonal antibody or portion thereof is humanized or chimeric PA-30.

[0044] The present invention provides a method for the treatment of HCV infection, comprising administering to an individual in need thereof a therapeutically effective amount of a monoclonal antibody according to the invention that blocks HCV entry into susceptible cells so as to thereby treat HCV infection. In an embodiment, the HCV infection blocking monoclonal antibody or portion thereof is humanized or chimeric. In an embodiment, the HCV infection blocking monoclonal antibody or portion thereof is PA-30. In an embodiment, the HCV infection blocking monoclonal antibody or portion thereof is humanized or chimeric PA-30.

[0045] The present invention further provides a method for reducing the occurrence of HCV infection in a population of individuals, in which the method comprises administering to the population of individuals in need thereof a therapeutically effective amount a monoclonal antibody of the invention that blocks HCV entry into susceptible cells so as to reduce the occurrence of HCV infection in the population. In an embodiment, the monoclonal antibody is humanized or chimeric. In an embodiment, the monoclonal antibody is PA-30. In an embodiment, the monoclonal antibody is humanized or chimeric PA-30.

[0046] The present invention provides a composition which includes a monoclonal antibody of the invention or a portion thereof that blocks HCV entry into susceptible cells. The composition may further include a carrier, excipient, vehicle, or diluent. The carrier, excipient, or diluent may be a physiologically acceptable or compatible carrier, excipient, vehicle, or diluent. In accordance with this aspect, the antibody or portion thereof may be labeled with a delectable marker, which may be one or more of a radioactive marker, a chemiluminescent marker, a luminescent marker, a calorimetric marker, or a fluorescent marker. In an embodiment, the monoclonal antibody is humanized or chimeric. In an embodiment, the monoclonal antibody is PA-30. In an embodiment, the monoclonal antibody is humanized or chimeric PA-30. In an embodiment, the composition includes at least one additive selected from the group consisting of antimicrobials, antioxidants, chelating agents and inert gases. In an embodiment, the composition includes one or more antiviral active agents, e.g., ribavirin, interferon-α, interferon-α-2β, or a combination thereof.

[0047] The present invention provides a pharmaceutical composition that includes a therapeutically effective amount of a monoclonal antibody of the invention or a portion thereof that blocks HCV entry into susceptible cells, and a pharmaceutically acceptable carrier, excipient, or diluent. In an embodiment, the monoclonal antibody is humanized or chimeric. In an embodiment, the monoclonal antibody is PA-30. In an embodiment, the monoclonal antibody is humanized or chimeric PA-30. hi accordance with this aspect, the antibody or portion thereof may be labeled with a detectable marker, which may be one or more of a radioactive marker, a chemiluminescent marker, a luminescent marker, a calorimetric marker, or a fluorescent marker. In an embodiment, the pharmaceutical composition includes at least one additive selected from the group consisting of antimicrobials, antioxidants, chelating agents and inert gases.

[0048J The present invention additionally provides a pharmaceutical composition which includes a therapeutically effective amount of a monoclonal antibody of the invention or a portion thereof that blocks HCV entry into susceptible cells, and a pharmaceutically acceptable carrier, in combination with one or more additional antiviral active ingredient selected from interferons, pegylated interferons, anti-HCV monoclonal antibodies, anti-HCV polyclonal antibodies, RNA polymerase inhibitors, protease inhibitors, JRES inhibitors, helicase inhibitors, anti sense compounds, anti-viral small molecules and ribozymes. hi an embodiment, the monoclonal antibody is humanized or chimeric. In an embodiment, the monoclonal antibody is PA-30. In an embodiment, the monoclonal antibody is humanized or chimeric PA-30. In an embodiment, the one or more other antiviral active agents include ribavirin, interferon-α, interferon-α-2β, or a combination thereof, hi an embodiment, the interferon is pegylated.

[0049J The present invention also provides a method of inhibiting HCV infection of a cell susceptible to HCV infection, in which the method comprises contacting the cell with a monoclonal antibody produced according to the present invention, in an amount and under conditions that inhibits HCV virion entry into the cell. Tn one aspect, the monoclonal antibody is humanized or chimeric. In one aspect, the monoclonal antibody is produced by a hybridoma cell line designated PA-30 (ATCC Accession No. PTA-7278), or a portion of antibody PA-30 that binds ligand on the surface of the cell susceptible to HCV infection. In another aspect, the monoclonal antibody is humanized or chimeric PA-30. [0050] The present invention also provides a method of inhibiting HCV infection of a cell susceptible to HCV infection, in which the method comprises contacting a cell susceptible to HCV infection with a monoclonal antibody produced by a hybridoma cell line designated PA-30 (ATCC Accession No. PTA-7278), or a portion of antibody PA-30 that binds ligand on the surface of the cell susceptible to HCV infection, in an amount and under conditions that inhibit HCV infection of the cell, hi an embodiment, the PA-30 monoclonal antibody is humanized or chimeric.

[0051] The present invention further provides a method for the treatment of HCV infections, comprising administering to an individual in need thereof a therapeutically effective amount of the PA-30 monoclonal antibody (ATCC Accession No. PTA-7278), or a pharmaceutical composition comprising a therapeutically effective amount of the PA-30 monoclonal antibody or a humanized or chimeric form thereof.

[0052] The present invention further provides a method for reducing the occurrence of HCV infection in a population of individuals, in which the method comprises administering to the population of individuals in need thereof a therapeutically effective amount of the PA-30 monoclonal antibody (ATCC Accession No. PTA-7278), or a portion thereof that reduces the occurrence of HCV infection in the population. In an embodiment, the PA-30 monoclonal antibody is humanized or chimeric.

[0053] The present invention provides a composition which includes a therapeutically effective amount of the PA-30 monoclonal antibody (ATCC Accession No. PTA-7278) and a pharmaceutically acceptable carrier, or a portion of the PA-30 monoclonal antibody that binds the ligand or epitopic region of ligand as does PA-30. In accordance with this aspect, the antibody may be labeled with a detectable marker, which may be one or more of a radioactive marker, a chemiluminescent marker, a luminescent marker, a calorimetrie marker, or a fluorescent marker. In an embodiment, the PA-30 monoclonal antibody is humanized or chimeric.

[0054] The present invention embraces the use of a monoclonal antibody or a portion thereof that binds the same cell types, cell ligand or epitopes thereof as does the PA-30 monoclonal antibody. In an embodiment, the portion of the monoclonal antibody binds the same cell types, ligand or epitopic region of the ligand as does the PA-30 monoclonal antibody. In an embodiment, the monoclonal antibody is humanized or chimeric.

[0055] The present invention provides a composition which includes a therapeutically effective amount of the PA-30 monoclonal antibody (ATCC Accession No. PTA-7278), or a comparably active portion thereof, a pharmaceutically acceptable carrier and also includes at least one additive selected from the group consisting of antimicrobials, antioxidants, chelating agents and inert gases. In an embodiment, the PA-30 monoclonal antibody is humanized or chimeric.

[0056] The present invention additionally provides a pharmaceutical composition comprising a therapeutically effective amount of the PA-30 monoclonal antibody (ATCC Accession No. PTA-7278), or a comparably active portion thereof, in combination with at least one additional anti-viral active ingredient selected from interferons, anti-HCV monoclonal antibodies, anti- HCV polyclonal antibodies, RNA polymerase inhibitors, protease inhibitors, IRES inhibitors, helicase inhibitors, antisense compounds, anti-viral small molecules and ribozymes, In an embodiment, the PA-30 monoclonal antibody is humanized or chimeric.

[0057] The present invention provides a method of inhibiting HCV infection of a cell susceptible to HCV infection which comprises contacting the cell with an amount of one or more monoclonal antibodies or a portion thereof according to the invention, in an amount effective to inhibit HCV entry or infection of an HCV-infectable cell, so as to thereby inhibit HCV infection of the cell susceptible to HCV infection. In an embodiment, the one or more monoclonal antibodies or a portion thereof in the presence of infectable HCV bind ligand present on cells susceptible to HCV infection. Additionally, the one or more monoclonal antibodies or a portion thereof inhibit infection of the susceptible cells by HCV. In an embodiment of this method, the monoclonal antibody is humanized or chimeric. In an embodiment of this method, the monoclonal antibody is PA-30 or a portion thereof. In an embodiment of this method, the monoclonal antibody is humanized or chimeric PA-30.

[0058] The present invention provides methods in which an HCV susceptible cell is present in a subject and the contacting is effected by administering one or more monoclonal antibodies or a portion thereof of this invention to the subject. The monoclonal antibody or a portion thereof may be administered prior to, during, or post-infection of a subject by HCV. In an embodiment, the monoclonal antibody is humanized or chimeric, hi an embodiment, the monoclonal antibody is PA-30 or a portion thereof. In an embodiment, the monoclonal antibody is humanized or chimeric PA-30.

[0059] The present invention provides a method of treating or preventing HCV infection in a subject which comprises inhibiting HCV infection of the subject's cells susceptible to HCV infection by a method described herein, wherein the inhibition is effected by administering one or more monoclonal antibodies or a portion thereof of this invention to the subject. In an embodiment of this method, the monoclonal antibody is humanized or chimeric. In an embodiment of this method, the monoclonal antibody is PA-30 or a portion thereof. In an embodiment of this method, the monoclonal antibody is humanized or chimeric PA-30 or a portion thereof.

[0060] The present invention provides a method of treating or preventing a liver disease or pathology in a subject which comprises administering to the subject an effective amount of one or more monoclonal antibodies of this invention or a portion thereof capable of inhibiting HCV entry or infection of a subject's cells susceptible to HCV infection, so as to thereby treat or prevent the liver disease or pathology in the subject. In an embodiment of this method, the monoclonal antibody is humanized or chimeric. In an embodiment of this method, the monoclonal antibody is PA-30 or a portion thereof. In an embodiment of this method, the monoclonal antibody is humanized or chimeric PA-30 or a portion thereof.

[0061] The present invention provides a method of treating or preventing hepatocellular carcinoma in a subject which comprises administering to the subject an effective amount of one or more monoclonal antibodies of this invention or a portion thereof capable of inhibiting HCV from entering or infecting the subject's hepatoma cells, so as to thereby treat or prevent hepatocellular carcinoma in the subject. In an embodiment of this method, the monoclonal antibody is humanized or chimeric. In an embodiment of this method, the monoclonal antibody is PA-30 or a portion thereof. In an embodiment of this method, the monoclonal antibody is humanized or chimeric PA-30 or a portion thereof.

[0062] The present invention provides a method of (i) treating or preventing hepatocellular carcinoma; (ii) preventing or treating a liver disease, more particularly, an HCV-induced liver disease, or (iii) treating an HCV associated disorder in a subject, which comprises administering to the subject a monoclonal antibody or a portion thereof, wherein the monoclonal antibody or portion thereof (i) binds human B lymphocytes, human liver cells, and mitogen-stimulated CD4+ and CD8+ T lymphocytes; and (ii) inhibits infection of susceptible cells by HCV of genotype 1, 2, or a combination thereof, in an amount effective to inhibit infection of the subject's HCV susceptible cells, thereby treating the liver disease in the subject. In an embodiment, the monoclonal antibody is PA-30 (ATCC Accession No. PTA-7278). In an embodiment, the monoclonal antibody, e.g., PA-30, is humanized or chimeric. Tn an embodiment, the monoclonal antibody or portion thereof binds the same ligand or epitope thereof as is bound by the PA-30 monoclonal antibody.

[0063] The invention provides a method of treating a liver disease, more particularly, an HCV-induced liver disease, in a subject, which comprises administering to the subject a monoclonal antibody or a portion thereof that (i) binds a ligand of approximately 45-65 kDa, more particularly, approximately 60 kDa, expressed by B cells, mitogen-stimulated T cells and liver cells and (ii) inhibits infection of susceptible cells by HCV of genotype 1, 2, or a combination thereof, in an amount effective to inhibit infection of the subject's HCV susceptible cells and to thereby treat the liver disease in the subject. In an embodiment of this method, the monoclonal antibody is humanized or chimeric. In an embodiment of this method, the monoclonal antibody is PA-30 (ATCC Accession No. PTA-7278) or a portion thereof. In an embodiment of this method, the monoclonal antibody is humanized or chimeric PA-30 (ATCC Accession No. PTA-7278) or a portion thereof.

[0064] The invention further provides a method of treating an HCV associated disorder in a subject, which comprises administering to the subject a monoclonal antibody or a portion thereof that (i) binds a ligand of approximately 45-65 kDa, expressed by B cells, mitogen-stimulated T cells and liver cells and (ii) inhibits infection of susceptible cells by HCV of genotype 1, 2, or a combination thereof, in an amount effective to treat the HCV associated disorder in the subject. In an embodiment, the ligand has a molecular weight of approximately 60 kDa. In an embodiment, the monoclonal antibody is humanized or chimeric. In an embodiment, the monoclonal antibody is PA-30 (ATCC Accession No. PTA-7278). In an embodiment, the monoclonal antibody is humanized or chimeric PA-30 (ATCC Accession No. PTA-7278).

[0065] The invention further provides a method of preventing HCV infection in a subject, wherein prevention is effected by inhibiting HCV entry into a target cell to which HCV fuses, which method comprises administering to the subject a monoclonal antibody or portion thereof according to the invention in an amount effective to inhibit HCV entry into the target cell so as to thereby prevent HCV infection. In an embodiment, the monoclonal antibody or portion thereof is humanized or chimeric. In an embodiment, the monoclonal antibody or portion thereof is PA-30 (ATCC Accession No. PTA-7278). In an embodiment, the monoclonal antibody or portion thereof is humanized or chimeric PA-30 (ATCC Accession No. PTA-7278). In an embodiment, the target cell is a liver cell or hepatocyte.

[0066] The invention also provides a method of reducing, preventing, or reversing inhibition of activity of HCV-specific B and T lymphocytes resulting from HCV infection of a subject, the method comprising administering to the subject a monoclonal antibody or a portion thereof that (i) binds a ligand of approximately 45-65 kDa, expressed by B cells, mitogen-stimulated T cells and liver cells; and (ii) inhibits infection of susceptible cells infection by HCV of genotype 1, 2, or a combination thereof; in an amount effective to reduce, prevent, or reverse the inhibition of activity of HCV-specific B and T lymphocytes. In an embodiment, the monoclonal antibody or portion thereof is PA-30 (ATCC Accession No. PTA-7278). In an embodiment, the PA-30 monoclonal antibody is humanized. [0067] The present invention further provides a protein ligand present on the surface of human B cells, including but not limited to primary B cells and Ramos cells, to which the PA-30 monoclonal antibody or a portion thereof binds and which is immunoprecipitated from human B cells, e.g., Ramos cells, by the PA-30 monoclonal antibody. In an embodiment, the ligand is an approximately 45-65 kDa, or more particularly, approximately 60 kDa, protein bound by the PA- 30 monoclonal antibody. In an embodiment, the PA-30 ligand is soluble, isolated and purified.

[0068] The present invention further provides a method of treating a subject infected with HCV involving administering to the subject the purified, soluble, approximately 60 kDa PA-30 ligand, or soluble peptide(s) thereof, in an amount effective to bind HCV and thereby treat HCV infection in the subject. In an embodiment, the purified, soluble PA-30 ligand, or soluble peptide(s) thereof, is contained in a pharmaceutically acceptable composition.

[0069] The present invention further provides a method of preventing HCV infection of a subject who is at risk of and/or susceptible to, infection by HCV. Such a method involves administering to the subject the purified, soluble, approximately 60 kDa PA-30 ligand, or soluble peptide(s) thereof, in an amount effective to bind HCV and thereby prevent HCV infection of the subject or of the cells of the subject. In an embodiment, the purified, soluble PA-30 ligand, or soluble peptide(s) thereof, is contained in a pharmaceutically acceptable composition.

[0070] Further aspects and advantages afforded by the present invention will be apparent from the detailed description and exemplification herein below.

BRIEF DESCRIPTION OF THE FIGURES

[0071 ] The appended figures are presented to further describe the invention and to assist in its understanding through clarification of its various aspects.

[0072] FIG. 1: Binding of PA-30 monoclonal antibody to whole cells as measured by flow cytometry assay. FIG. 1 presents a table showing the results of assays performed to assess the binding of PA-30 monoclonal antibody from hybridoma supernatant to various cell types by flow cytometry analysis as described in Example IF. An isotype-matched (i.e., IgG3. K) monoclonal antibody was used as a control, along with JS81, an anti-human CD81 monoclonal antibody (BD Biosciences, San Diego, CA). The data presented in the FIG. 1 table represent the mean fluorescent intensity (MFl) following binding of purified PA-30 or cell culture supernatant containing PA-30, incubation with secondary PE-labeled antibody and analysis via flow cytometry. The data presented in the table represent at least three experiments performed with similar results. As shown in FIG. 1, PA-30 binds at high levels to Ramos cells (B cell lymphoma, ATCC). PA-30 also specifically binds Daudi cells, primary human hepatocytes, Hep3b cells and Huh7 cells, which are infectable by HCVpp, and HepG2 cells, which are resistant to HCV infection due to the lack of CD81 expression. PA-30 recognizes a cell surface molecule that is highly expressed on Ramos cells (B cell lymphoma, ATCC), an which is also expressed on Daudi cells, Hep3b cells, Huh7 cells, HepG2 cells and primary human hepatocytes, as determined by PA-30 binding.

[0073] FIGS. 2A-2G show that the PA-30 monoclonal antibody binds primary human B cells and mitogen stimulated T cell populations, as further described in Example 2. Human peripheral blood mononuclear cells (hPBMC) obtained from an HIV^" HCV^~ normal human donor were isolated from a Ficoll gradient. For FIGS, 2A-2E, Ramos (B) cells were used as a control. 1x10⁶ hPBMC or 2x10⁵ Ramos cells were incubated with 10% heat-inactivated goat serum in staining buffer (0.25% BSA, 0.1% sodium azide in PBS) for 20min at 4°C. Varying amounts of PA-30 monoclonal antibody or an isotype-matched (i.e., IgG3, K) control monoclonal antibody were added to the cells for 30 minutes at 4⁰C in a volume of about lOOμl. After the cells were washed once with 2ml of staining buffer, secondary antibody, goat anti-mouse IgG (specific for H and L Ig chains) conjugated to allophycocyanin (APC) (i.e., APC-goat anti-mouse IgG) was added at lμl/sample. Cells and secondary antibody were incubated for 15 minutes at 4°C, followed by washing in staining buffer. Thereafter, anti-human CD4 conjugated to phycoerythrin (PE), i.e., anti-human CD4-PE, anti-human CD19 conjugated to AlexaFluor488, i.e., AlexaFluor488 -anti-human CD19, and anti-human CD20 conjugated to fluorescein isothiocyanate (FITC), i.e., anti-human CD20-FITC, or a combination of antibodies, was added to appropriate tubes. Following another incubation, the cells were washed and analyzed on a FACSCalibur instrument.

