WO2014033266A1

WO2014033266A1 - Anti-sr-bi antibodies for the inhibition of hepatitis c virus infection

Info

Publication number: WO2014033266A1
Application number: PCT/EP2013/068008
Authority: WO
Inventors: Thomas Baumert; Mirjam ZEISEL
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Université De Strasbourg
Priority date: 2012-08-31
Filing date: 2013-08-30
Publication date: 2014-03-06

Abstract

The present disclosure provides monoclonal antibodies that specifically bind to the N-terminal half of the extracellular domain of human SR-BI on the cell surface, thereby inhibiting HCV entry into susceptible cells during the post-binding steps and preventing HCV infection of these cells; and hybridoma cell lines which produce such monoclonal antibodies. Also provided are reagents that comprise such antibodies, combinations that comprise such antibodies, and pharmaceutical compositions comprising such antibodies. Methods of treating or preventing HCV infection by administration of an inventive monoclonal antibody, or a pharmaceutical composition thereof are also described.

Description

Anti-SR-BI Antibodies for the Inhibition of Hepatitis C Virus Infection

Related Patent Application

The present patent application claims priority to European Patent Application No. EP 12 306 037.8 filed on August 31, 2012. The European patent application is incorporated herein by reference in its entirety.

Background of the Invention

Hepatitis C virus (HCV) is a major global health problem, with an estimated 150-200 million people infected worldwide, including at least 5 million infected individuals within the European Union (Pawlotsky, Trends Microbiol., 2004, 12: 96- 102). According to the World Health Organization, 3 to 4 million new infections occur each year. The infection is often asymptomatic; however, the majority of HCV- infected individuals develop chronic infection (Hoofnagle, Hepatology, 2002, 36: S21-S2; Lauer et al, N. Engl. J. Med., 2001, 345: 41-52; Seeff, Semin. Gastrointest, 1995, 6: 20-27). Chronic HCV infection frequently results in serious liver disease, including fibrosis and steatosis (Chisari, Nature, 2005, 435: 930-932). About 20% of patients with chronic HCV infection develop liver cirrhosis, which progresses to hepatocellular carcinoma in 5% of the cases (Hoofnagle, Hepatology, 2002, 36: S21- S2).

Chronic HCV infection is the leading indication for liver transplantations (Seeff, Semin. Gastrointest., 1995, 6: 20-27). Unfortunately, liver transplantation is not a cure for hepatitis C; viral recurrence is an invariable problem and leading cause of graft loss (Brown, Nature, 2005, 436: 973-978). No vaccine protecting against HCV is yet available. Current therapies include administration of ribavirin and/or interferon-alpha (IFN-a), two non-specific anti-viral agents. Using a combination treatment of pegylated IFN-a and ribavirin, persistent clearance is achieved in about 50% of patients with chronic hepatitis C. However, a large number of patients have contraindications to one of the components of the combination, cannot tolerate the treatment, do not respond to IFN therapy at all or experience a relapse when administration is stopped. In addition to limited efficacy and substantial side effects such as neutropenia, haemolytic anemia and severe depression, current antiviral therapies are also characterized by high cost. Until recently, the development of more effective therapeutics to combat HCV infection has been hampered by the lack of a cell culture system supporting HCV replication. Robust production of infectious HCV in cell culture has now been achieved using a unique HCV genome derived from the blood of a Japanese patient with fulminant hepatitis C (JFH-1) Wakita et al, Nat. Med., 2005, 11: 791-796; Lindenbach et al, Science, 2005, 309: 623-626; Zhong et al., Proc. Natl. Acad. Sci. USA, 2005, 102: 9294-9299). The ability of the JFH-1 strain of HCV to release infectious particles in cell culture (HCVcc) and the development of retroviral HCV pseudoparticles (HCVpp) (Bartosch et al, J. Exp. Med., 2003, 197: 633-642; Hsu et al, Proc. Natl. Acad. Sci. USA, 2003, 100: 7271-727) have allowed studies on the mechanism of HCV entry and replication, that have led to the identification of potential therapeutic target biomolecules.