[0074] As seen in FIGS. 2A-2E, PA-30 bound to Ramos cells and primary B cells in a dose- dependent manner. Human PBMC having a CD4XD19⁺CD20⁺ phenotype, or identified as CD20+, correspond to primary B cells. FIG. 2 A shows the results of the FACS analysis of hPBMC stained with CD20-FITC and PA-30 and goat anti-mouse IgG-APC. As seen in FIG. 2A, human PBMC were stained with PA-30 (revealed by FITC-conjugated secondary antibody) and with anti-CD20 conjugated to APC. All of the CD20+ cells were also bound by PA-30 (note upper right quadrant). FIG. 2B shows the results of the FACS analysis of hPBMC stained with CD19-FITC and PA-30 and goat anti -mouse IgG-APC. FIG. 2C shows the results of the FACS analysis of hPBMC stained with CD4-PE and PA-30 and goat anti-mouse IgG-APC. FIG. 2D shows the results of a titration of the PA-30 monoclonal antibody on Ramos cells, using PA-30 in the amounts indicated in the Key. FIG. 2E shows the results of a titration of the PA-30 monoclonal antibody on hPBMCs, using PA-30 in the amounts indicated in the Key. The gray shading in FIGS. 2D and 2E indicates staining with secondary antibody only as a control.

[0075] For experiments involving T cell populations, 2 x 10⁷ PBMC/5 ml were stimulated overnight in phytohemagglutinin (PHA) as described in Example 2. FITC-conjugated anti-CD4 and anti-CD8 antibodies were used in conjunction with the PA-30 monoclonal antibody to assess binding of PA-30 to the CD4 and CD8 T cell populations. As seen in FIG. 2F, PHA-stimulated CD4+ cells exhibited an increased expression of ligand bound by PA-30 following PHA stimulation. FIG. 2G shows that expression of ligand bound by PA-30 is also upregulated on CD8+ T cells following PHA stimulation, albeit to a somewhat lesser extent for the CD4+ T cells. It was also determined that resting T cells do not bind PA-30.

[0076] FIGS. 3A-3E show the results of PA-30 monoclonal antibody binding to different cell types as analyzed by flow cytometry. (See also, FIG. 1 and Example 3). For the binding studies, 2xlO⁵ cells of each cell type (See Example IA) were incubated with 10% heat- inactivated goat serum in staining buffer (0.25% BSA, 0.1% sodium azide in PBS) for 20 minutes at 4⁰C. Thereafter, 0.2μg or 2μg of PA-30 monoclonal antibody was added to the cells for 30 minutes at 4°C in a volume of about lOOμl. The cells were washed once with 2 ml of staining buffer and secondary antibody (goat F(ab')₂ anti-mouse IgG-PE) was added at a concentration of 1 μl/sample. The cells were incubated with secondary antibody for 15 minutes at 4°C. After washing in staining buffer, the cells were analyzed on a FACSCalibur instrument. FIG. 3 A shows the results of the FACS analysis of Ramos cells stained with PA-30 in the amounts indicated in the Key. FIG. 3B shows the results of the FACS analysis of Namalwa cells stained with PA-30 in the amounts indicated in the Key. FIG. 3C shows the results of the FACS analysis of CEM NKR CCR5 cells stained with PA-30 in the amounts indicated in the Key. FIG. 3D shows the results of the FACS analysis of HeLa cells stained with PA-30 in the amounts indicated in the Key. For HeLa cells, virtually no staining is seen. FIG. 3E shows the results of the FACS analysis of Ll.2 cells stained with PA-30 in the amounts indicated in the Key. The gray shading in FIGS. 3A-3E indicates staining with secondary antibody only as a control.

[0077] FIG. 4: Schematic of vectors for generating HCV pseudoparticles (HC Vpp) for use in screening monoclonal antibodies in an HCV entry assay. The HCV E1E2 expression construct is in pcDNA3.1 (Invitrogen) and encodes amino acids 132-191 (aal 32-191) of the capsid C-terminus (AQ as well as full-length El (aal 92-383), E2 (aa384-746) and p7 (aa747- 809) when indicated. Tn the ΔC-E1*E2* construct, the "*" indicates that the putative splice acceptor sites were removed by conservative mutagenesis. The HIV-I based NL/«e+Δ299 vector encodes a packageable genome that expresses all structural and non-structural proteins, except for the envelope glycoproteins due to a 299 base pair deletion in env. Furthermore, this vector encodes luciferase instead of HlV-I nef. LTRMong terminal repeat; CMV=Cytomegalovirus promoter; SV40=Simian virus 40 early promoter; pA=poly A sequence; Ψ=packaging signal; /Mc=luciferase gene; black box indicates the 299 nucleotide deletion in HIV env gene.

[0078] FIG. 5: Monoclonal antibody PA-30 blocks entry of HCV pseudoparticles (HCVpp) of different genotypes into cells. Equal volumes of HCVpp and Hep3b cells were placed in 96 well culture plates. HCVpp were produced as described herein by cloning viral envelopes directly from patients' sera, thereby generating a test panel of viral genotypes for use in the described assays. In this case, a panel of HCVpp was derived from patient sera infected with HCV of genotype Ia (strain H77), genotype Ib (strain F7), genotype 2b, or genotype combinations (e.g., la/2b) by cloning into vectors (e.g., as shown in FIG. 4) to generate the HCV pseudoparticles as described herein. Purified PA-30 was added to the 96-well culture plates containing the Hep3b cells and HCVpp. The plates containing cells, HCVpp and PA-30 monoclonal antibody were incubated for 3 days after which time the HIV inhibition (luciferase) assay was performed, e.g., as described in Example ID. Virus inhibition curves and IC50 values were calculated using non-linear regression in GraphPad PRISM (GraphPad Software, Inc., San Diego, CA). IC50 values were determined for each patient's HCV isolate as shown in FIG. 5. For the samples listed in FIG. 5 under the heading "Patient Isolates, HCV Genotype", the initials of the patient follow the HCV genotype cloned from that patient. In some cases, e.g., IaMA, two clones (A02 and A06) were isolated from the patient's serum. "Delta C" represents a genotype Ia (HCV strain H77) HCVpp control used in the assay. The IC50 values represent the mean of three experiments performed using each isolate.

[0079] FIG. 6: Immunoprecipitation and Western blot of target ligand bound by PA-30 from surface-biotinylated Ramos cells. Ramos cells were harvested and washed twice with PBS-. Cells were resuspended at 2x10⁷ cells/ml in PBS- with Sulfo-NHS-LC-Biotin (Pierce), or in PBS- without Sulfo-NHS-LC-Biotin, and were incubated for 60 minutes at 4°C with constant agitation. After the cells were washed once with cold DMEM and twice with cold PBS-, 150 μl of lysis buffer (5OmM Tris-HCl, 15OmM NaCl, 0,5% NP-40, Complete Protease inhibitor cocktail (Roche Applied Science)) was added to the cell pellet and the cells and buffer w^rere mixed vigorously for 20 minutes at 4⁰C. The cell lysate was centrifuged for 10 minutes at 10,000 rpm at 4°C and the supernatant was saved for analysis. To reduce background, the lysate was pre-cleared with magnetic Protein G beads (Miltenyi Biotec) for 2 hours with constant agitation. The pre-cleared lysate was added to Protein G beads coated with 5μg of PA-30, or with an i so type-matched control immunoglobulin — mouse IgG3κ. The lysate and coated beads were incubated overnight at 4°C with constant agitation.

[0080] Antibody: ligand complex was eluted from the beads using magnetic separation, according to manufacturer's instructions. The eluted complex was separated on a 4-12% Bis- Tris gradient gel (Invitrogen) with MES buffer (Invitrogen). The gel contents were blotted onto nitrocellulose (Invitrogen). To reduce non-specific binding, the blot was blocked overnight in 5% milk, 0.1% Tween 20, PBS-. To visualize PA-30 binding to biotinylated cell surface protein, the nitrocellulose blot was incubated with horseradish peroxidase (HRP)-conjugated-Streptavidin (Amersham Biosciences). After washing, specific bands were visualized on the blot by developing with SuperSignal West Pico Chemiluminescent Substrate (Pierce) according to the manufacturer's instructions and exposure to film (Kodak, Rochester, NY).

[0081] FIG. 6 shows the blot incubated with Streptavidin and developed as described. Molecular weight standards (in kilodaltons, kDa) are marked at the left side of the blot. The samples with Sulfo-NHS-LC-Biotin (+ lane) and samples without Sulfo-NHS-LC-Biotin (- lane) were run in non-reduced form. A prominent band of approximately 60 kDa (approximately 45- 65 kDa) is identified in the lane which represents the cell protein or ligand that is bound and immunoprecipitated by the PA-30 monoclonal antibody. The lane containing an isotype- matched control antibody does not show any bands. As a positive control for the development process, a biotinylated monoclonal antibody (PA-25) was run as a control in parallel on the gel with the other samples. The PA-25 control lane shows a band of approximately 98 kDa representing non-reduced IgG heavy chain.

[0082] FIG. 7: PA-30 inhibits infection by HCVpp of genotype Ia. Infection of cells by HCVpp of genotype Ia was blocked by the PA-30 monoclonal antibody in a dose-dependent manner. As described in Example 4, purified PA-30 monoclonal antibody was serially diluted and added to Hep3b cells immediately prior to the addition of HCVpp derived from genotype Ia (strain H77) virus as indicated. Plates were incubated for 72 hours prior to measurement of luciferase activity. IC50 values were calculated by fitting the data to a 4-parameter logistic equation in GraphPad Prism (GraphPad Software, Inc., San Diego, CA). Against the H77 genotype, PA-30 demonstrated IC50 value of 6.4μg/ml and an IC90 value of 28.2μg/ml.

[0083] FIG. 8: Time course of inhibition of HCVpp infection by PA-30 MAb. Time course experiments were performed to evaluate the inhibition of HCV infection by the PA-30 monoclonal antibody at distinct time points. For these experiments, cold HCVpp were added to Hcp3b cells in wells of a 96 well plate. After cells and virus were centrifuged for one hour at 1000 rpm at 4°C, the plates and wells of cells were washed with cold PBS- to remove unbound virus. Purified MAbs (JS-81, PA-30, and isotype control) were prepared at a concentration of lOμg/ml in cell culture medium warmed to 37°C and were added to the wells containing Hep3b cells and HCVpp at specified time points. After the addition of the MAbs, plate was incubated at 37⁰C for 72h prior to measurement of luciferase activity.

[0084] FIG. 9: Deglycosylation of ligand bound by the PA-30 monoclonal antibody (the PA-30 ligand). Ramos cell lysate and antibody-coated Protein G beads were incubated together overnight. 150U of PNGaseF (NE Biolabs, Ipswich, MA) and 5mU of O-Glycosidase (Sigma) were added to the antigen: antibody complex for 1.5 hours at 37°C. The beads were washed and eluted with sample buffer according to conventional procedures. Identification of contents in Lanes: Lane 1 : biotin-labeled Molecular Weight markers; Lane 2: PA-30 monoclonal antibody (10 μg) and 1.3 x 10⁷ Ramos cells; Lane 3: PA-30 monoclonal antibody (40 μg) and 5 x 10⁷ Ramos cells; Lane 4: Same as Lane 2, incubated with PNGaseF; Lane 5: Same as Lane 3, incubated with PNGaseF; Lane 6: Same as Lane 2, incubated with O-glycosidase; Lane 7: Same as Lane 3, incubated with O-glycosidase; Lane 8: PA-30 monoclonal antibody with no cell lysate; Lane 9: PA-30 monoclonal antibody with no cell lysate; Lane 10: Isotype-matched control IgG3 monoclonal antibody, 25 ng. Bands in Lanes 3, 5, and 7 of the gel shown in FIG. 9 show similar intensities in the region of approximately 60 kDa, suggesting the presence of N- and O- linked sugars on the PA-30 ligand.

[0084] FIGS. 1 OA-I OE show the results of PA-30 monoclonal antibody binding to primary human hepatocytes as analyzed by flow cytometry and as described in Example 8 herein. (See also, FIGS. 1 OA-I OE).

DESCRIPTION OF THE DISCLOSURE AND EMBODIMENTS

[0085] The present invention relates to the production, identification and isolation of antibodies that inhibit HCV infection of cells. In particular, the invention relates to monoclonal antibodies that function to block or inhibit the entry of HCV into HCV susceptible cells. Such antibodies may be used both in active and passive immunotherapies to combat HCV infection, with the goal of inhibiting a broad spectrum of HCV genotypes, particularly in a particular geographical area. Antibodies such as those of the present invention that inhibit the mechanism of HCV entry into cells are particularly attractive as antiviral therapeutics and for use in antiviral treatments and therapies. An inhibitor of HCV entry does not need to cross the plasma membrane or be modified intracellularly. In addition, because viral entry is mediated by conserved structures on the viral and cellular membranes, monoclonal antibody inhibitors of viral entry can be very potent and less susceptible to the emergence of viral resistance.

[0086] The breadth of activity against multiple HCV genotypes and the ability to interfere with or otherwise affect the binding of HCV virions to susceptible cells are key attributes for a therapeutically useful virus neutralizing or inhibiting antibody. In addition, because of the potential for HCV to infect lymphoid cells, preferentially B lymphocytes, and monocytes/macrophages, there exists a serious risk of producing extra-hepatic reservoirs that can allow for the recurrence of virus infection or for the development of virus-associated pathologies and clinical manifestations related to the involvement of extra-hepatic cells and tissues in HCV infection. Thus, the generation and production of therapeutic and prophylactic antibodies, particularly, monoclonal antibodies, that are able to reduce or prevent the ability of HCV to infect susceptible cells and extra-hepatic cells and tissues are important advances for reducing and eradicating HCV and infection by HCV. In addition, the identification and elucidation of one or more cell surface structures, e.g., protein or ligands, on B cells, and/or other HCV susceptible cells, that are bound by HCV inhibitory agents, such as antibodies, or small molecule peptidyl (e.g., protein or peptide) or non-peptidyl (e.g., chemical) entities, are crucial to the development of useful and important drugs and clinical reagents to treat diseases and pathologies associated with HCV infection of hepatic and non-hepatic cells and tissues.

[0087] The present invention is directed to monoclonal antibodies (MAb) that inhibit Hepatitis C virus (HCV) entry and infection of susceptible cells, thus inhibiting or blocking the infection of the susceptible cells by HCV. In one embodiment, a monoclonal antibody of the invention binds a ligand present on human peripheral blood mononuclear cells (hPBMCs), including human B cells (B lymphocytes) and stimulated or activated T cells (T lymphocytes), and on liver or hepatic cells, including the hepatoma cell lines Hep3b, HepG2 and Huh-7. In an embodiment, the monoclonal antibody binds a ligand on primary human B cells having the surface phenotype CD4-CD19+CD20+. In an embodiment, the monoclonal antibody binds a ligand that is present on Ramos cells and on Daudi cells. In an embodiment, the monoclonal antibody binds primary human T cells and monocytes. The binding to primary human T cells increases following stimulation of the T cells with mitogen, e.g., phytohemagglutinin (PHA). f0088] According to one aspect of the invention, the ligand is a protein, not heavily glycosylated, expressed by the cell to which the monoclonal antibody binds. In an embodiment, the protein or ligand bound by the monoclonal antibody is a cell surface membrane receptor or co-receptor associated with HCV entry into the susceptible cell. In an embodiment, the protein or ligand bound by the monoclonal antibody is a receptor or co-receptor on a B cell (B lymphocyte). In an embodiment, the protein or ligand bound by the monoclonal antibody is upregulated on stimulated or activated T cells. In an embodiment, the protein or ligand bound by the monoclonal antibody is associated with B cell lymphoproliferative disorders, conditions, or pathologies and/or autoimmune disorders, conditions, or pathologies. In an embodiment, stimulation of the molecule bound by the monoclonal antibody of the invention leads to biochemical signal transduction, which results in one or more downstream, cellular, functional activities in B and T cells.

[0089] In an embodiment, a monoclonal antibody of this invention, or portion thereof, having the characteristics as described in the above embodiments and used in the methods described herein, is designated the PA-30 monoclonal antibody ("PA-30"). PA-30 is produced by a hybridoma cell line, also designated PA-30, which was deposited at the American Type Culture Collection (ATCC), 10801 University Boulevard, P.O. Box 1549, Manassas, VA 20110-2209 USA, on August 22, 2006 and assigned ATCC Deposit Designation or Accession No. PTA- 7828. The deposit was made pursuant to the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure (Budapest Treaty). Accordingly, the present invention provides monoclonal antibody PA-30 produced by a hybridoma cell line designated PA-30 (ATCC Accession No. PTA-7828), or a portion of the PA-30 monoclonal antibody. Both the monoclonal antibody according to the present invention and the hybridoma cell line that produces the monoclonal antibody are referred to as PA-30 herein.

[0090J The PA-30 monoclonal antibody as described and exemplified herein binds a ligand present on or expressed by liver cells. In addition, the PA-30 monoclonal antibody binds a ligand present on or expressed by human B cells. The ligand, a protein molecule, has an apparent molecular weight of approximately 60 kDa. As demonstrated in infection inhibition assays using HCV pseudoparticles as described herein, the PA-30 monoclonal antibody also blocks entry of HCV particles into susceptible cells, e.g., liver cells such as Hep3b cells to which PA-30 binds.

[0091] The ligand recognized by the PA-30 monoclonal antibody is present on the surface of the above-described cell types, for example, as determined by surface or membrane labeling of cells. It will be appreciated that other and synonymous terms may be used to describe that the ligand (cognate ligand) recognized and bound by the PA-30 monoclonal antibody is present on particular cell types. For example, the ligand present in the cell membrane and bound by PA-30 may be described as being "expressed by^", "expressed in", or "expressed on the surface of particular cell types, e.g., human B cells, stimulated T cells and liver cells. [0092] In accordance with an embodiment of the invention, the ligand recognized and bound by PA-30 is a receptor or co-receptor that is associated with HCV entry into a susceptible cell. In an embodiment, the ligand is a protein molecule. In a related embodiment, the protein or ligand is a cell receptor or co-receptor for HCV entry into a cell. Based on immunoprecipitation experiments and flow cytometry expression profile results, the PA-30 monoclonal antibody does not bind to CD81. Nor does the PA-30 monoclonal antibody bind SR-B 1 , which is a glycosylated protein having a molecular weight of 82 kDa and which is not expressed by B cells. In an embodiment, the receptor or co-receptor may be bound alone, or it may be complexed or associated with one or more proteins and/or glycoproteins expressed in a HCV susceptible cell. The one or more proteins or glycoproteins with which the PA-30 bindable 45-65 kDa ligand may associate or be complexed may reside within the cell membranes of cells. Such proteins may span the cell membrane and may have both intracellular (i.e., C-terminal) and extracellular (i.e., N-terminal) portions. In an embodiment, the one or more proteins associated or complexed with the PA-30 bindable ligand may comprise a molecular weight that is higher than that of the approximately 45-65 kDa molecular weight of the ligand, for example, the associated or complexed proteins may collectively have a molecular weight of approximately 160 kDa. In another embodiment, the ligand or co-ligand that is bound by PA-30 may be induced or stimulated to become complexed or associated with another protein or glycoprotein expressed in a HCV susceptible cell following binding, for example, by virus or by a monoclonal antibody of the invention, e.g., PA-30. In another embodiment, the receptor or co-receptor may be induced to undergo a conformational change either before or after binding, for example, by HCV or by a monoclonal antibody of the invention, e.g., PA-30.