HCV is a positive strand RNA virus classified in the Hepacivitus genus, within the Flaviviridae family. Translation of the major open reading frame of the HCV genome results in the production of an approximately 3000 amino acid long polyprotein, which is cleaved co- and post-translationally by the coordinated action of cellular and viral proteases into at least 10 mature proteins, including two envelope glycoproteins (El and E2). HCV initiates infection by attaching to molecules or receptors on the surface of hepatocytes. Current evidence suggests that at least three host cell molecules are important for HCV entry in vitro: the tetraspanin CD81 (Bartosch et al, J. Exp. Med., 2003, 197: 633-642; Hsu et al, Proc. Natl. Acad. Sci. USA, 2003, 100: 7271-727; Pileri et al, Science, 1998, 282: 938-941), the scavenger receptor class B type I (SB-RI) (Bartosch et al, J. Exp. Med., 2003, 197: 633-642; Grove et al, J. Virol., 2007, 81: 3162-3169; Kapadia et al, J. Virol., 2007, 81: 374- 383; Scarselli et al, EMBO J., 2002, 21: 5017-5025), and Claudin-1 (CLDN1), an integral membrane protein and a component of tight-junction strands (Evans et al, Nature, 2007, 446: 801-805). HCV glycoproteins have been reported to interact directly with CD81 and SR-BI (Cocquerel et al, J: Gen. Virol., 2006, 87: 1075-1084). Mutagenesis and antibody-blocking studies with tagged versions of CLDN1 suggest that the first extracellular loop is involved in interactions with HCV (Evans et al, Nature, 2007, 446: 801-805). However, the exact role played by each of the receptors is unclear. Identification of these receptors or co-receptors for HCV has opened up new avenues for the development of therapeutic and prophylactic agents as drug candidates for the prevention and/or treatment of HCV infection. For example, monoclonal antibodies raised against native human SR-BI have been shown to inhibit HCV E2 binding to SR-BI and to efficiently block HCVcc infection of hepatoma cells in a dose-dependent manner (Catanese et ah, J. Virol., 2007, 81: 8063-8071; WO 2006/005465). European patent application No. EP 1 256 348 discloses substances, including antibodies, with antiviral effects that inhibit binding of HCV E2 and CD81. International patent application WO 2007/130646 describes in vitro and cell-based assays for identifying agents that interfere with HCV interactions with Claudin-1 thereby preventing HCV infection. The present Applicants have generated monoclonal antibodies that efficiently inhibit HCV infection by targeting host entry factor Claudin-1 (EP 08 305 597 and WO 2010/034812).

Since the development of novel therapeutic approaches against HCV remains a high-priority goal, these studies are encouraging as they demonstrate that agents that affect HCV entry into susceptible cells may constitute an effective and safe alternative to current HCV therapies.

Summary of the Invention

The present invention relates to targeted systems and strategies for the prevention and/or treatment of HCV infection and HCV-related diseases. In particular, the present invention is directed to monoclonal antibodies that inhibit HCV infection and viral spread by interfering with HCV entry in one or more step(s) occurring following binding of the HCV envelope to the host cell membrane. The monoclonal antibodies of the present invention recognize the extracellular domain of human SR-BI, and more specifically the N-terminal half of the extracellular domain of human SR-BI. These antibodies can be used in the prophylactic or therapeutic treatment of HCV infection (acute or chronic HCV infection) and HCV-related diseases or disorders {e.g., liver inflammation, cirrhosis, and hepatocellular carcinoma). Monoclonal antibodies such as those provided herein that interfere with HCV entry into cells during post-binding steps are particularly attractive as antiviral therapeutics. An inhibitor of HCV entry does not need to cross the plasma membrane or to be modified intracellularly. In addition, because viral entry is mediated by conserved structures of the viral and cellular membranes, antibody inhibitors of viral entry can be very potent and less susceptible to the development of viral resistance. In particular, the Applicants have shown that the monoclonal antibodies of the present invention are effective at inhibiting HCV variants that are resistant to direct-acting antivirals currently in clinical use.