[0093] In an embodiment, the ligand bound by PA-30 is present on the surface of human B cells and liver cells and has a molecular weight of approximately 45-65 kDa, or of approximately 45-62 kDa, or of approximately 45-60 kDa, or of approximately 57-60 kDa, or of approximately 60 kDa, for example, as determined by methods known in the art, including without limitation, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), silver staining, 2-D gel analysis and the like. In an embodiment, the PA-30 monoclonal antibody binds and immimoprecipitates the ligand from human B cells, e.g., Ramos cells, as detected by Western blot. (See FIG. 6). The ligand recognized and bound by the PA-30 monoclonal antibody is distinct from known HCV ligands such as CD81, L-SIGN and SR-Bl.

[0094] The ligand recognized and bound by the PA-30 monoclonal antibody is highly expressed on B cells, including B-cell lymphomas, hi an embodiment of the invention in which B cells play a role in the pathogenesis of HCV infection, a monoclonal antibody of the invention, e.g., PA-30, is involved in the treatment of B cell pathogenesis. In an embodiment, the binding of the PA-30 monoclonal antibody to ligand on B cells in HCV-infected individuals may reduce, inhibit, or prevent the activation and/or proliferation of B cell clones, thereby reducing, inhibiting, or preventing the production of autoantibodies and cryoglobulins by such B cells. In an embodiment, a monoclonal antibody of the invention, e.g., PA-30, binds to ligand on innate B cells in HCV-infected liver to prevent the expansion of the innate B cell population in the liver and peripheral blood of HCV-infected patients and particularly in chronic HCV-infected patients.

[0095] In another embodiment of the invention, the PA-30 monoclonal antibody may act as an antagonist of immune system alterations in HCV-infected individuals by binding to ligand on B cells. In an embodiment, the binding of the PA-30 monoclonal antibody to ligand on B cells of HCV-infected patients may treat, or serve as a therapeutic for, a variety of autoimmune diseases, including B cell-related autoimmune diseases and disorders, and B cell pathologies and cancers, which diseases, disorders, pathologies and cancers may relate to a subject's infection by or exposure to HCV. hi an embodiment, the binding of the PA-30 monoclonal antibody to ligand on B cells may prevent the development of a variety of autoimmune diseases and B cell pathologies and cancers in HCV-infected individuals. The PA-30 monoclonal antibody may treat, ameliorate, reduce or eliminate various B-cell associated or immune system disorders and/or autoimmune diseases by binding the approximately 60 kDa target ligand expressed by B cells in HCV-infected individuals. Autoimmune disorders or conditions, or immune system disorders and pathologies, associated with HCV infection that may be treated, ameliorated, reduced, or eliminated by treatment with PA-30 include, but are not limited to, rheumatic conditions, rheumatoid arthritis, sicca syndrome, chronic polyarthritis, polydermatomyositis, fibromyalgia, autoimmune thyroiditis, lung fibrosis, MC, vasculitis and diabetes mellitus.

[0096] In another embodiment of the invention, the PA-30 monoclonal antibody may act as an agonist of immune system alterations in HCV-infected individuals by binding to ligand on B lymphocytes. In another embodiment of the invention, the PA-30 monoclonal antibody may act as an agonist of immune system alterations in HCV-infected individuals by binding to ligand on T lymphocytes. In an embodiment, the T lymphocytes are stimulated or upregulated to express ligand, for example, by exposure to mitogens, cytokines, or cell factors. In an embodiment, the T lymphocytes are CD4+ cells. In an embodiment, the T lymphocytes are CD8+ cells. In an embodiment, the binding of the PA-30 monoclonal antibody to the ligand expressed by B cells may stimulate the activity of B cells, for example, to produce a more active or effective immune response against HCV in a subject infected with or exposed to HCV. [0097J In another embodiment, and without wishing to be bound by theory, chronic or persistent HCV infection may trigger mechanisms of immune tolerance aimed at blocking or inhibiting the activity of virus-specific B and T cells. The stimulation of the target ligand bound by PA-30 monoclonal antibody on B and/or T lymphocytes may serve to reduce, prevent, or reverse such blocking or inhibition. As a result, the B and/or T lymphocytes may be activated or stimulated to provide an appropriate or a more potent immune response in an individual who is infected by or who has been exposed to HCV. Autoimmune disorders or conditions, or immune system alterations, disorders and pathologies associated with HCV infection include, but are not limited to, rheumatic conditions, rheumatoid arthritis, sicca syndrome, chronic polyarthritis, polydermatomyositis, fibromyalgia, autoimmune thyroiditis, lung fibrosis, MC, vasculitis and diabetes mellitus. The binding of target ligand on B and/or T lymphocytes by the monoclonal antibodies of the invention, including PA-30, may activate a response in an individual to quell or overcome the autoimmune disorder or condition or to eliminate HCV infected cells.

[0098] The present invention further relates to antibody-based therapies for the treatment of HCV infection. In an embodiment, the invention relates to a monoclonal antibody, e.g., PA-30, that specifically blocks HCV entry into susceptible cells, also called target cells herein, for example, human liver cells and human B cells. Without wishing to be bound by theory, the binding of the HCV inhibiting monoclonal antibody, or portion thereof, of this invention to the ligand present on human B cells and liver cells may allow the antibody to interfere with HCV infection of susceptible cells, for example, by blocking or inhibiting the ability of the virus to bind, enter, or otherwise infect cells that are susceptible to infection by HCV.

[0099] As described herein, embodiments of the present invention encompass a monoclonal antibody, e.g., the PA-30 monoclonal antibody, and a portion thereof. Preferably, the portion of the antibody has functional activity. By functional activity is meant binding activity, HCV infection-blocking or inhibiting activity, or both, or another activity characteristic of the antibody of the invention. Functional activity may also encompass downstream events that occur in an HCV-susceptible cell, which events are caused by binding of the antibody to ligand, to epitope(s) therein, or by the virus infection inhibiting activity of the antibody. It is to be understood that the methods involving a monoclonal antibody according to this invention, or more specifically the PA-30 monoclonal antibody of the invention, encompass the use of the complete antibody molecule, or a portion or fragment of the antibody that exhibits and maintains a functional activity. Illustratively, a portion or fragment of the monoclonal antibody may comprise a light chain of the antibody, a heavy chain of the antibody, a Fab portion of the antibody, an F(ab')₂ portion of the antibody, an Fd portion of the antibody, an Fv portion of the antibody, a variable domain of the antibody, or one or more CDR domains of the antibody. It is to be understood that a bindable portion of a monoclonal antibody of this invention is a functional portion of the antibody as encompassed in the methods and utilities described for the monoclonal antibodies of the present invention, even if not expressly stated or unless otherwise indicated. A bindable portion of a monoclonal antibody of the invention, e.g., PA-30, binds one or more epitopes recognized by the antibody on a ligand that is expressed by a human B cell, a liver cell, a stimulated T cell and/or other HCV susceptible cells. Ligand epitopes may be linear or conformational. Accordingly, both a complete monoclonal antibody and a portion thereof may bind ligand and/or inhibit or block entry of HCV into a cell that is susceptible to HCV infection. A functional portion of the PA-30 monoclonal antibody is also able to bind ligand and/or inhibit HCV infection of susceptible target cells.

[0100] A monoclonal antibody of the present invention blocks entry of one or more genotypes of HCV so as to inhibit HCV infection of susceptible cells. In an embodiment, inhibition of HCV infection by the monoclonal antibody is dose dependent. Without limitation, the monoclonal antibody inhibits infection by HCV of genotypes 1, 2 and 3-6, as well as others that may become known. HCV genotype subtypes include, without limitation, Ia, Ib, 2a, 2b and 2c, as well as combinations of subtypes, e.g., la/lb, la/2b, la/2a, lb/2a, lb/2b, etc. In an embodiment, the PA-30 monoclonal antibody inhibits the ability of HCV virions or particles of various genotypes, e.g., 1, 2, Ia, Ib and la/2b, to enter HCV susceptible cells, such as liver cells, as described, e.g., in Example 4 herein.

[0101 J The PA-30 monoclonal antibody according to the present invention was generated and screened as described in Example IB herein. PA-30 is an immunoglobulin of the IgG3 heavy (H) chain and kappa (K) light (L) chain isotypes. PA-30 was produced utilizing methods practiced by those having skill in the pertinent art and were selected for their ability to inhibit (i.e., block) different HCV pseudoparticle or pseudovirion (HCVpp) genotypes from infecting susceptible cells. Screening using HCVpp as described herein (See, e.g., Example 1C/1D and U.S. Patent Application No. 20050266400 to J. Dumonceaux et al., the contents of which are hereby incorporated by reference in their entirety) provides a rigorous assay for selecting those monoclonal antibodies that inhibit cell infection by HCVpp of diverse genotypes. In an embodiment, the ability of PA-30 to inhibit HCV infectivity of cells is specifically assessed using HCVpp of genotypes 1 and 2, namely, Ia, Ib and la/2b, as described in Example 4.

[0102] Other methods for testing the specificity of anti-HCV or HCV inhibitory monoclonal antibodies include, without limitation, flow cytometry analysis, Western blot analysis, ELISA and inhibition of binding assays involving ligand / receptor binding by the antibody. These assays can be utilized both for testing supernatants from hybridomas producing HCV infection inhibiting monoclonal antibodies and for testing the activity of purified HCV infection-inhibiting monoclonal antibodies. For specific binding of monoclonal antibody to cell surface ligand, flow cytometry analysis can be used, such as described herein in Example IF. Binding specificity testing includes binding assays using the monoclonal antibody against a panel of cells, e.g., human cells, including, without limitation, liver cell lines (Hep3b, Huh-7, or HepG2), embryonic kidney cells (293T), fibroblasts (HeLa), B cells (Daudi and Ramos, a population of primary B cells enriched from PBMCs), T cells (Sup-Tl, Molt-4, or Hut-78), monocytic cells (THP-I), astrocytic cells (U87), hepatoma cells (PLC/PRF/5) or other liver cell types, e.g., the liver adenocarcinoma SkHepl, human peripheral blood cells and various fractionated subtypes thereof including lymphocytes and monocytes. Various combinations of the foregoing cell types may be employed in binding analyses. Flow cytometry analysis can reveal binding specificities of an anti-HCV or HCV inhibitory monoclonal antibody for cell surface molecules on various cell types.

[0103] In an embodiment of the present invention, antibodies, either polyclonal or monoclonal, having a binding pattern and specificity highly similar to or the same as that of the PA-30 monoclonal antibody are encompassed by the invention. In an embodiment, the epitopes bound by the antibody, e.g., the PA-30 monoclonal antibody, comprise the target ligand expressed on human B cells and liver cells, e.g., primary B cells and Ramos cells, and Hep3b, HepG2 and Huh-7 cells, respectively. In an embodiment, the epitopic specificity is associated with a ligand that is upregulated on stimulated human T cells, e.g., T cells stimulated with mitogen, e.g., PHA. In one embodiment, the PA-30 monoclonal antibody binds and precipitates a ligand having an apparent molecular weight (MW) of approximately 60 kDa from biotin- labeled surface molecules present on Ramos B cells and from membrane preparations of detectably labeled Ramos cells, which have been determined to exhibit a high level of expression of the PA-30 ligand. (See, e.g., FIG. 1 and FIG. 6). Accordingly, monoclonal antibodies or portions thereof that have the same or virtually the same binding specificity, or binding specificity for ligand, or ligand epitopes, as does the PA-30 monoclonal antibody, are encompassed by this invention.

[0104] Inhibition of viral entry into cells and specificity of the inhibition represent screening and selection criteria for the monoclonal antibodies of the present invention. (See, e.g., Examples 2 and 4 herein). In accordance with the present invention, the PA-30 monoclonal antibody blocks HC Vpp entry into susceptible cells and exhibits a median IC50 value of less than 10 μg^/ml (< 10 μg/ml), or a median IC50 value of 5 μg/ml or less (5 μg/ml or less), or a median IC50 value of 2 μg/ml or less (2 μg/ml or less), or a median ΪC50 value of 1 μg/ml or less (1 μg/ml or less), or a median IC50 value of 0.5 μg/ml or less (0.5 μg/ml or less), or a median 1C50 value of 0.1 μg/ml or less (0.1 μg/ml or less), against HCV of different genotypes, e.g., genotypes 1 and 2, as indicative of HCV infection inhibiting potency. PA-30 shows no measurable activity against unrelated viruses, e.g., HIV-I, VSV and MLV pseudoviruses, wherein no measurable activity is typically represented by an IC50 of > 100 μg/ml. As is common in this field of art, IC50 values, which, in the present case, represent the concentration of antibody required for 50% inhibition of viral infectivity, provide meaningful and significant quantitative criteria, as IC50 values are in the dynamic range of the dose-response curve and may be considered to be a most reliable indicator of infection inhibiting activity. Additionally, at higher concentrations, e.g., 10 μg/ml or greater, HCV infection inhibiting monoclonal antibodies mediate essentially complete inhibition of HCV entry into cells, as evaluated by HCVpp as well as HCVcc (cell culture) inhibition assays.

[0105] In an embodiment, a monoclonal antibody of this invention, e.g., PA-30, inhibits infection by HCV of genotypes 1 and 2, including subtypes thereof, which are responsible for 80-100% of all HCV infections in the United States, western Europe and Japan. In another embodiment, a monoclonal antibody inhibits infection by HCV of diverse genotypes, including 1, 2 and 3-6, subtypes thereof, and/or combinations thereof. A monoclonal antibody with a broad spectrum profile of HCV infection inhibition may be widely used in many demographic areas without regard to the infecting strain of HCV. The invention further encompasses the use of a combination of HCV infection inhibiting monoclonal antibodies, e.g., two or more HCV infection inhibiting monoclonal antibodies in combination, to achieve a broad antivirus spectrum against a number of HCV genotypes. For example, a combination of two or more HCV infection inhibiting monoclonal antibodies may achieve an increased breadth of activity against genotypes 3-6, which are common in regions of the world outside of the United States, western Europe and Japan.

[0106] In an embodiment, a monoclonal antibody of this invention, e.g., PA-30, inhibits infection of susceptible cells by HCV, for example, as assayed using HCVpp (FIGS. 5 and 7), in a dose-dependent manner. In an embodiment, the monoclonal antibody inhibits infection of susceptible cells at a level of 50% or more. In an embodiment, the monoclonal antibody inhibits infection of susceptible cells at a level of 60% or more. In an embodiment, the monoclonal antibody inhibits infection of susceptible cells at a level of 70% or more. In an embodiment, the monoclonal antibody inhibits infection of susceptible cells at a level of 80% or more. In an embodiment, the monoclonal antibody inhibits infection of susceptible cells at a level of 90% or more. In an embodiment, the monoclonal antibody inhibits infection of susceptible cells at a level of 95% or more. In an embodiment, the monoclonal antibody inhibits infection of susceptible cells at a level of 99%. In an embodiment, the monoclonal antibody inhibits infection of susceptible cells at a level of 100%.

[0107] The present invention also encompasses a monoclonal antibody, e.g., PA-30, in a composition with other ingredients, excipients, diluents, or carriers and the like. In an embodiment, a monoclonal antibody, e.g., PA-30 is included in an effective amount in a pharmaceutical composition in combination with other pharmaceuticals, pharmaceutically acceptable carriers, excipients, or diluents, therapeutics, drugs, or immune-enhancing or stimulating agents, including small molecules, antivirals, therapeutic DNA or RNA molecules, oligonucleotides, proteins, peptides, polypeptides, nucleosides, nucleoside analogs, for use in compositions, e.g., pharmaceutically acceptable compositions, and in methods of treating, preventing, or treating and preventing HCV infection. The pharmaceutical compositions of the invention may comprise a therapeutically effective amount of one or more of the monoclonal antibodies of the invention, e.g., the PA-30 monoclonal antibody (ATCC Accession No. PTA- 7278), or a portion thereof, in combination with at least one additional antiviral active ingredient selected from interferons, anti-HCV monoclonal antibodies, anti-HCV polyclonal antibodies, RNA polymerase inhibitors, protease inhibitors, IRES inhibitors, helicase inhibitors, antisense compounds, anti-viral small molecule inhibitors, such as HCV polymerase and protease inhibitors, and ribozymes.

[0108] In accordance with the present invention, the PA-30 monoclonal antibody, produced by the PA-30 hybridoma cell line (ATCC Accession No. PTA-7278), is an isotype IgG3, K (kappa) immunoglobulin molecule. In an embodiment, the present invention encompasses a monoclonal or polyclonal antibody or a fragment or portion of such antibody that binds the same epitope(s) or ligand as monoclonal antibody PA-30 produced by the hybridoma cell line designated PA-30 (ATCC Accession No. PTA-7278). In a related embodiment, the invention encompasses a composition comprising a carrier and a monoclonal antibody or a fragment or portion of such antibody that binds the same epitope(s) or ligand as monoclonal antibody PA-30 produced by the hybridoma cell line designated PA-30 (ATCC Accession No. PTA-7278).

[0109] As appreciated by those skilled in the relevant art, monoclonal antibodies may be produced by mammalian cell culture (hybridomas), murine myeloma cell lines, or synthetically, in recombinant form, in recombinant cell lines such as Chinese hamster ovary (CHO) cells. The monoclonal antibody technology is well-known to those skilled in the art (e.g., Kohler and Milstcin, 1975). Bacterial, yeast, plant and insect cell lines can also be used to produce monoclonal antibodies or fragments thereof. In addition, methods exist to produce monoclonal antibodies in transgenic animals or plants (Pollock et al., 1999; Russell, 1999).

[0110] In an embodiment, a monoclonal antibody of the invention, e.g., PA-30, is a chimeric antibody in which the carboxy terminus is replaced with that of a human immunoglobulin molecule. In another embodiment, the antibody is humanized or chimeric. In another embodiment, the antibody is a human antibody. In a specific embodiment, the monoclonal antibody is PA-30 that is chimeric or humanized according to established procedures in the art. In another embodiment, the monoclonal antibodies of the invention, e.g., PA-30, are single chain antibodies, including chimeric or CDR-grafted single chain antibodies, which may be produced using techniques routinely practiced in the art. The chimeric, humanized, CDR-grafted, or single chain antibodies will have activity or function, e.g., binding and/or virus infection inhibitory activity or function, that is essentially the same as, equal to, or greater than that of the original murine monoclonal antibody, e.g., the murine monoclonal antibody PA-30.

[0111] In the humanized form of the antibody, some, most, or all of the amino acids outside the CDR regions (i.e., in the framework region) are replaced with amino acids from human immunoglobulin molecules, while some, most, or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible so long as they do not abrogate the ability of the antibody to bind a given antigen. Suitable human "replacement" immunoglobulin molecules include IgGl, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgA, IgM, IgD or IgE molecules. A humanized antibody retains similar, highly similar, or essentially the same antigenic specificity and/or function as the original antibody. In accordance with the present invention, specificity and/or function reflect the ability of the antibody to bind ligand on human B cells and liver cells and to block HCV entry into susceptible target cells so as to inhibit or prevent infection of target cells by the virus. Accordingly, in an embodiment of the present invention, a humanized form of the PA-30 immunoglobulin, or portion thereof, can compete with the murine PA-30 monoclonal antibody, or portion thereof, for binding and activity.