More specifically, in one aspect, the present invention provides hybridoma cell lines which secrete monoclonal antibodies that specifically bind to the N-terminal half of the extracellular domain of human SR-BI. In particular, the present Applicants have deposited four of such hybridoma cell lines at the CNCM (Collection Nationale de Culture de Microorganismes, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France) on August 1, 2012. They were assigned Accession Numbers CNCM 1-4662, CNCM 1-4663, CNCM 1-4664 and CNCM 1-4665. The deposit was made pursuant to the provisions of the Budapest Treaty on the International recognition of the Deposit of Microorganism for the Purpose of Patent Procedure (Budapest Treaty).

In another aspect, the present invention provides a monoclonal antibody that is secreted by any one of the hybridoma cell lines deposited under Accession Numbers CNCM 1-4662, CNCM 1-4663, CNCM 1-4664 and CNCM 1-4665. The monoclonal antibody may or may not be isolated and/or purified from hybridoma cultures. In certain embodiments, the monoclonal antibody is an immunoglobulin of the rat IgG2 heavy (H) chain and kappa light (L) chain isotype. In other embodiments, the monoclonal antibody is an immunoglobulin of the mouse IgG2 heavy (H) chain and kappa light (L) chain isotype.

As demonstrated by the Applicants, monoclonal antibodies secreted by the deposited hybridoma cell lines specifically bind to the extracellular domain of human SR-BI and more specifically to the N-terminal half of the extracellular domain of human SR-BI. The Applicants have also shown that these monoclonal antibodies efficiently inhibit HCV infection in vitro by interfering with HCV entry during post- binding steps. The present invention also encompasses any biologically active fragment of the inventive monoclonal antibodies, i.e., any fragment or portion that retains the ability of the monoclonal antibody to interfere with HCV-host cells interactions during post-binding steps, and/or to specifically bind to the extracellular domain of human SR-BI, in particular the N-terminal half of this extracellular domain, and/or to inhibit or block HCV entry into HCV- susceptible cells, and/or to inhibit or block HCV viral spread, and/or to reduce or prevent HCV infection of susceptible cells. More generally, the present invention encompasses any molecule that comprises an inventive anti-SR-BI monoclonal antibody or a fragment thereof, including chimeric antibodies, humanized antibodies, de-immunized antibodies and antibody- derived molecules comprising at least one complementary determining region (CDR) from either a heavy chain or light chain variable region of an inventive anti-SR-BI monoclonal antibody as secreted by a hybridoma cell line, including molecules such as Fab fragments, F(ab')₂ fragments, Fd fragments, Fab fragments, Sc antibodies (single chain antibodies), diabodies, individual antibody light single chains, individual antibody heavy chains, chimeric fusions between antibody chains and other molecules, and antibody conjugates, such as antibodies conjugated to a diagnostic agent (detectable moiety) or therapeutic agent, so long as these antibody-related molecules retain at least one biologically relevant property of the inventive monoclonal antibody from which it is "derived". The biologically relevant property may be the ability to interfere with HCV-host cells interactions during post-binding steps, to specifically bind to the extracellular domain of human SR-BI, in particular to the N-terminal half thereof, to inhibit or block HCV entry into HCV-susceptible cells, to inhibit or block HCV viral spread, and/or to reduce or prevent HCV infection of susceptible cells.

The monoclonal antibodies and antibody-related molecules of the present invention can find application in a variety of prophylactic and therapeutic treatments. Accordingly, in another aspect, the inventive monoclonal and antibody-related molecules are provided for use in preventing HCV infection of a cell (e.g., a susceptible cell or a population of susceptible cells); preventing or treating HCV infection or a HCV-related disease in a subject; and preventing HCV recurrence in a liver transplantation patient. The HCV infection may be due to HCV of a genotype selected from the group consisting of genotype 1, genotype 2, genotype 3, genotype 4, genotype 5 and genotype 6, or more specifically of a subtype selected from the group consisting of subtype la, subtype lb, subtype 2a, subtype 2b, subtype 2c, subtype 3a, subtype 4a-f, subtype 5a, and subtype 6a. In other embodiments, the HCV infection is due to a HCV that is resistant to at least one direct-acting antiviral. The at least one direct-acting antiviral may be a protease inhibitor, such as boceprevir or telaprevir.

In a related aspect, the present invention provides a method of reducing the likelihood of a susceptible cell of becoming infected with HCV as a result of contact with HCV, which comprises contacting the susceptible cell with an effective amount of an inventive antibody or antibody-related molecule. Also provided is a method of reducing the likelihood of a subject' s susceptible cells of becoming infected with HCV as a result of contact with HCV, which comprises administering to the subject an effective amount of an inventive antibody or antibody-related molecule. The present invention also provides a method of treating or preventing HCV infection or a HCV-associated disease (e.g. , a liver disease or pathology) in a subject in need thereof which comprises administering to the subject an effective amount of an inventive antibody or antibody-related molecule. Also provided is a method of preventing HCV recurrence in a liver transplantation patient, which comprises administering to the patient an effective amount of an inventive antibody or antibody-related molecule. Administration of an inventive antibody or antibody-related molecule to a subject may be by any suitable route, including, for example, parenteral, aerosol, oral and topical routes. The inventive antibody or antibody-related molecule may be administered alone or in combination with a therapeutic agent, such as an anti- viral agent.

The inventive monoclonal antibodies and antibody-related molecules may be administered per se or as pharmaceutical compositions. Accordingly, in another aspect, the present invention provides for the use of an inventive monoclonal antibody or antibody-related molecule for the manufacture of medicaments, pharmaceutical compositions, or pharmaceutical kits for the treatment and/or prevention of HCV infection and HCV-associated diseases.

In a related aspect, the present invention provides a pharmaceutical composition comprising an effective amount of an inventive antibody or antibody-related molecule and at least one pharmaceutically acceptable carrier or excipient. In certain embodiments, the pharmaceutical composition is adapted for administration in combination with an additional therapeutic agent, such as an antiviral agent. In other embodiments, the pharmaceutical composition further comprises an additional therapeutic agent, such as an antiviral agent. Antiviral agents suitable for use in methods and pharmaceutical compositions of the present invention include, but are not limited to, interferons (e.g. , interferon-alpha, pegylated interferon-alpha), ribavirin, anti-HCV (monoclonal or polyclonal) antibodies, RNA polymerase inhibitors, protease inhibitors, IRES inhibitors, helicase inhibitors, antisense compounds, ribozymes, and any combination thereof.

In yet another aspect, the present invention provides a combination of at least one anti-SR-BI monoclonal antibody as described herein and:

at least one anti-HCV envelope antibody selected from anti-El antibodies, anti- E2 antibodies, and anti-HCV IgGs from individuals chronically or previously infected with HCV, or

at least one protein kinase inhibitor selected from erlotinib and dasatinib, or at least one direct acting antiviral selected from telaprevir, boceprevir, danoprevir, TMC-435, daclatasvir, mericitabine, and GS-7977; or

at least one interferon selected from IFNcc-2a and IFNcc-2b, or

at least one host-targeting agent such as alisporivir or an anti-CLDNl monoclonal antibody,

wherein the anti-SR-BI monoclonal antibody and anti-HCV envelope antibody, or the anti-SR-BI monoclonal antibody and protein kinase inhibitor, or the anti-SR-BI monoclonal antibody and direct acting antiviral, or the anti-SR-BI monoclonal antibody and host-targeting agent act in synergy to inhibit HCV infection.

In certain embodiments, the combination is used for the treatment of HCV infection or a HCV-related disease in a subject; or for the control of chronic HCV infection in a subject; or for the prevention of HCV re-infection and recurrence in a liver transplantation patient. HCV infection, chronic HCV infection and HCV reinfection may be due to HCV of any of the major genotypes or subtypes, as described above. In certain embodiments, HCV infection, chronic HCV infection and HCV reinfection are due to a HCV that is resistant to at least one direct-acting antiviral. The at least one direct-acting antiviral may be a protease inhibitor, such as boceprevir or telaprevir. In certain embodiments, the HCV infection, HCV-related disease or HCV reinfection is caused by a Hepatitis C virus that is resistant to a direct acting antiviral and/or that is transmitted by cell-cell transmission.