[0112] One skilled in the art would know how to make the humanized antibodies of the present invention. Various publications, several of which are hereby incorporated by reference into this application, also describe how to make humanized antibodies. For example, the methods described in U.S. Patent No. 4,816,567 enable the production of chimeric antibodies having a variable region of one antibody and a constant region of another antibody. U.S. Patent No. 5,225,539 describes another approach for the production of a humanized antibody. In this approach, recombinant DNA technology is used to produce a humanized antibody in which the CDRs of a variable region of one immunoglobulin are replaced with the CDRs from an immunoglobulin with a different specificity, such that the humanized antibody would recognize the desired target, but would not be recognized in a significant way by a human subject's immune system. Specifically, site directed mutagenesis is used to graft the CDRs of the heavy and light chain variable regions of the immunoglobulin molecule onto the framework region.

[0113] Other approaches for humanizing an antibody are described in U.S. Patent Nos. 5,585,089 and 5,693,761 and in WO 90/07861, which describe methods for producing humanized immunoglobulins. The described immunoglobulins have one or more CDRs and possible additional amino acids from a donor immunoglobulin and a framework region from an accepting human immunoglobulin. The patents describe a method to increase the affinity of an antibody for the desired antigen. Some amino acids in the framework are chosen to be the same as the amino acids at those positions in the donor rather than in the acceptor. Specifically, these patents describe the preparation of a humanized antibody that binds to a receptor by combining the CDRs of a mouse monoclonal antibody with human immunoglobulin framework and constant regions. Human framework regions can be chosen to maximize homology / identity with the mouse sequence. A computer model can be used to identify those amino acids in the framework region that are likely to interact with the CDRs or the specific antigen. Thereafter, mouse amino acids can be used at these positions to create the humanized antibody.

[0114] The variable regions of the humanized antibody may be linked to at least a portion of an immunoglobulin constant region of a human immunoglobulin. In one embodiment, the humanized antibody contains both light chain and heavy chain constant regions. The heavy chain constant region usually includes the CHl, hinge, CH2, CH3 and sometimes the CH4 regions.

[0115] Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci (See, e.g., U.S. Patent Nos. 5,591,669; 5,598,369; 5,545,806; 5,545,807; 6,150,584 and references cited therein, the contents of which are incorporated herein by reference). The transgenic animals have been genetically modified such that there is a functional deletion in the production of endogenous (e.g., murine) antibodies. These animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals results in the production of fully human antibodies. Following immunization of these mice (e.g., XenoMouse®, Abgcnix, Fremont, CA; HuMab-Mouse®, Medarex/GenPharm, Princeton, NJ.), monoclonal antibodies are prepared according to standard hybridoma technology. [0116] In vitro methods also exist for producing human antibodies. These include phage display technology (e.g., U.S. Patent Nos. 5,565,332 and 5,573,905, the contents of which are incorporated herein by reference) and in vitro stimulation of human B cells (e.g., U.S. Patent Nos. 5,229,275 and 5,567,610, the contents of which are incorporated herein by reference).

[0117] The cell lines of the present invention, e.g., the hybridoma cell line producing the PA- 30 monoclonal antibody, have uses other than for the production of the monoclonal antibody. For example, the cell lines of the present invention can be fused with other cells (such as suitably drug-marked human myeloma, mouse myeloma, human-mouse heteromyeloma or human lymphoblastoid cells) to produce additional hybridomas, and thus provide for the transfer of the genes encoding the monoclonal antibodies. In addition, the cell lines can be used as a source of nucleic acids encoding antigen-specific immunoglobulin chains, which can be isolated and expressed, such as upon transfer to other cells using any suitable technique (See, e.g., U.S. Patent No. 4,816,567 to Cabilly et al.; U.S. Patent No. 5,225,539 to Winter). For instance, clones comprising a rearranged immunoglobulin light or heavy chain can be isolated (e.g., by PCR) or cDNA libraries can be prepared from mRNA isolated from the cell lines, and cDNA clones encoding an antigen-specific immunoglobulin chain can be isolated. Thus, nucleic acids encoding the heavy and/or light chains of the antibodies or portions thereof can be obtained and used in accordance with recombinant DNA techniques for the production of the specific immunoglobulin, immunoglobulin chain, or variants thereof (e.g., humanized immunoglobulins) in a variety of host cells or in an in vitro translation system. For example, the nucleic acids, including cDNAs, or derivatives thereof encoding variants such as a humanized immunoglobulin or immunoglobulin chain, can be placed into suitable prokaryotic or eukaryotic vectors (e.g., expression vectors) and introduced into a suitable host cell by an appropriate method (e.g., transformation, transfection, electroporation, infection), such that the nucleic acid is operably linked to one or more expression control elements (e.g., in the vector or integrated into the host cell genome). For production, host cells can be maintained under conditions suitable for expression (e.g., in the presence of inducer, suitable media supplemented with appropriate salts, growth factors, antibiotic, nutritional supplements, etc.), whereby the encoded immunoglobulin polypeptide is produced. If desired, the encoded protein can be recovered and/or isolated (e.g., from the host cells, medium, milk). The method of production may also encompass expression in a host cell of a transgenic animal. (See e.g., WO 92/03918, published Mar. 19, 1992, GcnPharm International).

[0118] The present invention additionally encompasses one or more nucleic acid molecules encoding a monoclonal antibody of the invention, e.g., PA-30, that specifically binds a Iigand (i.e., cognate or target ligand) expressed by human B cells (e.g., primary B cells or Ramos cells) or human liver cells (e.g., Hep3b, HepG2, or Huh-7)). In an embodiment, the one or more nucleic acid molecules encode a monoclonal antibody (e.g., the heavy and light chains of the antibody) that specifically binds ligand, which is a protein receptor or co-receptor for HCV. In an embodiment, the encoded monoclonal antibody is chimeric. In an embodiment, the encoded monoclonal antibody is humanized. In another embodiment, the encoded monoclonal antibody is fully human.

[0119] The nucleic acid molecule as described above can be RNA, DNA or cDNA. The nucleic acid molecule may encode the light chain or the heavy chain of an antibody (immunoglobulin (Ig)) molecule. Alternatively, the nucleic acid encodes both the heavy and light chains of an immunoglobulin molecule. In an embodiment, one or more nucleic acid molecules encode the Fab portion. In an additional embodiment, one or more nucleic acid molecules encode CDR portions. In another embodiment, the nucleic acid molecule encodes the variable (V) domain of the Ig light (L) or heavy (H) chain, e.g., V_L or Vi₁, respectively. In a further embodiment, the nucleic acid molecule encodes the variable domain and one or more constant domains of the immunoglobulin L or H chain.

[0120] In accordance with the present invention, a recombinant PA-30 immunoglobulin (rPA- 30) is produced by cloning the genes encoding the H and L chains of the PA-30 immunoglobulin from the PA-30 hybridoma cell line, inserting the genes into a suitable expression vector and introducing the vector into suitable cell lines which express recombinant PA-30 antibody. In one embodiment the rPA-30-expressing cell line is a stable CHO cell line (ATCC Accession No. CCL-61). Recombinant PA-30 antibody is comprised of a kappa (K) light chain and a heavy chain of the IgG3 isotype.

[0121] In one embodiment, recombinant PA-30 antibody is isolated and purified from the CHO cell line using procedures known in the art to obtain a purified rPA-30 for a variety of utilities described herein. The amino acid sequence of the variable region of the H chain (Vn) of the rPA-30 antibody and its encoding nucleic acid are obtained. In an embodiment, the signal sequence, complementarity determining regions (CDRl, CDR2 and CDR3), framework regions (FRl, FR2. FR3 and FR4) of the H chain V region, and the IgG constant region of the rPA-30 monoclonal antibody are obtained. The amino acid sequence of the light (L) chain of the rPA-30 antibody and its encoding nucleic acid sequence are obtained. In one embodiment, the signal sequence, complementarity determining regions (CDRl , CDR2 and CDR3), framework regions (FRl, FR2. FR3 and FR4) of the L chain variable (V) region, and the L chain constant (C) region of rPA-30 monoclonal antibodv are obtained. [0122] In an embodiment of the present invention, the rPA-30 antibody is humanized to generate a humanized immunoglobulin using techniques described hereinabove and as known in the art. Accordingly, the humanized immunoglobulin has binding specificity for a ligand (i.e., a protein of approximately 60 kDa) present on or expressed by a human B cell or liver cell. In an embodiment, the humanized immunoglobulin comprises a ligand-binding region of non-human, i.e., murine, origin and at least a portion that is of human origin, i.e., a human framework region, a human constant region or portion thereof, or a combination thereof. Additionally, an antibody of the present invention comprising human Ig sequences may refer to a chimeric immunoglobulin in which the variable region, or portion thereof, of requisite binding specificity is of non-human (murine) origin and the constant region comprises immunoglobulin sequences of human origin, the variable and constant regions being joined together chemically by conventional techniques (e.g., synthetic) or prepared as a contiguous polypeptide using genetic engineering techniques (e.g., DNA encoding the protein portions of the antibody can be expressed to produce a contiguous polypeptide chain). This type of "humanized" antibody may also be referred to as a chimeric antibody.

[0123] Another example of a humanized antibody according to the present invention is an immunoglobulin containing one or more immunoglobulin chains comprising a CDR of non- human origin (e.g., one or more CDRs of the antibody are derived from an antibody of non- human origin, e.g., PA-30 CDRs, and a framework region derived from a light and/or heavy chain of human origin (e.g., CDR-grafted antibodies with or without framework changes). In one embodiment, the humanized immunoglobulin molecule can compete with the PA-30 monoclonal antibody (or rPA-30) for binding a protein or ligand present on or expressed by a human B cell or liver cell. In another embodiment, the antigen-binding region of the humanized immunoglobulin is derived from the PA-30 monoclonal antibody to produce a humanized immunoglobulin comprising CDRl, CDR2 and CDR3 of the PA-30 light chain and CDRl, CDR2 and CDR3 of the PA-30 heavy chain. Chimeric or CDR-grafted single chain antibodies are embraced by the term humanized immunoglobulin. The production of single chain antibodies is known and practiced in the art, for example, as described in U.S. Patent No. 4,946,778 to Ladner et al., U.S. Patent No. 5,476,786 to Huston and R.E. Bird et aL 1988, Science, 242:423-426.

[0124] Humanized antibodies of the present invention can be produced using synthetic and/or recombinant nucleic acids to prepare genes, e.g., cDNA, encoding the desired humanized immunoglobulin protein chain. For example, nucleic acid, e.g., DNA, sequences coding for humanized variable regions can be constructed using PCR mutagenesis methods to alter DNA sequences encoding a human or humanized immunoglobulin chain, such as a DNA template from a previously humanized variable region. See, for example, M. Kanunan et al., 1989, Nucl Acids Res., 17:5404; K, Sato et al., 1993, Cancer Res., 53:851-856; B.L. Daugherty et al., 1991, Nucl. Acids Res., 19(9):2471-2476; A.P. Lewis and J.S. Crowe, 1991, Gene, 101 :297-302. Using these or other suitable techniques, variants can also be readily produced. In one embodiment, cloned variable regions can be mutagenized and sequences encoding variants with the desired binding specificity can be selected, for example, from a phage library. See, e.g., U.S. Patent No. 5,514,548 to Krebber et al.; WO 93/06213, Inventor Hoogenboom et al., published Apr. 1, 1993.

[0125] A monoclonal antibody in accordance with this invention, e.g., PA-30, serves as an agent that inhibits entry of HCV into a cell or a target cell susceptible to infection by HCV. Cells susceptible to HCV infection include cells having or expressing receptor proteins, glycoproteins, ligands, or molecular structures to which HCV binds. Susceptible cells may include, without limitation, primary cells, dendritic cells, placental cells, endometrial cells, lymph node cells, lymphoid cells (B and T cells), placenta cells, peripheral blood mononuclear cells, monocytes/macrophages, or liver or hepatic cells. Hepatic cells, i.e., liver cells, may include, but are not limited to, hepatocytes, liver sinusoidal cells, a Hep3b cell, a HepG2 cell, SK-HEPl cell, C3A cell or an Huh-7 cell. In one embodiment, the hepatic cell is a primary hepatic cell. In another embodiment, the hepatic cell is a hepatoma or abnormal liver cell or hepatocyte.

[0126] In accordance with this invention, a monoclonal antibody, e.g., PA-30, may be used in its native form, or it may be truncated (e.g., via enzymatic cleavage and the like) to provide immunoglobulin fragments or portions, in particular, fragments or portions that bind ligand and/or that possess functional inhibiting or blocking activity against HCV. Illustratively, the ligand binding or functional antibody fragment or portion may be an Fab fragment or portion, an F(ab')₂ fragment or portion, a variable domain or one or more CDR domains of the antibody. In an embodiment, the fragment or portion of the monoclonal antibody may derive from the carboxy] portion or terminus of antibody protein and may comprise an Fc fragment, an Fd fragment or an Fv fragment. In some embodiments, the Fc fragment may be engineered to improve the functional properties of the antibody, e.g., following binding to ligand. Such Fc engineered antibodies may provide more effective interactions with components of the complement cascade.

[0127] A further aspect of this invention encompasses a method of producing a monoclonal antibody that is an inhibitor of HCV entry into susceptible cells. The method involves immunizing a subject, e.g., a recipient animal, such as a rodent, e.g., mouse or rat, with cells that express functional HCV receptor and/or co-receptor molecules. In an embodiment, the cells used as immunogen are replication-competent for HCV and are permissive for HCV entry and infection, for example, HepG2, Hep3b, Huh-7, Huh-7.1, or Huh-7.5.1. In an embodiment, the cells used as immunogen, e.g., HepG2, may be molecularly engineered to express a receptor or co-receptor molecule, for example, CD81 , that renders the cells susceptible to HCV entry; HepG2 transfected with an expression vector harboring CD81 -encoding nucleic acid is an example of this latter embodiment.

[0128] hi an embodiment, the invention provides a method of producing a monoclonal antibody which binds cells susceptible to HCV infection and inhibits entry of HCV into the HCV susceptible cells, which involves immunizing a subject with cells which (i) express functional HCV receptor and/or co-receptor molecules; (ii) are replication-competent for HCV and (iii) are permissive for HCV entry and infection; producing hybridoma cells comprising immune B cells from the immunized subject of (a); and assaying antibody generated by the hybridoma cells for the antibody's ability to bind HCV susceptible cells and inhibit entry of HCV into the HCV susceptible cells. Cells suitable for use in the method comprise, without limitation,

[0129] In brief, in an exemplary embodiment, the method involves immunizing mice (e.g., Balb/c) every three weeks via intraperitoneal injection with 2-5x10⁶ permissive hepatoma cells or cell lines as described above over a time period of from 6-15 months. For example, different cohorts of mice are immunized with HepG2 cells, Hep3b cells, or Huh-7 hepatoma cell lines that are permissive for HCV (or HCVpp) entry. Three days before harvesting spleens from the immunized animals for fusion, the mice are boosted intravenously with 2x10⁶ of the cells used as the initial immunogen. Adjuvants, such as Quil A, may be used to increase the magnitude of the immune response. Following the immunization and boosting regimen, splenocytes from immunized animals are fused with cells of a myeloma cell line, e.g., Sp2/0.904 (ATCC, Manassas, VA), or an equivalent myeloma cell line, using methods and techniques that are standard in the art. Other immune cells, such as lymph node, may be fused to myeloma cells, as would be appreciated by the skilled practitioner. The resulting hybridoma cells are suspended in RPMI-1640 medium supplemented to contain 10% FBS, 10% BM Condimed-Hl (Roche Applied Science. Indianapolis. IN) and 24 μM beta-mercaptoethanol, and the cells are plated into 96-wcll flat-bottomed tissue culture plates (BD Biosciences). Selective pressure is applied for seven days using hypoxanthine, azaserine and thymidine. In the primary screen, hybridoma supernatants are tested for their ability to inhibit HCV entry into Hep3b cells, for example, employing an HCV pseudoparticle assay. Hybridomas that produce supernatants containing monoclonal antibody having the highest inhibiting activity are twice cloned by limiting dilution. The monoclonal antibodies may be purified from the hybridoma supernatant by methods known in the art,

[0130] The monoclonal antibody or portion thereof of this invention, e.g., PA-30, may be used in therapeutic and prophylactic methods to treat and prevent HCV infection and to treat and prevent a liver disease, or a pathological condition affecting susceptible cells, such as liver cells or hepatocytes, lymphoid cells, or monocytes/macrophages. The monoclonal antibody according to the present invention binds to a cell surface ligand and can also inhibit and/or prevent infection of cells susceptible to HCV infection.

[0131] In one embodiment of the methods described herein, the susceptible cell is present in a subject and a monoclonal antibody or portion thereof according to the present invention is administered to a subject to treat the subject who has become infected by HCV or afflicted with HCV or a condition related to HCV infection. As used herein, "afflicted with or infected by HCV" means that the subject has at least one cell which has been infected by HCV. As used herein, "treating" means slowing, reducing, stopping, alleviating, or reducing the progression of an HCV disorder, or reversing the progression to the point of eliminating the disorder. Treating an HCV disorder also relates to the reduction of the number of viral infections, reduction of the number of infectious viral particles, reduction of the number of virally infected cells, or the amelioration of symptoms associated with HCV.

[0132] Another application of the present invention relates to preventing a subject from contracting HCV, such that a subject is prevented from becoming infected with HCV; thus, the genetic information of HCV cannot replicate in and/or incorporate into the host cells. Another application of the present invention is to treat a subject who has become infected with HCV. As used herein, "HCV infection" refers to the introduction of HCV genetic information into a target cell, such as by fusion of the target cell membrane with HCV or an HCV envelope glycoprotein- positive cell. One beneficial application of the monoclonal antibodies of the present invention is to inhibit or block HCV infection. As used herein, "inhibiting or blocking HCV infection" refers to reducing the amount of HCV genetic information introduced into a target cell population as compared to the amount that would be introduced without the presence of the antibodies of the invention.

[0133] In accordance with this invention, a target cell or an HCV susceptible cell may be a body cell of a subject. An HCV susceptible or afflicted subject means any animal, preferably mammalian, or artificially modified animal capable of becoming HCV-infected. Such subjects include, but are not limited to, a human being, a primate, equine, ovine, avian, bovine, porcine, canine, feline or murine subjects. Artificially modified animals include, but are not limited to, SCTD mice with human immune systems. In particular, the animals include but are not limited to mice, rats, dogs, pigs, goats, guinea pigs, ferrets, rabbits, llamas, horses, and chimpanzees.

[0134] A monoclonal antibody of the present invention, e.g., PA-30, finds use in compositions for prophylactic therapy and/or for treating HCV infection by reducing viral load, by inhibiting binding of the virus to its target protein(s), by inhibiting virus mediated fusion with a target cell, and/or by interfering with conformational changes in the viral envelope proteins necessary for cell infectivity. The composition used can include a monoclonal antibody directed to linear epitopes, or to a conformational epitope, or a mixture of complementary monoclonal antibodies that recognize distinct conformational epitopes. The binding of a monoclonal antibody of the invention, e.g., PA-30, to a cell receptor or co-receptor of HCV may serve to inhibit or otherwise block infection of the cell by HCV. Binding of PA-30 to its cell receptor or co-receptor may, in turn, stimulate other immune cell and immune system functions.