These and other objects, advantages and features of the present invention will become apparent to those of ordinary skill in the art having read the following detailed description of the preferred embodiments.

Brief Description of the Drawing

Figure 1. Binding of monoclonal anti-SR-BI antibodies to human hepatocytes and inhibition of HCV infection. (A) Huh7.5.1 cells and (B) primary human hepatocytes (PHH) were incubated with anti-SR-BI mAbs and antibody binding was assessed using flow cytometry. Results are expressed as net mean fluorescence intensity (AMFI) of a representative experiment. (C) Inhibition of HCVcc infection by anti-SR-BI mAbs. Huh7.5.1 cells were preincubated for 1 hour at 37°C with anti-SR-BI or control mAbs (100 μg/mL) before infection with HCVcc (Luc-Jcl) for 4 hours at 37 °C. HCV infection was assessed by lucif erase activity in lysates of infected Huh7.5.1 cells 72 hours post-infection. Results are expressed as means + SD % HCVcc infectivity in the absence of antibody of three independent experiments. (D) Dose-dependent inhibition of HCVcc infection by anti-SR-BI mAbs. Huh7.5.1 cells were preincubated for 1 hour at 37°C with anti-SR-BI or control mAbs at the indicated concentrations before infection with HCVcc (Luc-Jcl) for 4 hours at 37°C. HCV infection was assessed by luciferase activity in lysates of infected Huh7.5.1 cells 72 hours post-infection. Results are expressed as mean + SD % HCVcc infectivity in the absence of antibody of three independent experiments performed in triplicate. *, P<0.01. Figure 2. Monoclonal anti-SR-BI antibodies do not interfere with HCV binding to SR-BI but inhibit HCV entry at post-binding steps. (A) To assess the effect of anti-SR-BI mAbs on viral binding, Huh7.5.1 cells were pre-incubated with heparin (100 μg/mL), anti-SR-BI or control (CTRL) serum (1:50) or anti-SR-BI or control (CTRL IgG) mAbs (20 μg/mL) for 1 hour prior to incubation with HCVcc (Jcl) at 4°C in the presence of compounds. Non-bound HCVcc were removed by washing of cells with PBS and HCVcc binding was then quantified by RT-PCR of cell bound HCV RNA. Results are expressed as mean + SD of one representative experiment performed in quintuplicate. (B) Schematic drawing of the experimental setup. To discriminate between virus binding and post-binding events, HCVcc (Luc- Jcl) binding to Huh7.5.1 cells was performed in the presence or absence of anti-CD81 (5 μg/mL), anti-SR-BI (20 μg/mL) or control mAbs (20 μg/mL) or proteinase K (50 μg/mL) for 1 hour at 4°C, before cells were washed and incubated for 4 hours at 37°C with compounds added at different time-points during infection. Compounds were then removed and cells were cultured for an additional 48 hours. Dashed lines indicate the time intervals where compounds were present. (C) HCV entry kinetics. Time-course of HCVcc infection of Huh7.5.1 cells following addition of the indicated antibodies at different time-points during infection is shown. HCV infection was assessed by luciferase activity in lysates of infected Huh7.5.1 cells 48h post-infection. Results are expressed as mean % HCVcc infectivity in the absence of antibody of three independent experiments performed in triplicate. *, P<0.01 Figure 3. The SR-BI post-binding function is relevant for HCV cell-to-cell transmission and viral spread. (A) Quantification of HCV-infected target cells (Ti) after co-cultivation with HCV (Jcl) producer cells (Pi) during incubation with control or anti-SR-BI mAbs (10 μg/mL) in the presence of E2-neutralizing antibody AP33 (25 μg/mL) by flow cytometry. Data are expressed as % infected target cells and represent means + SD of three independent experiments. (B) Quantification of HCV cell-to-cell transmission in parental target cells compared to target cells overexpressing mouse (m) or human (h) SR-BI. Data are expressed as means + SD from three different experiments. (C-D) Long-term analysis of HCVcc (Luc-Jcl) infection in the presence or absence of control (10 μg/mL) or anti-SR-BI mAb (C) QQ-4G9-A6 or (D) NK-8H5-E3 at the indicated concentrations. Antibodies were added 48 hours after HCVcc infection and control medium or medium containing antibodies were replenished every 4 days. Luciferase activity was determined in cell lysates every 2 days. Data are expressed as LoglO RLU and represent means + SD of one representative out of three different experiments performed in duplicate. (E-F) Cell spread in the presence or absence of anti-SR-BI mAbs. Antibodies were added 48 hours after HCVcc (Jcl) infection and control medium or medium containing antibodies were replenished every 4 days. HCV-infected cells were visualized 7 days post-infection by immunofluorescence using (E) anti-NS5A or (F) anti-E2 (CBH23) antibodies. The percentage of infected cells was calculated as the number of infected cells relative to the total number of cells as assessed by DAPI staining of the nuclei. *, P<0.01