[0135] Treatment of hepatitis C virus (HCV) infection may also be accomplished using pharmaceutical compositions comprising a monoclonal antibody of the present invention. The monoclonal antibody is present in the pharmaceutical composition in an effective amount or in a therapeutically effective amount. Suitable formulations for delivery of the antibodies are found in Remington's Pharmaceutical Sciences (1985 or more current version). These pharmaceutical compositions are suitable for use in a variety of drug delivery systems (See, Langer, 1990). Monoclonal antibodies in compositions are suitable for single administration or in a series of inoculations (e.g., an initial immunization followed by subsequent inoculations to boost the HCV inhibitory immune response). Pharmaceutical compositions or formulations comprising a monoclonal antibody according to this invention, e.g., PA-30, may include other reagents, substances, excipients, carriers, vehicles and diluents as described herein. The determination of a therapeutically or prophylactically effective amount of the antibody and compositions can be made by the skilled practitioner in the art, typically based on animal data using routine computational methods. The effective amount is based upon, among other things, the size, form, biodegradability, bioactivity and bioavailability of the antibody as described below.

[0136] As guidance for the amount of the monoclonal antibody or portion thereof for administration to the subject, a dose or amount would be one in sufficient quantities to either inhibit HCV infection, treat HCV infection, treat the subject, or prevent the subject from becoming infected with HCV. This amount may be considered an effective amount. The skilled practitioner in the art can perform simple titration experiments to determine what amount is required to treat the subject. The dose of the composition of the invention will vary depending on the subject (e.g., physical size, mass, weight and health status) and upon the particular route of administration used. In one embodiment, the dosage can range from about 0.1 to about 100,000 g/kg body weight of the subject. Based upon the composition, the dose can be delivered continuously, such as by continuous pump, or at periodic intervals, for example, on one or more separate occasions. Desired time intervals of multiple dosing of a monoclonal antibody or a particular composition thereof can be readily determined by the skilled practitioner in the art.

[0137] In one embodiment of the methods described herein, the effective amount of the monoclonal antibody, e.g., PA-30, a portion thereof, or an antibody-containing composition as described, is between about 0.5 mg and about 50 mg per kg body weight of the subject. In other embodiments, the effective amount is between about 1 mg and about 50 mg per kg body weight of the subject; or between about 2 mg and about 40 mg per kg body weight of the subject; or between about 3 mg and about 30 mg per kg body weight of the subject; or between about 4 mg and about 20 mg per kg body weight of the subject; or between about 5 mg and about 10 mg per kg body weight of the subject. In other embodiments, the effective amount of the monoclonal antibody, a portion thereof, or an antibody-containing composition as described, may comprise from about 0.000001 mg/kg body weight to about 100 mg/kg body weight; or from about 0.001 mg/kg body weight to about 50 mg/kg body weight; or from about 0.01 mg/kg body weight to about 10 mg/kg body weight.

[0138] The effective amount may be based upon, among other things, the form, size, biodegradability, bioactivity and the bioavailability of the monoclonal antibody, a portion thereof, or an antibody-containing composition as described. If the active, i.e., the monoclonal antibody, a portion thereof, does not degrade quickly, is bioavailable and highly active, a smaller amount will be required to be effective. Through knowledge possessed by the skilled practitioner and routine methods, the effective amount of antibody will be known to one of skill in the art. One of skill in the art could also routinely perform empirical activity tests for an antibody or other active substance to determine the bioactivity in bioassays and thus determine the effective amount.

[0139] In an embodiment of the above methods, the effective amount of the monoclonal antibody, e.g., PA-30, a portion thereof, or an antibody-containing composition as described comprises from about 1.0 ng/kg to about 100 mg/kg body weight of the subject. In other embodiments, the effective amount of the monoclonal antibody, a portion thereof, or an antibody-containing composition as described comprises from about 100 ng/kg to about 50 mg/kg body weight of the subject; or from about 1 μg/kg to about 10 mg/kg body weight of the subject; or about 100 μg/kg to about 1 mg/kg body weight of the subject. [0140] The skilled practitioner in the art can determine when to administer the monoclonal antibody or antibody-containing composition in accordance with the present invention. The administration may be constant for a certain period of time or periodic and at specific intervals. The monoclonal antibody or composition may be delivered hourly, daily, weekly, monthly, yearly (e.g., in a time release form) or as a one time delivery. Alternatively, the delivery may occur at multiple times during a given time period, e.g., two or more times per week; two or more times per month, and the like. The delivery may be continuous delivery for a period of time, e.g. intravenous delivery. In embodiments of the methods described herein, the monoclonal antibody or composition is administered at least once per day; daily; every other day; every 6 to 8 days; or weekly.

[0141] Methods for treating a subject afflicted with HCV infection or an HCV-associated disorder, including liver disease, and methods for inhibiting in a subject the onset of HCV infection or an HCV-associated disorder, including liver disease, may include the administration of at least one conventional antiviral agent in conjunction with at least one monoclonal antibody, e.g., PA-30, according to the present invention. It will be appreciated that the antibody may be chimeric or humanized. Such antiviral agents include, but are not limited to, interferon-alpha, interferon-alpha-2B and ribavirin. The one or more antiviral agents may be administered to a patient in need thereof either before, at the same time as, or following administration of one or more monoclonal antibodies of the present invention, e.g., PA-30.

[0142] A monoclonal antibody or a pharmaceutical composition comprising the monoclonal antibody or portion thereof according to the present invention, may be administered using any of the methods known to the skilled practitioner in the art. The antibody and/or composition may be administered by various routes including but not limited to aerosol, intravenous, oral, or topical route. The administration may comprise intralesional, intraperitoneal, subcutaneous, intramuscular or intravenous injection; infusion; liposome-mεdiatcd delivery; topical, intrathecal, gingival pocket, rectal, intrabronchial, nasal, transmucosal, intestinal, oral, ocular or otic delivery. Additionally, the administration includes intrabronchial administration, anal, intrathecal administration or transdermal delivery. The monoclonal antibody of the invention may be delivered locally via a capsule, which allows sustained release of the antibody over a period of time. Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). Also embraced by the present invention are particulate compositions coated with polymers (e.g., poloxamcrs or poloxamines). Other modes of administration of the antibodies and compositions of this invention incorporate particulate forms protective coatings, protease inhibitors, and/or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal and oral. Carriers included in the compositions may be a diluent, an aerosol, a topical carrier, an aqueous solution, a nonaqueous solution or a solid carrier.

[0143] Parenteral administration may be preferentially directed to the patient's liver, such as by catheterization to hepatic arteries or into a bile duct. For parenteral administration, the compositions can include the monoclonal antibody suspended in a suitable sterile carrier such as water, aqueous buffer, 0.4% saline solution, 0.3% glycine, hyaluronic acid or emulsions of nontoxic nonionic surfactants as is well known in the art. The carrier may be a pharmaceutically acceptable carrier. The compositions may further include substances to approximate physiological conditions, such as buffering agents and wetting agents, e.g., NaCl, KCl, CaCl₂, sodium acetate and sodium lactate. Aqueous suspensions containing a monoclonal antibody can be lyophilized for storage and can be suitably recombined with sterile water before administration. Solid compositions including a monoclonal antibody in conventional nontoxic solid carriers may be used. For oral administration of solid compositions, the monoclonal antibody may comprise 10% to 95%, or 25% to 75%, of the composition.

[0144] Pharmaceutically acceptable carriers are well known to those skilled in the art. Such pharmaceutically acceptable carriers may include, but are not limited to, aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers are typically sterile and include water, alcoholic/aqueous solutions, emulsions or suspensions, saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like

[0145] The present invention provides a method of treating or preventing a liver disease or pathology in a subject, which comprises administering to the subject an effective amount of an antibody, e.g., PA-30, of the invention which inhibits HCV from entering or infecting a subject's cells, so as to thereby treat or prevent the liver disease or pathology in the subject. In an embodiment of the methods described herein, the liver disease is hepatitis. In an embodiment of the methods described herein, the liver disease is cirrhosis. In an embodiment, the method involves the use of the PA-30 monoclonal antibody or portion thereof, alone or in combination with other suitable antibodies. [0Ϊ 46] The present invention provides a method of treating or preventing hepatocellular carcinoma in a subject which comprises administering to the subject an effective amount of a monoclonal antibody of the invention which inhibits (or blocks) HCV from entering or infecting a subject's cells, so as to treat or prevent hepatocellular carcinoma in the subject. In an embodiment, the method involves the use of the PA-30 monoclonal antibody or portion thereof.

[0147] A monoclonal antibody of the present invention, e.g., PA-30, may also be used for passive immunization therapies or other in vivo therapies. See, for example, Piazzi, et al., 1997; Farci, et al., 1996; al-Hemsi, et al., 1996; Krawczynski, et al., 1996). For such therapeutic uses, the monoclonal antibody may be formulated in any convenient way for injection or intravenous administration as described herein. Various media may be used, such as phosphate buffered saline, saline, or the like. The amount of the monoclonal antibodies used may be varied depending on the level of infection, the affinity of the antibodies, the manner of administration, the frequency of administration, the response of the patient, the use of other therapeutics, and the like. In general, the amount of monoclonal antibody administered is in the range of about 0.1 to 50 mg/kg, or 0.1 to 20 mg/kg, or 0.1 to 15 mg/kg, or 0.1 to 5 mg/kg. See, for example, Andrus et al., 1998; Kreil et al., 1998.

[0148] The chimpanzee is an accepted animal model for screening HCV vaccines and therapeutics. See, e.g., Farci, et al., 1996; Farci, et al., 1994; Farci, et al, 1992; Krawczynski, et al., 1996; Bassett, et al., 1998. Other animal models, e.g., mice as described herein, are also available for testing anti-HCV and HCV inhibitory monoclonal antibodies, as well as other HCV inhibitory agents, (e.g., WO2005/067709). In chimpanzee studies, the effectiveness of the monoclonal antibodies can be determined by monitoring for the presence and titer of HCV RNA using quantitative PCR methods. A successful reduction of viral load, or prevention of infection in a test animal or subject, is reflected as a reduction or elimination of HCV RNA in serum. Enzymatic tests such as measurement of alanine aminotransferase and/or the use of sequential punch needle liver biopsies are further used to test effectiveness of a therapeutic or treatment, where improvement in the rating of either (or both) measurement(s) indicates a reduction in viral-induced liver damage.

[0149] The present invention encompasses an article of manufacture comprising a solid support having operably affixed thereto a monoclonal antibody, e.g., PA-30. or a portion thereof that is capable of specifically binding target ligand, e.g., protein or glycoprotein expressed by human B cells and liver or hepatic cells. The solid support may be any solid support known in the art to which the antibody can be operably affixed. Operably affixed refers to the antibody (or other agent) being affixed in a manner permitting the formation of a complex between the affixed antibody (or agent) and the domain present on the ligand, e.g., protein or glycoprotein, recognized by the antibody. Methods by which an antibody (or agent) may be operably affixed to a solid support are well known in the art. Solid supports include, by way of example, natural or synthetic polymers. Synthetic polymers include, by way of example, polystyrene, polyethylene and polypropylene. Natural polymers include, by way of example, latex. The solid support includes a bead, a receptacle, or a filter. Solid supports in the form of beads are widely used and readily available to those skilled in the art. Beads include, for example, latex and polystyrene beads. Solid supports in the form of filters are widely used and readily available to those skilled in the art. Filters include, for example, polyester filters (e.g., polyester leukofiltration devices) and cellulose acetate filters.

[0150] In an embodiment of the above methods, the domain present on the cell ligand bound by the monoclonal antibody is a conserved domain, which may be defined as a protein or glycoprotein ligand domain that is present on, and whose structure is invariant among, those cells expressing the ligand. In an embodiment, the ligand is a receptor or co-receptor of HCV. In an embodiment, the ligand is a cell receptor or co-receptor for the entry of HCV into a susceptible cell.

[0151] The present invention further encompasses an aqueous-soluble monoclonal antibody, e.g., PA-30, or portion thereof that blocks HCV infection of cells and binds a domain present on a cell surface ligand. The monoclonal antibody or portion thereof further comprises a moiety capable of specifically forming a complex with a known binding member, which moiety permits the removal of the antibody or portion thereof from a sample via contact with an immobilized form of the known binding member. As used herein, "aqueous-soluble" means capable of existing in soluble form in water at 4°C at a concentration of at least 1 pM. The use of a moiety capable of specifically forming a complex with a known binding member is commonly referred to in the art as "molecular tagging." The moiety may be selected, for example, from the group consisting of a small molecule and a protein. The binding member includes, but is not limited to, for example, a metal ion, a small molecule, a peptide, or a protein. Specific examples of moiety/binding member combinations include, but are not limited to, (a) oligohistidine/nickel ion, (b) glutathione S-transferase/glutathione, (c) biotin/streptavidin, and (d) the HA peptide/anti- HA peptide antibody. The HA peptide is composed of nine contiguous amino acids, wherein the first amino acid (aa) residue is tyrosine (Y), the second aa residue is proline (P), the third aa residue is tyrosine (Y), the fourth aa residue is aspartic acid (D), the fifth aa residue is valine (V), the sixth aa residue is proline (P), the seventh aa residue is aspartic acid (D), the eighth aa residue is tyrosine (Y), and the ninth aa residue is alanine (A). This peptide moiety may be attached by any means known to one skilled in the art, such as for example, chemically or genetically,

[0152] The present invention further provides a method of substantially reducing the likelihood of a subject's susceptible cells from becoming infected with HCV as a result of contact with a sample containing HCV, which comprises the steps of (a) contacting the susceptible cells with a suitable amount of an aqueous-soluble monoclonal antibody or a portion thereof according to the present invention capable of binding a domain present on a ligand associated with HCV entry or infection and being present on susceptible cells, thereby forming a complex between the monoclonal antibody and the ligand; whereby entry or infection of the susceptible cell by HCV is blocked or inhibited via the binding of ligand by the antibody, so as to thereby reduce the likelihood of a subject's becoming infected with HCV as a result of contact with the sample. An embodiment of the method involves the use of the PA-30 monoclonal antibody or portion thereof.

[0153] Illustratively, as used in the described methods, substantially reducing the likelihood of the subject's becoming infected with HCV, refers to reducing the likelihood of the subject's becoming infected with HCV by at least two-fold. For example, if a subject has a 1% chance of becoming infected with HCV, a two-fold reduction in the likelihood of the subject's becoming infected with HCV would result in the subject's having a 0.5% chance of becoming infected with HCV. hi one embodiment, substantially reducing the likelihood of the subject's becoming infected with HCV means reducing the likelihood by at least ten-fold. In another embodiment, substantially reducing the likelihood of a subject's becoming infected with HCV means reducing the likelihood by at least 100-fold. The amount of aqueous-soluble monoclonal antibody or portion thereof suitable to substantially reduce the likelihood of a subject's becoming infected with HCV may be determined according to methods known to those skilled in the art. In one embodiment, a suitable amount is an amount between about 1 pM and about 10 niM. In another embodiment, the suitable amount is an amount between about 1 pM and about 10 μM.

[0154] This invention encompasses a method of treating HCV infection in a subject which comprises inhibiting HCV infection of the subject's cells susceptible to HCV infection by a method described herein, wherein the contacting is effected by administering to the subject one or more of the monoclonal antibodies or active portion thereof of the invention. The invention also encompasses a method of preventing HCV infection of a subject which comprises inhibiting HCV infection of the subject's cells susceptible to HCV infection by a method described herein, wherein the contacting is effected by administering to the subject a monoclonal antibody or active portion thereof of the invention. In an embodiment, the method involves the use of an effective amount of the PA-30 monoclonal antibody or portion thereof which is administered to the subject. In an embodiment, the PA-30 monoclonal antibody is chimeric or humanized.

[0155] The present invention also provides a method of inhibiting HCV infection of a cell, e.g., new infection or established infection, which involves contacting a cell susceptible of infection by HCV with an effective amount of the monoclonal antibody, e.g., PA-30, or a portion thereof, which binds a susceptible target cell ligand and inhibits HCV infection of the cell. In an embodiment, the PA-30 monoclonal antibody is chimeric or humanized.

[0156] This invention further encompasses a method of preventing a cell or cells of a subject from becoming infected with HCV, which method includes administering to the subject a monoclonal antibody according to the invention, e.g., PA-30, in an amount effective to bind ligand on the surface of the subject's cells and block infection by HCV so as to thereby prevent the subject's cell or cells from becoming infected with the virus. The present invention provides a method of treating a subject whose cells are infected with HCV, which method comprises administering to the subject a monoclonal antibody or portion thereof according to the invention in an amount effective to bind ligand or co-ligand on the surface of the subject's cells and inhibit infection by HCV so as to treat the subject. The subject may be a human, a non-human primate, or other animal, such as a SCID-BNX mouse (Galun et al., 1995). In an embodiment, the method involves the use of an effective amount of the PA-30 monoclonal antibody or portion thereof. In an embodiment, the PA-30 monoclonal antibody is chimeric or humanized.

[0157] In an embodiment of the above methods, the subject is infected with HCV prior to administering the monoclonal antibody to the subject. In another embodiment of the above methods, the subject is not infected with HCV prior to administering the antibody to the subject. In another embodiment of the above methods, the subject is not infected with, but has been exposed to, HCV.

[0158] This invention further provides a method for inhibiting in a subject the onset of a hepatitis C virus (HCV)-associated disorder, the inhibition of which is effected by immunizing the subject, which method comprises: injecting into the subject a pharmaceutical composition comprising a monoclonal antibody of the invention; thereby eliciting a protective immune response in the subject. Such a method further embraces the administration of at least one conventional antiviral agent, including, but not limited to, interferon-alpha, interferon-alpha-2B and ribavirin. In an embodiment, the method involves the use of the PA-30 monoclonal antibody or portion thereof. In an embodiment, the PA-30 monoclonal antibody is chimeric or humanized. [0159] A monoclonal antibody described herein, e.g., PA-30, can be used prophylactically as a vaccine to prevent HCV infection or a liver disease. Accordingly, this invention also provides a method for preventing a hepatitis C virus (HCV) infection or liver disease in a subject, the prevention of which is effected by immunizing the subject, which method comprises: (a) injecting into the subject a pharmaceutical composition comprising a monoclonal antibody of the invention; and (b) eliciting a protective HCV immune response in the subject. In an embodiment, this method involves the use of an effective amount of the PA-30 monoclonal antibody or portion thereof in the pharmaceutical composition. Tn an embodiment, the PA-30 monoclonal antibody is chimeric or humanized.

[0160] A vaccine containing a monoclonal antibody of the invention, e.g., PA-30, contains an immunogenically effective amount of the monoclonal antibody, or an active portion thereof, admixed with a pharmaceutically acceptable carrier such as those described above. The vaccine may further include other carriers known in the art such as, for example, thyroglobulin, albumin, tetanus toxoid, polyamino acids such as polymers of D-lysine and D-glutamate, inactivated influenza virus and hepatitis B recombinant protein(s). The vaccine may also include any well known adjuvants such as, without limitation, incomplete Freund's adjuvant, alum, aluminum phosphate, aluminum hydroxide, monophosphoryl lipid A (MPL, GlaxoSmithKline), a saponin, CpG oligonucleotides. (Krieg et al., 1995), montanide, vitamin E and various water-in-oil emulsions prepared from biodegradable oils such as squalene and/or tocopherol, Quil A, Ribi Detox, CRL-1005, L-121 and combinations thereof. As a result of administration of a monoclonal antibody of the invention, HCV entry into cells is ultimately reduced, inhibited, or prevented. A heightened immune response on the part of the patient may include generation of a cellular immune response (e.g., activation of cytotoxic T lymphocytes or CTL).