Figure 4. Monoclonal anti-SR-BI antibodies block HCV cell-to-cell transmission and spread. (A-B) Quantification of HCV-infected target cells (Ti) after co-cultivation with HCV producer cells (Pi) during incubation with (A) control or anti-SR-BI mAb QQ-4G9-A6 (10 μg/mL) or (B) control or anti-SR-BI mAb QQ- 2A10-A5 (10 μg/mL) in the presence of E2-neutralizing antibody AP33 (25 μg/mL) by flow cytometry. (C) Cell viability after long-term exposure to anti-SR-BI mAbs. Cell viability was assessed using MTT assay after incubation of Huh7.5.1 cells for 14 days in the presence or absence of control or anti-SR-BI mAbs at 1, 10, or 100 μg/mL. Control medium and medium containing antibodies were replenished every 4 days. Data are expressed as % cell viability relative to cells incubated in the absence of mAb and represent means + SD from one experiment. Figure 5. Anti-SR-BI mAbs do not interfere with HDL binding but partially inhibit lipid transfer. (A) HDL binding to BRL3-hSR-BI cells. BRL3- hSR-BI cells were incubated in the presence or absence of anti-SR-BI mAbs (20 μg/mL) or polyclonal serum (1:50) or respective controls, prior to Cy5-HDL binding for 1 hour at 4°C. Bound Cy5-HDL was quantified using flow cytometry. Results represent mean + SD of three different experiments performed in duplicate. (B) Lipid uptake by Huh7 cells. Huh7 cells were incubated with a mixture of anti-SR- BI mAbs (20 μg/mL) and H-CE-labeled HDL for 5 hours before incubation with unlabelled HDL for 30 minutes. Selective uptake was calculated from the known specific radioactivity of radiolabeled HDL-CE and is denoted in μg HDL-CE^g cell protein. Results represent mean + SD of three different experiments performed in sixtuplate. (C) Cholesterol efflux from Huh7 cells. Huh7 cells were first incubated with H-cholesterol for 48 hours and then with BSA (0.5%) for 24 hours. Subsequently, cells were first incubated with anti-SR-BI mAbs (20 μg/mL) for 1 hour and then with unlabeled HDL for 4 hours. Fractional cholesterol efflux was calculated as the amount of the label obtained in the medium divided by the total label in each well regained after lipid extraction from cells. Results represent mean + SD of three different experiments performed in triplicate. *, P<0.01 Figure 6. Genotype-independent inhibition of HCVpp and HCVcc infection by monoclonal anti-SR-BI antibodies. (A-E) Inhibition of entry into Huh7.5.1 cells of HCVpp bearing envelope glycoproteins from genotypes 1-4. Huh7.5.1 cells were pre-incubated with control (CTRL IgG) or anti-SR-BI mAbs (50 μg/mL) for 1 hour at 37 °C before infection with HCVpp bearing envelope glycoproteins of strains H77 (la), HCV-J (lb), JFH1 (2a), UKN3A1.28 (3a) or UKN4.21.16 (4) and VSV-Gpp. Means + SD from 3 experiments performed in triplicate are shown. HCVpp infection was analyzed by luciferase reporter gene expression. Results are expressed as % HCVpp entry and represent means + SD from 3 independent experiments performed in triplicate. *, P<0.01