[0161] Accordingly, a vaccine composition containing a monoclonal antibody according to the present invention, e.g., PA-30, is administered to a patient in an immunogenically effective amount to elicit a protective immune response against HCV. The immunogenically effective amount will vary depending on the composition of the vaccine (e.g., whether or not it contains adjuvant), the manner of administration, the weight and general health of the patient and the judgment of the prescribing health care provider. For initial vaccination, the general range of antibody, or portion thereof, in the administered vaccine is about 100 μg to about 1 mg per 70 kg patient; subsequent inoculations to boost the immune response include antibody, or a portion thereof, in the range of 100 μg to about 1 mg per 70 kg patient. Single or multiple boosting immunizations are administered over a period of about two weeks to about six months from the initial vaccination. The prescribing health care provider may determine the number and timing of booster immunizations based on well known immunization protocols and the individual patient's response to the immunizations (e.g., as monitored by assaying for viral load, infected cells and the like).

[0162] For treatment of a patient infected with HCV, the amount of monoclonal antibody, or portion thereof, to be delivered will vary with the method of delivery, the number of administrations and the state of the person receiving the composition (e.g., age, weight, severity of HCV infection, active or chronic status of HCV infection and general health status). Before therapeutic administration, the patient will already have been diagnosed as being HCV-infected and may or may not be symptomatic. Generally, a therapeutically effective amount of the monoclonal antibody or portion thereof will be in the range of about 1 mg to about 10 gm per day, or about 50 mg to about 5 gm per day, or about 100 mg to 1 gm per day for a 70 kg patient. The monoclonal antibody or portion thereof may be administered as a prime and/or boost, alone or in various prime/boost combinations with other agents as described herein.

[0163] The monoclonal antibody or portion thereof according to this invention, e.g., PA-30, may be used in combination with one or more additional antiviral agents, e.g., in compositions, which include, without limitation, a non-nucleoside HCV RNA-dependent RNA polymerase (RdRP) inhibitor, a nucleoside HCV RNA-dependent RNA polymerase (RdRP) inhibitor, a non- nucleoside HCV RNA protease inhibitor, a nucleoside HCV RNA protease inhibitor, non- nucleoside reverse transcriptase inhibitors (NNRTIs), a nucleoside reverse transcriptase inhibitor, a viral entry inhibitor, interferon, PEG-interferon, ribavirin and combinations thereof. It will be understood that the nucleoside and non-nucleoside inhibitors include analogs of nucleoside and non-nucleoside molecules. The polymerase inhibitors can target HCV NS5B and NS5 A; the protease inhibitors can target HCV NS3 and NS4.

[0164] Illustrative nucleoside analog inhibitors of NS5B that may be used in the compositions of the present invention include, without limitation, valopicitabine (NM283, Idem^'x/Novartis), a prodrug of nucleoside analog 2'-C-methylcytosine; JTK 103 (Japan Tobacco/AKROS); R04048 (Pharmasset/Roche); R-1479/R-1626 (Pharmasset/Roche), nucleoside analog of 4'-azidocytosine and prodrug thereof; and R-7128 (Pharmasset/Roche). Illustrative non-nucleoside analog inhibitors (NNRTI) that may be used in the compositions of the present invention include, without limitation, HCV- 796 (ViroPharma/'Wyeth), a benzofuran HCV polymerase inhibitor; GL60667 or "667" (Genelab Technologies. Inc./Gilead Sciences. Inc.); and XTL-2125 (XTL Biopharmaceutϊcals, Inc.). Illustrative serine protease inhibitors of NS3/4A of HCV that may be used in the compositions of the present invention include, without limitation, VX-950 (Vertex/Janssen-Tibotec); SCH-503034 (Schering-Plough); ACH-806/GS-9132 (Achillion/Gilead); and BILN-2061 (Boehringer Ingleheim).

[0165] For a combination therapy in which the monoclonal antibody or portion thereof of the present invention, e.g., PA-30, is used in conjunction with one or more conventional antiviral compounds or agents, the antibody may be provided to the subject prior to, subsequent to, or concurrently with the one or more conventional antiviral compounds or agents.

[0166] The present invention also provides isolated, purified ligand of approximately 60 kDa, or a peptide thereof, which is capable of being bound by the PA-30 monoclonal antibody. In an embodiment, the ligand or peptide thereof is soluble. In an embodiment, the purified, soluble ligand, or soluble peptide thereof, is used as a therapeutic or prophylactic agent, e.g., a drug, for the treatment and/or prevention of infection of cells by HCV. Without wishing to be bound by theory, purified, soluble PA-30 ligand according to the invention may serve as a therapeutic or prophylactic agent to prevent HCV binding to cells following administration of an effective amount of the purified soluble ligand, or a pharmaceutical composition containing the ligand, to a subject infected with HCV, or susceptible to or at risk of, infection by HCV. In particular, the approximately 60 kDa protein ligand bound by monoclonal antibody PA-30 (ATCC Accession No. PTA-7278), or by an antibody, e.g., monoclonal antibody, or portion thereof that binds the same ligand epitope(s) as does PA-30, or by an antibody or portion thereof which competes with PA-30 for binding the ligand, is isolated and purified.

[0167] The invention further embraces a method of treating a subject infected with HCV, which involves administering to the subject a pharmaceutically-acceptable composition containing a carrier or the like and the purified soluble PA-30 ligand, or a soluble peptide thereof, in an amount effective to treat the HCV-infected subject. The effective amount of the ligand, or soluble peptide thereof, may bind HCV such that HCV cannot, or does not, enter and/or infect susceptible cells. The invention further embraces a method of preventing HCV infection of a subject, which involves administering to the subject a pharmaceutically-acceptable composition containing a carrier or the like and the purified soluble PA-30 ligand. or a soluble peptide thereof, in an amount effective to prevent HCV infection of the cells of the subject. These methods may also include one or more additional antiviral agents as described herein.

[0168] The monoclonal antibodies, e.g., PA-30, may be further employed in diagnosis and screening applications and methods. For example, antibodies such as PA-30 may be used in HCV neutralization assays against which other anti-HCV antibodies or chemical compounds are compared for the extent of inhibition of virus entry into cells. [0169] In an embodiment, monoclonal antibodies such as PA-30 may be used in assays for HCV antibodies in a serum sample or body fluid sample. A body fluid is any fluid which is present in a subject's body and is capable of containing HCV in an HCV-infected subject. Body fluids include, but are not limited to, whole blood or derivatives thereof (e.g., red blood cell and platelet preparations), saliva, cerebrospinal fluid, tears, vaginal secretions, semen, urine, alveolar fluid, synovial fluid, pleural fluid and bone marrow. In addition, body fluid samples such as whole blood may further comprise exogenous substances added thereto for clinical or storage purposes. Such exogenous substances include, by way of example, anticoagulants (e.g., citrate) and preservatives (e.g., dextrose). Lysates or homogenates of body cells, tissues and organs may also be considered as body fluids. The foregoing assay may involve the steps of allowing competitive binding between putative antibodies in the sample and a known amount of the known monoclonal antibody, such as PA-30, for binding to cells to which PA-30 binds, and measuring the amount of the known monoclonal antibody bound. For example, cells to which the monoclonal antibody, e.g., PA-30, binds may be immobilized on a solid support, such as in an ELISA or RIA format. The known monoclonal antibody, e.g., PA-30, is appropriately labeled, for example, with an enzymatic, chemiluminescent, or fluorescent label. In such an assay, competitive binding between sample antibodies and the antibody of the invention for binding to the cell surface results in the bound antibody being a measure of antibodies in the sample.

EXAMPLES EXAMPLE 1

Materials and methods related to the production and characterization of the PA-30 monoclonal antibody

A. Cell lines and antibodies

[0170] The human hepatoma derived cell line, Hep3b, the human hepatoma derived cell line HepG2 and the human embryonic kidney cell line, 293T, as well as SK-Hepl, PLC/PRF/5 and HeLa cells, were purchased from the American Type Culture Collection, ATCC, (Manassas, VA) and maintained in DMEM with 10% fetal bovine serum (FBS) and ImM L-glutamine. The Ramos, Namalwa and Molt-4 cell lines, also purchased from the ATCC, were maintained in RPMI- 1640 medium supplemented to contain 10% FBS and ImM L-glutaminc. CEM NKR CCR5 cells obtained from the NIH AIDS Research and Reference Reagent Program were also maintained in RPMT-164U medium supplemented to contain 10% FBS and ImM L-glutamine. Cell culture reagents were purchased from Invitrogen (Carlsbad, CA), unless otherwise noted. JS-81, a murine anti-human CD81 antibody and murine IgG3.κ isotype control antibody were purchased from BD Biosciences (San Diego, CA). Antibodies directed against various CD antigens, e.g., anti-CD4, anti-CD8, anti-CD19, anti-CD20 antibodies, were purchased from Bccton Dickinson, Mountain View, CA).

B. Isolation and purification of PA-30 monoclonal antibody (MAb)

[0171] The PA-30 MAb was generated against a cellular immunogen by immunizing mice with Hep3b cells. Female Balb/c mice (Charles River Labs, Wilmington, MA) received seven intraperitoneal injections at intervals of at least 3 weeks with 5x10⁶ Hep3b cells. One mouse was boosted with 2x10⁶ Hep3b cells three days prior to splenectomy and then splenocytes were fused with the Sp2/0.904 myeloma cell line (ATCC, Manassas, VA) using standard methods. The resulting hybridomas were suspended in RPMI- 1640, 10% FBS, 10% BM Condimed-Hl (Roche Applied Science, Indianapolis, IN), containing 24μM beta-mercaptoethanol and plated into 96- well flat-bottomed tissue culture plates (BD Biosciences). Selective pressure was applied for seven days using hypoxanthine, azaserine, and thymidine. In the primary screen, 1750 hybridoma supernatants were tested for the ability to block HCVpp entry into Hep3b cells, as described below. Hybridomas that produced supematants having the highest HCV infection blocking or inhibitory activity were cloned twice by limiting dilution. One screened hybridoma line that was cloned by limiting dilution demonstrated stable and specific inhibition of HCVpp in the virus infection inhibition assay. This hybridoma line was designated PA-30. The isotype of the PA-30 cell line, i.e., IgG3 K, was determined using IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche Applied Science).

[0172] The cloned PA-30 hybridoma cell line was seeded in T-150 flasks (BD Biosciences) according to the manufacturer's instructions using RPMI- 1640 supplemented to contain 10%FBS and 10% BM Condimed-Hl, containing 24 μM beta-mercaptoethanol. Cell culture supernatant from the PA-30 cell line was harvested and centrifuged at 2000 rpm for 10 minutes to remove cellular debris. PA-30 was purified to homogeneity by protein A chromatography and resuspended in 0.1 M Sodium Acetate with 0.005% Tween®80.

C. Production of HCV pseudoparticjes (HCVpp) as used in virus inhibition assays

[0173] HCV pseudoparticles (HCVpp) were generated as described previously (E. Cormier et al., 2004). Briefly, using Lipofectamine 2000 (Invitrogen), 293T cells (5x10⁶) were plated in 10 cm' culture dishes (BD Falcon, Bedford, MA) and cotransfccted with 4 μg of NLluc^'env^' reporter vector (for obtaining a readout of relative light units, RLU), (R.I. Conner et al., 1995) and 8 μg of the F.1/E2 expression vector, which is described in detail by J. Dumonceaux et al., 2003 and E. Cormier ct al., 2004; and depicted in FlG. 2. Forty-eight hours after transfection. supernatants containing the HCVpp were removed and clarified by low speed centrifugation for 5 minutes.

D. Blocking HCVpp entry into susceptible cells

[0174] Hybridoma supernatants containing the PA-30 monoclonal antibody were tested for the ability to inhibit the entry of HCV pseudoparticles (HCVpp) of different genotypes into hepatic cell lines. HCVpp-containing supernatants were stored at -80⁰C and then thawed at 25⁰C for thirty minutes prior to use in the virus infection inhibition assay. Equal volumes (40 μl) of HCVpp and Hep3b or Huh-7 cells (ATCC), (8000 cells/well), were plated in solid white 96 well plates (Perkin Elmer) in DMEM/10%FCS. Hybridoma supernatant samples (20μl) or control samples (20 μl) were added to the HCVpp and Hep3b or Huh-7 cells. After incubating the plates at 37°C for 3 days, medium was removed from the wells and equal volumes of PBS and Bright- GIo (Promega, Madison WI) (50 μl) were added. Luciferase activity (Relative Light Units, R.L.U.) was measured by a luminescence plate reader (Victor2, Perkin Elmer). Percent inhibition of virus entry was calculated from the R.L.U. values using the following formula: [(cellsrvirus without sample-cells: virus with sample)/(cells:virus without sample-cells with no virus)]xlOO. Neutralization/Inhibition curves and IC50 calculations were performed by nonlinear regression in GraphPad PRISM.

E. Cloning infectious HCV E1/E2 envelope glycoproteins from patient sera

[0175] Genotype specific primers and RT-Nested-PCR were used to amplify the El /E2 gene from sera of individuals infected with HCV genotype Ia, Ib, or 2b, as described elsewhere (D. Lavillette et al., 2005). Briefly, viral RNA was isolated from 150 μL of infected patient serum using the QIAamp Viral RNA mini Kit (QIAGEN). Viral RNA was then reverse transcribed using the Superscript™ III First-Strand Synthesis System for RT-PCR (Invitrogen). The resulting DNA served as template for a first round of amplification with genotype-specific outer primers followed by a second round of amplification with genotype-specific inner primers. The forward inner primer contained a 5' CACC sequence to allow directional cloning into the pcDNA3.1 TOPO vector (Invitrogen). Both rounds of amplification were performed with the High fidelity Platinum Pfx DNA polymerase (Invitrogen).

F. Flow cytometry binding of different cell types by PA-30 monoclonal antibody

[0176] Test cells (2x1 Obtest) were incubated with 10% heat-inactivated goat serum in staining buffer (0.25% BSA, 0.1% sodium azide in PBS) for 20 minutes at 4°C and treated with 0.2μg or 2μg of hybridoma supernatant containing PA-30 monoclonal antibody for 30 minutes at 4⁰C. Cells were washed once with 2 ml of staining buffer. Secondary antibody (goat F(ab')₂ anti- mouse IgG-PE, Invitrogen) was added at a concentration of 1 μl/sample and the cells and secondary antibody were incubated for 15 minutes at 4⁰C. The cells were washed once and resuspended in staining buffer. Samples were analyzed on a FACSCalibur analyzer (BD Biosciences). For two-color staining, human peripheral blood mononuclear cells (hPBMC) were obtained from a HIV^~ HCV^" normal human donor. The cells were isolated from a Ficoll gradient and cryopreserved in liquid nitrogen. Thawed hPBMC (10⁶/test) or Ramos cells (2xlO⁵/test) were stained as described above. After staining with secondary antibody, the cells were washed. Anti-human CD4-PE, anti-human CD19-AlexaFluor488, anti-human CD20-FITC (BD Biosciences), or a combination of antibodies was added to the appropriate tubes and the cells were incubated with these reagents for 30 minutes at 4°C. After washing one time and resuspending in staining buffer, the cell samples were analyzed on the FACSCalibur.

EXAMPLE 2

PA-30 MAb binds primary human B cells and mitogen-stimulated T cells

[0177] Example 2 describes an analysis to determine if the PA-30 monoclonal antibody binds primary B cells and T cells isolated from human peripheral blood.

[0178] Human peripheral blood mononuclear cells (hPBMC) were obtained from a HIV^" HCV^" normal human donor. The cells were subjected to a Ficoll gradient, collected and cryopreserved in liquid nitrogen. Prior to use, the hPBMCs were thawed and counted. Ramos cells were used as a control. 1 x 10⁶ hPBMC or 2χ 10⁵ Ramos cells were incubated with 10% heat-inactivated goat serum in staining buffer (0.25% BSA, 0.1% sodium azide in PBS) for 20 minutes at 4⁰C. The cells were then treated with varying amounts of PA-30 or with an isotype- matched (i.e., IgG3, K) control monoclonal antibody for 30 minutes at 4°C in a volume of about lOOμl. The cells were washed once with 2 ml of staining buffer and then secondary antibody (goat anti-mouse IgG (H+L)-APC) was added at 1 μl/sample. The cells and secondary antibody were incubated for 15 minutes at 4⁰C, after which the cells were washed again in staining buffer. Thereafter, anti-human CD4-PE, anti-human CD19-AlexaFluor488, anti-human CD20-FITC, or a combination of these detection antibodies was added to appropriate tubes. The cells were incubated with the detection antibodies for 30 minutes at 4⁰C, washed and analyzed on a FACSCalibur instrument.

[0179] As observed from FIGS. 2A-2E, the PA-30 monoclonal antibody bound to (stained) Ramos cells and primary B cells (FIGS. 2D and 2E) in a dose-dependent manner. PA-30 also stained CD4 CD19 CD20^""" cells having a phenotype that corresponds to B cells. [0180] For analysis of primary T cells and PHA-stimulated T cells, PBMC were stimulated in overnight culture at a concentration of 2 x 10⁷ cells/5 ml with 1 μg/ml of PHA. As a control, an aliquot of the cells at the same concentration were not mitogen-stimulated. Cells were stained with anti-CD4-F!TC or with anti-CD8-FITC conjugated antibodies, with mouse IgG3 isotype control antibody and with PA-30 monoclonal antibody. Non-fluorescent antibodies were detected by staining with secondary anti-mouse IgG conjugated to PE. In FIG. 2F, it is observed that PHA-stimulated CD4+ cells showed a significant increase in expression of the ligand bound by the PA-30 monoclonal antibody compared with unstimulated control cells and staining with the IgG3 isotype control antibody. As seen in FIG. 2G, PHA-stimulated CD8+ cells showed enhanced expression of the ligand bound by the PA-30 monoclonal antibody, but to a lesser extent than that of the CD4+ cells. It is possible that the kinetics of upregulation of the PA-30 ligand may differ between the CD4+ and CD8+ T cell populations such that a longer period of stimulation or exposure may be required for expression of the ligand on CD8+ T cells.

EXAMPLE 3

Cell type binding specificity of PA-30 MAb

[0181] In this Example, several different cell lines were screened by flow cytometry for PA- 30 binding. For the binding studies, 2χlO⁵ cells of each cell type (See Example IA) were incubated with 10% heat-inactivated goat serum in staining buffer (0.25% BSA, 0.1% sodium azide in PBS) for 20 minutes at 4°C. Thereafter, 0.2μg or 2μg of PA-30 monoclonal antibody was added to the cells and the cells and PA-30 incubated for 30 minutes at 4°C in a volume of about lOOμl. The cells were washed once with 2 ml of staining buffer and secondary antibody (goat F(ab^')₂ anti-mouse IgG-PE) was added at a concentration of lμl/sample. The cells were incubated with secondary antibody for 15 minutes at 4°C. After washing in staining buffer, the cells were analyzed on a FACSCalibur instrument as presented in FIGS. 3A-3E. The staining results revealed that the PA-30 monoclonal antibody bound human B cells in a dose-dependent manner (FIG. 3A, Ramos cells and FlG. 3B, Namalwa cells), thus indicating the binding specificity of the PA-30 monoclonal antibody compared with the results observed with isotype- matched control antibody.