Figure 7. Genotype-independent inhibition of HCVpp infection by monoclonal anti-SR-BI antibodies. Inhibition of entry into Huh7.5.1 cells of HCVpp bearing envelope glycoproteins from genotypes 5 and 6. Huh7.5.1 cells were pre-incubated with control or anti-SR-BI mAbs (50 μg/mL) for 1 hour at 37°C before infection with HCVpp bearing envelope glycoproteins of strains UKN5.14.4 (5) or UKN6.5.340 (6) and VSV-Gpp. HCVpp entry was analyzed by luciferase reporter gene expression. Results are expressed as % HCVpp entry and represent means + SD from 3 independent experiments performed in triplicate.

Figure 8. Synergy between anti-SR-BI and neutralizing antibodies in inhibiting HCV infection. Patient derived HCVpp P02VJ (A-C) or HCVcc (Luc- Jcl) (D) were pre-incubated with (A) anti-El or (B) anti-E2 mAbs or (C-D) purified heterologous anti-HCV IgG (1 or 10 μg/mL) obtained from an unrelated chronically infected subject or isotype control IgGs for 1 hour at 37°C and added to Huh7.5.1 cells pre-incubated with increasing concentrations of control or anti-SR-BI mAbs (NK-8H5-E3). HCVpp and HCVcc infection was analyzed by luciferase reporter gene expression. Results are expressed as mean % HCVpp entry or HCVcc infection from a representative experiment. (E) Effects of antibody combinations on HCVcc infection were evaluated using the method of Prichard and Shipman. The antiviral assay was performed as described above except that the compound dilutions were added in a checkerboard format. Combination studies for each pair of compounds were performed in triplicate. The theoretical additive effect is calculated from the dose-response curves of individual compounds by the equation Z=X+Y(1-X) where X and Y represent the inhibition produced by the individual compounds and Z represents the effect produced by the combination of compounds. The theoretical additive surface is subtracted from the actual experimental surface, resulting in a horizontal surface that equals the zero plane when the combination is additive. A surface that lies higher than 20% above the zero plane indicates a synergistic effect of the combination and a surface lower than 20% below the zero plane indicates antagonism.

Figure 9. Competition of monoclonal anti-SR-BI antibodies for cellular binding. Huh7.5.1 cells were incubated with 0.1 μg/mL of biotinylated anti-SR-BI mAb (A) QQ-4A3-A1, (B) QQ-2A10-A5, (C) QQ-4G9-A6 or (D) NK-8H5-E3, together with increasing concentrations of unlabeled control or anti-SR-BI mAb (QQ- 4A3-A1, QQ-2A10-A5, QQ-4G9-A6, NK-8H5-E3) as competitors. Following washing of cells with PBS, binding of labelled mAbs was determined by flow cytometry and is shown % binding relative to biotinylated mAb incubated in the absence of antibody.