EXAMPLE 4

PA-30 MAb inhibits entry of HCVpp into susceptible cells

[0182} The PA-30 monoclonal antibody was tested for its ability to inhibit infection of susceptible cells by various genotypes of HCV in a virus infection inhibition assay employing HCVpp as described. The assay involved a panel HCVpp representing genotypes Ia, Ib and la/2b. The monoclonal antibody PA-29, that binds the HCV E1E2 envelope glycoprotein heterodimer and potently neutralizes HCV of diverse genotypes, was used as a reference and was tested in parallel with PA-30 for inhibition of HCVpp (virus) entry into Hep3b cells.

[0183] Purified PA-30 was serially diluted and added to Hep3b cells immediately prior to the addition of HCVpp derived from different genotypes, e.g., genotype Ia (HCV strain H77) or genotype 2b (HCV strain 2bCD), as described in Example 1. Plates were incubated for 72 hours prior to measurement of luciferase activity. IC50 values were calculated by fitting the data to a 4-parameter logistic equation in GraphPad Prism (GraphPad Software, Inc., San Diego, CA). As presented in the table shown in FIG. 5, PA-30 inhibited entry of HCVpp and inhibited the infection of Hep3b cells by HCVpp of different genotypes, i.e., HCV genotypes Ia, Ib and la/2b. FIG. 7 shows that inhibition of HCVpp (genotype Ia) infection of susceptible cells by PA-30 is concentration dependent. As demonstrated in FIG. 7, PA-30 exhibited an IC50 value of 6.4 μg/ml and an IC90 value of 28.2 μg/ml.

EXAMPLE 5

Immunoprecipitation of PA-30 Iigand

[0184] In this Example, experiments conducted to elucidate the cell Iigand recognized and bound by the PA-30 monoclonal antibody are described. For these studies, Ramos cells were harvested and washed three times with PBS buffer. The cells were resuspended at a concentration of 2.5 x 10⁷ cells/ml in PBS- (Phosphate buffered saline without calcium or magnesium) containing NHS-PECvBiotin (Pierce) and were incubated for 60 minutes at 20⁰C with constant agitation. After washing the cells three times with cold PBS-, the cells were centrifuged and 375 μl of lysis buffer [5OmM Tris-HCl, 15OmM NaCl, 1.0% NP-40, Complete Protease inhibitor cocktail (Roche Applied Science)] was added to the cell pellet. The cells and buffer were mixed vigorously for 20 minutes at 4°C. The cell lysate was centrifuged for 10 minutes at 10,000 rpm at 4°C and the supernatant was saved for analysis. To reduce background, the lysate was pre-cleared with 125μl of Protein G beads (GE Healthcare) for 2 hours at 4°C with constant agitation. PA-30 monoclonal antibody or isotype-matched control antibody (mouse IgG3, K). (60μg). were added to l OOμl of magnetic Protein G beads (Miltenyi Biotec. Auburn, CA) according to the manufacturer^'s instructions. Pre-cleared lysate was added to the Protein G beads coated with MAb, PA-30 monoclonal antibody or with the isotype- matched control antibody and the bead-antibody mixture was incubated overnight at 4°C with constant agitation.

[0185] The Iigand was eluted from the beads by heating the beads to 95°C for 5 minutes with LDS sample buffer (Invitrogen). Eluted complex was separated on a 4-12% Bis-Tris gel (Invitrogen) with MES buffer (Invitrogen) and blotted onto nitrocellulose (Invitrogen). The blot was blocked overnight in 5% bovine serum albumin, 0.1% Tween 20, PBS-. After washing and incubating with Streptavidin conjugated to horseradish peroxidase (GE Healthcare), the blot was developed with SuperSignal West Pico Chemiluminescent Substrate (Pierce) and exposed to film (Kodak, Rochester, NY).

[0186] From FlG. 6 it can be seen that a band of approximately 60 kDa (45-65 kDa) is specifically immunoprecipitated from Ramos cells by the PA-30 monoclonal antibody. The isotype-matched control does not immunoprecipitate a band of this size. The result shows that the PA-30 monoclonal antibody, which has been demonstrated to inhibit HCV entry into cells, also specifically recognizes, binds and immunoprecipitates at least one protein or ligand that is highly expressed on the surface of Ramos cells. The molecular weight of this ligand, a putative HCV receptor or co-receptor, approximately 60 kDa. A control monoclonal antibody, PA-25, was included to observe the size of non-reduced Ig heavy chain.

EXAMPLE 6

Time course of inhibition of HCVpp infection by PA-30.

[0187] Experiments were conducted to a time course of inhibition of HCVpp infection of cells by the PA-30 monoclonal antibody. In these experiments, cold HCVpp (genotype Ia) were added to Hep3b cells in wells of a 96 well plate. The cells and virus pseudoparticles were centrifuged for one hour at 1000 rpm at 4°C and the plates and wells of cells and virus particles were washed with cold PBS- to remove unbound virus particles. Purified MAbs (JS-81 , PA-30, and isotype-matched control antibody) were prepared at a concentration of lOμg/ml in cell culture medium warmed to 37°C. The monoclonal antibodies were added to the wells containing Hep3b cells and HCVpp at specified time points. After the addition of the MAbs, plate was incubated at 37°C for 72 hours prior to measuring luciferase activity. As observed in FIG. 8, the PA-30 monoclonal antibody (filled diamonds) acts differently from the JS-81 monoclonal antibody (filled circles) in the time course inhibition assay. JS-81 is seen to block virus entry at early time points after addition to cells, indicating that JS-81 functions as a post-attachment virus blocking agent, acting to inhibit virus infection of target cells subsequent to the attachment of virus particles to the target cells. In contrast, the inhibition profile of PA-30 suggests that this monoclonal antibody optimally acts to inhibit HCV infection at a time prior to attachment of the virus to the cell surface target receptor or co-receptor.

EXAMPLE 7

Deglycosylation of ligand bound by the PA-30 monoclonal antibody (the PA-30 ligand). [0188] Experiments were performed to investigate glycosylation of the approximately 60 kDa ligand bound by the PA-30 monoclonal antibody. In these studies, Ramos cell lysate and PA-30 MAb-coated Protein G beads were incubated together overnight. Thereafter, 150U of PNGaseF (NE Biolabs, Ipswich, MA) and 5mU of O-Glycosidase (Sigma) were added to the antigen: antibody complex for 1.5 hours at 37°C. The beads were washed and eluted with sample buffer prior to electrophoresis on SDS-PAGE, blotting onto nitrocellulose and probed with Streptavidin according to conventional procedures. Bands in Lanes 3, 5 and 7 of the blot shown in FIG. 9 exhibit similar intensities in the region of approximately 60 kDa, suggesting that there is a minimal number of N- and O- linked sugars present on the PA-30 ligand. The FIG. 9 blot represents a few second exposure so that intensity of the bands in the above-mentioned lanes with 5 x 10⁷ cells was not overexposed. Upon slightly longer exposure, a similar pattern was observed in the lanes (Lanes 2, 4 and 6) having fewer cells/lane (1.3 x 10⁷ cells). The activity of the PNGaseF and O-Glycosidase enzymes was confirmed in parallel control experiments in which the enzymes removed N-and O-linked sugars, respectively, following incubation with HIV-I gpl20.

EXAMPLE 8

PA30 binds primary human hepatocytes

[0189J Four vials of frozen primary human hepatocytes, obtained from a single female donor (lot# IJJ), a single male donor (lot# UOE), or from 10 pooled donors (lot# FRG and GCA), were purchased from Celsis International, PLC. Each vial contained approximately 5x10⁶ cells. On the day that the cells were used in an experiment, each vial was removed from storage at -15O⁰C and thawed at 37⁰C for about 2 minutes. The contents of each vial were then transferred to a 15ml tube and 4ml of 4°C Thawing Solution A (1 : 1 TCM and heat-inactivated FBS) were added drop-wise, followed by the drop-wise addition of 5ml of 4⁰C TCM (1 :1 RPMI 1640:Alpha- Medium supplemented with 100ml heat-inactivated FBS, 10ml each IM HEPES buffer, MEM non-essential amino acids solution, GlutaMAX™ Supplement, sodium pyruvate, Peniciilin/Streptomycin, and ImI (55mM) 2-mercaptoethanol), Each tube was then centrifuged for Sminutes at about 200χg at 4°C. The supernatant in each tube was decanted and cells were gently mixed by tapping each tube to resuspend the remaining contents. 10ml of Thawing Solution B (3: 1 TCM and Thawing Solution A) were slowly added, and each tube was centrifuged again. Following removal of the supernatant, cells were resuspended in TCM for counting via Vi-CeIl XR machine (Beckman-Coulter),

[0190] Hep3b cells were rinsed with PBS and released from the flask using Cell Dissociation Solution (Sigma). Cells were resuspended into medium for counting via Vi-CeIl XR machine. 1 x 10⁶ viable hepatocytes from each lot or 1 x 10⁶ Hep3b cells were washed once in 2ml of Staining Buffer [0.25% BSA, 0.1% sodium azide in PBS]. Cells were centrifuged for 5minutes and, following removal of the supernatant, were incubated with 10% heat-inactivated goat serum in Staining Buffer for 15min at 4°C. Cells received either lμg of mlgGl MAb (isotype control; BD Biosciences lot# 44883), anti-CD81 (clone JS-81; BD Biosciences lot# 67835), or PA30 (lot 5/15/07 VP in 0.1M sodium acetate pH 5.0) in Staining Buffer. Cells were incubated for 40 minutes at 4⁰C in a volume of about lOOμl. Cells were washed once with 2ml of Staining Buffer. Secondary antibody (goat anti-mouse IgG (H+L)-Alexa Fluor 488; Invitrogen lot# 51163A) was then added at lμl/sample and were incubated for 15 minutes at 4°C. Cells were washed again in Staining Buffer and analyzed on a FACSCalibur instrument. The results of the studies showed that the PA30 monoclonal antibody bound to both individual and pooled primary human hepatocytes with minimal donor to donor variation. (See, FIGS. 10A- 10E).

EXAMPLE 9

HCV therapy and prophylaxis using monoclonal antibodies of the invention in an in vivo animal model

[0191] The monoclonal antibodies according to the present invention, e.g., the PA-30 monoclonal antibody, are employed as therapeutic and prophylactic agents for treating animals as demonstrated via a mouse model of in vivo HCV infection and treatment. In this Example, the in vivo model of HCV infection uses SCID mice carrying a plasminogen activator transgene under control of the albumin promoter (Alb-uPA), (Kneteman, N.M. et al., 2003; Mercer, D. F. et al., 2001; Kneteman, N.M. et al., 2005; Meuleman, P. et al., 2005). SCID mice are homozygous for a mutation that impairs the recombination of gene segments (V, D and J) that code for the variable (antigen-binding) regions of antigen receptors (Ig molecules) in lymphocytes. Such mice lack mature, functional lymphocytes from both the T and B cell lineages. The transgene directs overproduction of urokinase in the liver resulting in accelerated death of hepatocytes. Engraftment of human liver cells into these mice rescues the animals from liver failure.

[0192] The integrity of human liver tissue grafts is monitored by assessing human alpha- 1 antitrypsin (hAAT). The human liver graft can be infected with HCV in vivo. SCID/Alb-uPA mice engrafted with human liver tissue are infected by inoculation of HCV positive human serum. Following the establishment of infection, viral load in the animals ranges from 10⁴-10' RNA copies/ml (based on Amplicor test, Roche) and infection can be maintained in these animals for up to 4 months. In this system, the animals are treated with a candidate molecule (e.g., an HCV inhibitor, such as an HCV inhibitory monoclonal antibody) before and/or after exposure to HCV in order to examine the prophylactic and therapeutic effectiveness of the inhibitor.

[0193] In a treatment study according to this invention, the liver engrafted SClD animals are infected with HCV-positive human serum and then plasma HCV viral load is determined. Animals with viral loads of 10⁴- 10⁷ RNA copies/ml are randomized by HCV RNA and injected intraperitoneally in groups of 3-6 with the PA-30 monoclonal antibody, with an isotype-matched control monoclonal antibody (JS-81), or with vehicle (PBS) control weekly for 4 weeks. Typical dose levels are 0.25 mg and 1.0 mg per dose. Blood samples are collected from the mice prior to administration of test antibody, control antibody, or vehicle and then weekly (w) thereafter, (i.e., -Iw, Ow, Iw, 2 w, 3 w, 4w, 5w, 6w, 7w, 8w) and are analyzed for the presence and/or levels of HCV viral RNA (viral load, VL) and hAAT.

[0194J To assess prophylactic or preventative effects of the compounds, animals are injected with test antibody (e.g., monoclonal and/or humanized) or vehicle at a predetermined time prior to administration of the HCV-positive human serum, for example, at 7 days, 5 days, 3 days, and/or 2 days prior to such administration. HCV viral load is monitored periodically thereafter to determine a prophylactic or preventative effect of the test antibody on the reduction of viral load in the HCV infected animals.

[0195] Viral load (VL) data are analyzed for individual mice. Changes in VL are assessed by the log 10 change in HCV RNA from baseline, as a function of time post-treatment and of the number of animals that achieve undetectable levels of HCV RNA. Cohort means and medians are determined and compared using parametric (e.g., t-tests) and non-parametric (e.g., rank-sum) methods.

[0196] The contents of all patent applications and issued patents mentioned, referred to, or described herein are incorporated by reference herein in their entireties. The contents of all references and publications mentioned, referred to, or described herein are incorporated by reference herein in their entireties. REFERENCES

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Claims

WHAT IS CLAIMED IS:

1. A monoclonal antibody or a portion thereof which (i) inhibits entry of Hepatitis C Virus (HCV) into cells susceptible to infection by HCV and (ii) binds human B lymphocytes, human liver cells, and mitogen-stimulated CD4+ and CD8+ T lymphocytes.

2. The monoclonal antibody or portion thereof according to claim 1 , wherein the human B lymphocytes are selected from the group consisting of peripheral blood mononuclear cells having a CD4TD8XD19⁺CD20⁺ phenotype, Ramos cells and Daudi cells.

3. The monoclonal antibody or portion thereof according to claim 1 , wherein the liver cells are Hep3b cells, HepG2 cells, or primary human hepatocytes.

4. The monoclonal antibody or portion thereof according to claim 1, wherein the mitogen- stimulated T lymphocytes are CD4+ T lymphocytes.

5. The monoclonal antibody or portion thereof according to claim 1, wherein the mitogen- stimulated T lymphocytes are CD8+ T lymphocytes.

6. The monoclonal antibody or portion thereof according to claim 1, wherein HCV is of genotype 1, genotype 2, or a combination thereof.

7. The monoclonal antibody or portion thereof according to claim 1 , wherein HCV is of one or more genotypes selected from the group consisting of Ia, Ib, 2b and la/2b.

8. The monoclonal antibody or portion thereof according to claim 7, wherein the monoclonal antibody or portion thereof inhibits entry of genotype Ia HCV into cells.

9. The monoclonal antibody or portion thereof according to claim 7, wherein the monoclonal antibody or portion thereof inhibits entry of genotype Ib HCV into cells.

10. The monoclonal antibody or portion thereof according to claim 7, wherein the monoclonal antibody or portion thereof inhibits entry of genotype 2b HCV into cells.

1 1. The monoclonal antibody or portion thereof according to claim 7, wherein the monoclonal antibody or portion thereof inhibits entry of genotype la^;2b HCV into cells.

12. The monoclonal antibody or portion thereof according to claim 1 , wherein the monoclonal antibody or portion thereof inhibits entry of HCV into cells susceptible to HCV infection as determined by a median IC50 value of 10 μg/mL or less.

13. The monoclonal antibody or portion thereof according to claim 1, wherein the monoclonal antibody or portion thereof inhibits entry of HCV into cells susceptible to HCV infection as determined by a median IC50 value of 5 μg/mL or less.

14. The monoclonal antibody or portion thereof according to claim 1, wherein the monoclonal antibody or portion thereof blocks or prevents HCV entry into a cell susceptible to infection by HCV.

15. Monoclonal antibody PA-30 produced by a hybridoma cell line designated PA-30 (ATCC Accession No. PTA-7278), or a portion of monoclonal antibody PA-30, which binds human B lymphocytes, human liver cells, and CD4+ and CD8+ T lymphocytes following stimulation with mitogen.

16. The monoclonal antibody PA-30 or the portion of monoclonal antibody PA-30 according to claim 15, which binds a ligand of approximately 45-65 kDa expressed by the human B lymphocytes.

17. The monoclonal antibody PA-30 or the portion of monoclonal antibody PA-30 according to claim 15, which binds a ligand of approximately 60 kDa expressed by the human B lymphocytes.

18. The PA-30 monoclonal antibody or portion thereof according to claim 15, wherein the monoclonal antibody or portion thereof inhibits one or more of HCV entry into a cell susceptible to infection by HCV or HCV infection of a cell susceptible to infection by HCV.

19. The PA-30 monoclonal antibody or portion thereof according to claim 18, wherein the monoclonal antibody or portion thereof inhibits entry or infection by HCV of genotype 1, genotype 2, or a combination thereof.

20. The PA-30 monoclonal antibody or portion thereof according to claim 18, wherein the monoclonal antibody or portion thereof inhibits entry or infection by HCV of one or more genotypes selected from the group consisting of I a, Ib, 2b, la/2b and a combination thereof.

21. The PA-30 monoclonal antibody or portion thereof according to claim 20, wherein the monoclonal antibody or portion thereof inhibits entry or infection by HCV of genotype I a.

22. The PA-30 monoclonal antibody or portion thereof according to claim 20, wherein the monoclonal antibody or portion thereof inhibits entry or infection by HCV of genotype Ib.

23. The PA-30 monoclonal antibody or portion thereof according to claim 20, wherein the monoclonal antibody or portion thereof inhibits entry or infection by HCV of genotype 2b.

24. The PA-30 monoclonal antibody or portion thereof according to claim 20, wherein the monoclonal antibody or portion thereof inhibits entry or infection by HCV of genotype la/2b.

25. The PA-30 monoclonal antibody or portion thereof according to claim 18, wherein the monoclonal antibody or portion thereof inhibits entry of HCV into cells susceptible to HCV infection as determined by a median IC50 value of 10 μg/mL or less.

26. The PA-30 monoclonal antibody or portion thereof according to claim 18, wherein the monoclonal antibody or portion thereof inhibits entry of HCV into cells susceptible to HCV infection as determined by a median TC50 value of 5 μg/mL or less.

27. A hybridoma cell line designated PA-30 (ATCC Accession No. PTA-7278) which produces a monoclonal antibody designated PA-30.

28. A cell which expresses a monoclonal antibody designated PA-30 (ATCC Accession No.

PTA-7278).

29. Monoclonal antibody PA-30 produced by a hybridoma cell line designated PA-30 (ATCC Accession No. PTA-7278), or a portion of the PA-30 monoclonal antibody.

30. An antibody or portion thereof that binds the same B-cell expressed ligand or epitope thereof as is bound by monoclonal antibody PA-30 (ATCC Accession No. PTA-7278), or a portion thereof.

31. The antibody or portion thereof according to claim 30, wherein the antibody is a monoclonal antibody.

32. A monoclonal antibody or portion thereof that binds the same B-cell expressed ligand or epitope thereof as is bound by the monoclonal antibody produced by the hybridoma cell line according to claim 28.

33. The monoclonal antibody or portion thereof according to claim 32, wherein the B-cell expressed ligand has a molecular weight of approximately 45 to 65 kDa.

34. The monoclonal antibody or portion thereof according to claim 33, wherein the B-cell expressed ligand has a molecular weight of approximately 60 kDa.

35. An antibody or binding portion thereof which competes with monoclonal antibody PA-30 (ATCC Accession No. PTA-7278) for binding one or more of human B lymphocytes, human liver cells, and mitogen-stimulated CD4+ and CD8+ T lymphocytes.

36. The antibody or binding portion thereof according to claim 35, which antibody or binding portion thereof competes with monoclonal antibody PA-30 (ATCC Accession No. PTA- 7278) or a portion thereof for binding an approximately 60 kDa protein expressed by human B lymphocytes.

37. The antibody or portion thereof according to claim 35, wherein the antibody is a monoclonal antibody.

38. The monoclonal antibody or portion thereof according to any one of claims 1, 15, 29, or 37, wherein the monoclonal antibody or portion thereof is humanized.

39. The monoclonal antibody or portion thereof according to claim 38, comprising complementarity determining regions (CDRs), or portions thereof, derived from the PA- 30 monoclonal antibody.

40. The monoclonal antibody or portion thereof according to claim 39, comprising one or more framework regions, or portions thereof, derived from a human immunoglobulin molecule.

41. The monoclonal antibody or portion thereof according to claim 40, wherein the human immunoglobulin molecule is selected from the group consisting of IgGl, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgA, IgM, IgD and IgE.

42. The monoclonal antibody or portion thereof according to any one of claims 1, 15, 29, or 37, wherein the monoclonal antibody or portion thereof is a chimeric antibody.

43. The monoclonal antibody or portion thereof according to claim 42, wherein the constant region of the chimeric antibody is derived from a human immunoglobulin molecule.

44. The monoclonal antibody or portion thereof according to claim 43, wherein the human immunoglobulin molecule is selected from the group consisting of IgGl, IgG2, IgG2a, IgG2b, IgG3, ϊgG4, IgA, IgM, IgD and IgE.

45. A method of inhibiting HCV infection of a cell susceptible to HCV infection, comprising: contacting the cell with the monoclonal antibody or portion thereof according to any one of claims 1, 15, 29, or 37 in an amount and under conditions to inhibit HCV infection of the susceptible cell.

46. The method according to claim 45, wherein the monoclonal antibody or portion thereof is humanized or chimeric.

47. A method for treating HCV infection, comprising administering to an individual in need thereof the monoclonal antibody or portion thereof according to any one of claims 1, 15, 29, or 37 in an amount effective to inhibit HCV infection of susceptible cells so as to thereby treat the infection.

48. The method according to claim 47, wherein the monoclonal antibody or portion thereof is humanized or chimeric.

49. A method of reducing the occurrence of HCV infection in a population of individuals, comprising administering to the population of individuals in need thereof the monoclonal antibody or portion thereof according to any one of claims 1, 15, 29, or 37 in an amount effective to reduce the occurrence of HCV infection in the population.

50. The method according to claim 49, wherein the monoclonal antibody or portion thereof is humanized or chimeric.

51. A composition which comprises the monoclonal antibody or a portion thereof according to any one of claims 1, 15, 29, or 37 and a carrier, excipient, or diluent.

52. The composition according to claim 51 , wherein the monoclonal antibody or portion thereof is humanized or chimeric.

53. A composition which comprises a monoclonal antibody designated as PA-30 (ATCC Accession No. PTA- 7278), or a portion thereof, and a carrier, excipient, or diluent.

54. The composition according to claim 51 , wherein the monoclonal antibody is labeled with a detectable marker.

55. The composition according to claim 53, wherein the monoclonal antibody is labeled with a detectable marker.

56. The composition according to claim 54 wherein the detectable marker is a radioactive marker, a chemiluminescent marker, a luminescent, a calorimetric. or a fluorescent marker.

57. The composition according to claim 55 wherein the detectable marker is a radioactive marker, a chemiluminescent marker, a luminescent, a calorimetric, or a fluorescent marker.

58. The composition according to claim 51, wherein the composition further comprises at least one additive selected from the group consisting of antimicrobials, antioxidants, chelating agents and inert gases.

59. The composition according to claim 53, wherein the composition further comprises at least one additive selected from the group consisting of antimicrobials, antioxidants, chelating agents and inert gases.

60. A pharmaceutical composition comprising a therapeutically effective amount of the monoclonal antibody according to any one of claims 1, 15, 29, or 37 in combination with at least one additional antiviral active ingredient selected from the group consisting of interferons, anti-HCV monoclonal antibodies, anti-HCV polyclonal antibodies, HCV RNA polymerase inhibitors, HCV protease inhibitors, IRES inhibitors, helicase inhibitors, antisense compounds, anti-viral small molecules and ribozymes.

61. A pharmaceutical composition comprising a therapeutically effective amount of the antibody according to claim 38 in combination with at least one additional anti-viral active ingredient selected from the group consisting of interferons, anti-HCV monoclonal antibodies, anti-HCV polyclonal antibodies, RNA polymerase inhibitors, protease inhibitors, IRES inhibitors, helicase inhibitors, antisense compounds, anti-viral small molecules and ribozymes.

62. The composition according to claim 60, wherein the at least one antiviral active ingredient is selected from ribavirin, interferon-α, interferon-α-2β, or a combination thereof.

63. The composition according to claim 61 , wherein the at least one antiviral active ingredient is selected from ribavirin, interferon-α, interferon-α-2β, or a combination thereof.

64. A method of inhibiting HCV infection of a cell susceptible to HCV infection, comprising contacting the cell with the monoclonal antibody or portion thereof according to any one of claims 1, 15, 29, or 37 in an amount and under conditions to inhibit HCV virion entry into the HCV susceptible cell.

65. The method according to claim 64, wherein the monoclonal antibody or portion thereof is humanized or chimeric.

66. The method according to claim 65, wherein the cell is present in a subject and the contacting is effected by administering the monoclonal antibody or portion thereof in an amount effective to inhibit HCV infection in the subject.

67. The method according to claim 66, wherein the cell is a liver cell, a B lymphocyte, or a mitogen-stimulated T lymphocyte.

68. The method according to claim 66, wherein the monoclonal antibody or portion thereof is administered orally, intravenously, subcutaneously, intramuscularly, topically or by liposome-mediated delivery.

69. The method according to claim 66, wherein the effective amount of the monoclonal antibody or portion thereof is between 1 mg and 50 mg per kg body weight of the subject.

70. The method according to claim 69, wherein the effective amount of the monoclonal antibody or portion thereof is between 2 mg and 40 mg per kg body weight of the subject.

71. The method according to claim 70, wherein the effective amount of the monoclonal antibody or portion thereof is between 3 mg and 30 mg per kg body weight of the subject.

72. The method according to claim 71, wherein the effective amount of the monoclonal antibody or portion thereof is between 4 mg and 20 mg per kg body weight of the subject.

73. The method according to claim 72, wherein the effective amount of the monoclonal antibody or portion thereof is between 5 mg and 10 mg per kg body weight of the subject.

74. The method according to claim 66, wherein the monoclonal antibody or portion thereof is administered at least once per day.

75. The method according to claim 66, wherein the monoclonal antibody or portion thereof is administered daily.

76. The method according to claim 66, wherein the monoclonal antibody or portion thereof is administered every other day.

77. The method according to claim 66, wherein the monoclonal antibody or portion thereof is administered every 6 to 8 days.

78. The method according to claim 77, wherein the monoclonal antibody or portion thereof is administered weekly.

79. A method of treating a liver disease in a subject, which comprises administering to the subject a monoclonal antibody or a portion thereof, wherein the monoclonal antibody or portion thereof (i) binds human B lymphocytes, human liver cells, and CD4 t~ and CD8+ T lymphocytes following stimulation with mitogen; and (ii) inhibits infection of susceptible cells by HCV of genotype 1 , 2, or a combination thereof, in an amount effective to inhibit infection of the subject's HCV susceptible cells, thereby treating the liver disease in the subject.

80. The method according to claim 79, wherein the monoclonal antibody is PA-30 (ATCC Accession No. PTA-7278), or a portion thereof.

81. The method according to claim 79, wherein the monoclonal antibody or portion thereof binds the same ligand or epitope thereof as is bound by the PA-30 monoclonal antibody.

82. The method according to claim 79 or claim 80, wherein the monoclonal antibody or portion thereof is humanized or chimeric.

83. A method of treating an HCV associated disorder in a subject, which comprises administering to the subject a monoclonal antibody or a portion thereof, wherein the monoclonal antibody or portion thereof (i) binds human B lymphocytes, human liver cells, and CD4+ and CD8+ T lymphocytes following stimulation with mitogen; and (ii) inhibits infection of susceptible cells by HCV of genotype 1, 2, or a combination thereof, in an amount effective to inhibit infection of the subject's HCV susceptible cells, thereby treating the liver disease in the subject.

84. The method according to claim 83, wherein the monoclonal antibody is PA-30 (ATCC Accession No. PTA-7278), or a portion thereof.

85. The method according to claim 83, wherein the monoclonal antibody or portion thereof binds the same ligand or epitope thereof as is bound by the PA-30 monoclonal antibody.

86. The method according to claim 83 or claim 84, wherein the monoclonal antibody or portion thereof is humanized or chimeric.

87. A method of preventing HCV infection in a subject, wherein prevention is effected by inhibiting HCV entry into a target cell to which HCV fuses, which comprises administering to the subject a monoclonal antibody or portion thereof according to any one of claims 1, 15, 29, or 37 in an amount effective to inhibit HCV entry into the target cell, thereby preventing HCV infection.

88. The method according to claim 87, wherein the monoclonal antibody or portion thereof is PA-30 (ATCC Accession No. PTA-7278).

89. The method according to claim 87, wherein the monoclonal antibody or portion thereof binds the same ligand or epitope thereof as is bound by the PA-30 monoclonal antibody.

90. The method according to claim 87 or claim 88, wherein the monoclonal antibody or portion thereof is humanized or chimeric.

91. A method of preventing or treating HCV infection in a subject, wherein prevention or treatment is effected by inhibiting HCV entry into a target cell to which HCV fuses, which comprises administering to the subject an antibody or portion thereof according to claim 38 in an amount effective to inhibit HCV entry into the target cell, thereby preventing HCV infection.

92. The method according to claim 87, wherein the target cell is a liver cell or hepatocyte.

93. The method according to claim 91, wherein the target cell is a liver cell or hepatocyte.

94. A method of reducing, preventing, or reversing inhibition of activity of HCV-specific B and T lymphocytes resulting from HCV infection of a subject, which comprises: administering to the subject a monoclonal antibody or a portion thereof, wherein the monoclonal antibody or portion thereof (i) binds human B lymphocytes, human liver cells, and mitogen-stimulated CD4+ and CD8+ T lymphocytes; and (ii) inhibits infection of cells susceptible to infection by HCV of genotype 1, 2, or a combination thereof, in an amount effective to reduce, prevent, or reverse the inhibition of activity of HCV-specific B and T lymphocytes.

95. The method according to claim 94, wherein the monoclonal antibody or portion thereof is PA-30 (ATCC Accession No. PTA-7278).

96. The method according to claim 94, wherein the monoclonal antibody or portion thereof binds the same ligand or epitope thereof as is bound by the PA-30 monoclonal antibody.

97. The method according to claim 94 or claim 95, wherein the monoclonal antibody or portion thereof is humanized or chimeric.

98. An isolated protein having a molecular weight of approximately 45-65 kDa expressed by B cells, stimulated T cells, and liver cells, and being bindable by monoclonal antibody PA-30 (ATCC Accession No. PTA-7278) or by an antibody or a portion thereof which competes with PA-30 (ATCC Accession No. PTA-7278) for binding the approximately 45-65 kDa protein.

99. An isolated protein having a molecular weight of approximately 60 kDa expressed by B cells, stimulated T cells and liver cells, and being bindable by monoclonal antibody PA- 30 (ATCC Accession No. PTA-7278) or by an antibody or a portion thereof which competes with PA-30 (ATCC Accession No. PTA-7278) for binding the approximately

60 kDa protein.

100. A method of treating a subject infected with HCV. comprising administering to the subject the isolated protein according to claim 98 or claim 99. or a soluble peptide thereof, in an amount effective to treat the subject.

101. A monoclonal antibody or portion thereof which (i) inhibits entry of Hepatitis C Virus (HCV) into cells susceptible to infection by HCV; (ii) binds a ligand of approximately 45-65 kDa expressed by human B cells; and (iii) binds human liver cells.

102. The monoclonal antibody or portion thereof according to claim 101, wherein the B cell- expressed ligand is approximately 60 kDa.

103. The monoclonal antibody or portion thereof according to claim 101 or claim 102, wherein the ligand bound by the monoclonal antibody is expressed by human B cells selected from the group consisting of peripheral blood mononuclear cells having a CD4^~ CD8 CD39⁺CD2O⁺ phenotype, Ramos cells and Daudi cells.

104. The monoclonal antibody or portion thereof according to claim 101 or claim 102, wherein the liver cells are Hep3b cells, HepG2 cells, or primary human hepatocytes.

105. The monoclonal antibody or portion thereof according to claim 101 or claim 102, further wherein the ligand bound by the monoclonal antibody or portion thereof is expressed by CD4+ and CD8+ T lymphocytes following stimulation with mitogen.

106. The monoclonal antibody or portion thereof according to claim 105, wherein the mitogen-stimulated T lymphocytes are CD4+ T lymphocytes.

107. The monoclonal antibody or portion thereof according to claim 101 or claim 102, wherein the monoclonal antibody or portion thereof neutralizes HCV of genotype 1, genotype 2, or a combination thereof.

108. The monoclonal antibody or portion thereof according to claim 101 or claim 102, wherein the monoclonal antibody or portion thereof neutralizes HCV of one or more genotypes selected from the group consisting of Ia, Ib, 2b and la/2b.

109. The monoclonal antibody or portion thereof according to claim 101 or claim 102, wherein the monoclonal antibody or portion thereof inhibits entry of HCV into cells susceptible to HCV infection as determined by a median TC50 value of 10 μg/mL or less.

110. The monoclonal antibody or portion thereof according to claim 109, wherein the monoclonal antibody or portion thereof inhibits entry of HCV into cells susceptible to HCV infection as determined by a median IC50 value of 5 μg/mL or less.

111. The monoclonal antibody or portion thereof according to claim 101 or claim 102, wherein the monoclonal antibody or portion thereof inhibits HCV infection of a cell susceptible to infection by HCV.

112. A method of preventing or treating a liver disease in a subject, which comprises administering to the subject a monoclonal antibody or a portion thereof, wherein the monoclonal antibody or portion thereof (i) binds a ligand of approximately 60 kDa expressed by B cells, mitogen-stimulated T cells and liver cells; and (ii) inhibits infection of susceptible cells by HCV of genotype 1, 2, or a combination thereof, in an amount effective to inhibit infection of the subject's HCV susceptible cells, thereby treating the liver disease in the subject.

113. The method according to claim 112, wherein the monoclonal antibody is PA-30 (ATCC Accession No. PTA-7278), or a portion thereof.

114. The method according to claim 112, wherein the monoclonal antibody or portion thereof binds the same ligand or epitope thereof as is bound by the PA-30 monoclonal antibody.

115. The method according to claim 112 or claim 113, wherein the monoclonal antibody or portion thereof is humanized or chimeric.

116. A method of treating an HCV associated disorder in a subject, which comprises administering to the subject a monoclonal antibody or a portion thereof, wherein the monoclonal antibody or portion thereof (i) binds a ligand of approximately 60 kDa expressed by B cells, mitogen-stimulated T cells and liver cells; and (ii) inhibits infection of susceptible cells by HCV of genotype 1, 2, or a combination thereof, in an amount effective to treat the HCV associated disorder in the subject.

117. The method according to claim 116, wherein the monoclonal antibody is PA-30 (ATCC Accession No. PTA-7278), or a portion thereof.

118. The method according to claim 116, wherein the monoclonal antibody or portion thereof binds the same ligand or epitope thereof as is bound by the PA-30 monoclonal antibody.

1 19. The method according to claim 116 or claim 117, wherein the monoclonal antibody or portion thereof is humanized or chimeric.

120. A method of reducing, preventing, or reversing inhibition of activity of HCV-specific B and T lymphocytes resulting from HCV infection of a subject, which comprises: administering to the subject a monoclonal antibody or a portion thereof, wherein the monoclonal antibody or portion thereof (i) binds a ligand of approximately 45-65 kDa expressed by B cells, stimulated T cells and liver cells and (ii) inhibits infection of cells susceptible to infection by HCV of genotype 1, 2, or a combination thereof, in an amount effective to reduce, prevent, or reverse the inhibition of activity of HCV-specific B and T lymphocytes.

121. The method according to claim 120, wherein the monoclonal antibody is PA-30 (ATCC Accession No. PTA-7278), or a portion thereof.

122. The method according to claim 120, wherein the monoclonal antibody or portion thereof binds the same ligand or epitope thereof as is bound by the PA-30 monoclonal antibody, or portion thereof.

123. The method according to claim 120 or claim 121, wherein the monoclonal antibody or portion thereof is humanized or chimeric.

124. The method according to claim 120, wherein the stimulated T cells are CD4+ T cells or CD8+ T cells.

125. The method according to claim 120 or claim 124, wherein the T cells are mitogen stimulated T cells.

126. The method according to any one of claims 112, 116, or 120, wherein HCV is of a genotype selected from the group consisting of Ia, Ib, 2a, 2b, la/2b and a combination thereof.

127. The method according to claim 120, wherein the ligand is approximately 60 kDa.

128. A method of producing a monoclonal antibody which binds cells susceptible to HCV infection and inhibits entry of HCV into the HCV susceptible cells, comprising:

(a) immunizing a subject with cells which (i) express functional HCV receptor and/or co-receptor molecules; (ii) are permissive for HCV entry and infection; and (iii) are replication-competent for HCV;

(b) producing hybridoma cells comprising immune B cells from the immunized subject of (a); and

(c) assaying antibody generated by the hybridoma cells for the antibody's ability to bind HCV susceptible cells and inhibit entry of HCV into the HCV susceptible cells.

129. The method according Io claim 128, wherein the eel Js of step (a) are selected from Hep3b, Huh-7, Huh-7.5, or Huh-7.5.1.

130. The method according to claim 128, wherein the cells of step (a) are HepG2 cells transfected with CD81 -encoding nucleic acid, thereby resulting in cell surface CD81 expression.

131. The method according to claim 128, wherein the antibody of step (c) inhibits entry of HCV into a susceptible cell as measured by an IC50 value of lϋ μg/mL or less. The method according to claim 131, wherein the antibody of step (c) inhibits entry of HCV into a susceptible cell as measured by an IC50 value of 5 μg/mL or less.