Figure 10. Binding of monoclonal anti-SR-BI antibodies to human, mouse or chimeric mouse and human SR-BI. (A-B) Schematic representations of three human/murine SR-BI chimeras that were generated through PCR by swapping three SR-BI domains between amino-acid positions 38-215 (region 1), 216-398 (region 2) and 399-432 (region 3), respectively. While the HHH and MMM SR-BI constructs refer to the wild-type human (H) and murine (M) SR-BI molecules, respectively, the human/mouse SR-BI chimeras were denominated according to the origin of either SR- BI domain, e.g., HMM bears the region 1 from human SR-BI and the regions 2 and 3 from murine SR-BI.15 (C) BRL3A cells engineered to express human (HHH), mouse (MMM) or chimeric mouse and human (HMM, MHM, MMH) SR-BI were first incubated with monoclonal anti-SR-BI antibodies (20 μg/mL) for 1 hour at room temperature before bound antibodies were detected using PE-labelled secondary antibodies. Results are expressed as means + SD net mean fluorescence intensity (AMFI). (D) BRL3A cells engineered to express wild-type human SR-BI (SR-BI wt) or human SR-BI point mutants (Q420R, Q402R, E418R, and Q402R-E418R) were first incubated with monoclonal anti-SR-BI antibodies (20 μg/mL) for lh at RT before bound antibodies were detected using PE-labelled secondary antibodies. Results are expressed as means + SD net mean fluorescence intensity (AMFI). Figure 11. Western blot analysis of anti-SR-BI mAb binding to endogenous SR-BI expressed in Huh7.5.1 cells. Lysates of Huh7.5.1 cells were subjected to SDS-PAGE. Immunoblotting was performed using anti-SR-BI mAbs (5 μg/mL) and AP-labelled secondary antibodies. The presence or absence of SR-BI and actin and their respective molecular weight are indicated on the right.

Figure 12. Antiviral Activity of the anti-SR-BI mAb with IFN-a and DAAs.

Huh7.5.1 cells were pre-incubated for 1 hour with serial concentrations of (A) IFN- a2a or (B) IFN-a2b or (C) protease inhibitors telaprevir, boceprevir, TMC-435 or danoprevir, NS5A inhibitor daclatasvir or polymerase inhibitors mericitabine or GS- 7977and 0.01 μg/ml anti-SRBI mAb before incubation with HCVcc Luc-Jcl in the presence of both compounds. HCVcc infection was analyzed by lucif erase reporter gene expression. The CI for an IC₅₀ was calculated and is indicated in Table 2. Dotted lines at combination values of 0.9 and 1.1 indicate the boundaries of an additive interaction. Figure 13. Antiviral synergy of the anti-SR-BI mAb with the NSA5 inhibitor, daclatasvir. (A) Combination of daclatasvir and the anti-SR-BI mAb resulted in a high shift in the IC₅₀. Means + SEM from at least three independent experiments performed in triplicate are shown. (B) Synergy was confirmed using the method of Prichard and Shipman. One representative experiment is shown. Figure 14. Antiviral synergy of the anti-SR-BI mAb with the polymerase inhibitor, GS-7977. Combination of GS-7977 and the anti-SR-BI mAb decreased the IC₅₀ of GS-7977 up to 210 fold. Means + SEM from at least three independent experiments performed in triplicate are shown.

Figure 15. Antiviral synergy of the anti-SR-BI mAb with the cyclophilin inhibitor, alisporivir, and the protein kinase inhibitors, erlotinib and dastinib.

(A) Huh7.5.1 cells were pre-incubated with serial concentrations of alisporivir and 0.01 μg/ml of anti-CD81, anti-SRBI or anti-CLDNl mAbs or 0.1 μΜ erlotinib or dasatinib before incubation with HCVcc Luc-Jcl in the presence of both compounds.

(B) Huh7.5.1 cells were pre-incubated with serial concentrations of anti-CD81, anti- SR-BI or anti-CLDNl mAbs and 0.1 μΜ erlotinib or dasatinib before incubation with

HCVcc Luc-Jcl in the presence of both compounds. Means + SEM from at least three independent experiments performed in triplicate are shown. Figure 16. Functional characterization of protease inhibitor-resistant viruses in HCV infection and their sensitivity to HTEIs. Huh7.5.1 cells were pre- incubated for 1 hour with serial concentrations of (A) telaprevir, (B) boceprevir, or (C) SR-BI- specific mAb or respective control reagents before incubation with HCVcc-Jcl-Luc containing the DAA-resistant mutations NS3 R155K in the presence of each compound. HCV infection was analysed 72 hours post-infection by luciferase reporter gene expression in cell lysates. Means + SEM from at least three independent experiments performed in triplicate are shown.

Figure 17. Functional characterization of NS5A inhibitor-resistant viruses in HCV infection and their sensitivity to HTEIs. Huh7.5.1 cells were pre-incubated for 1 hour with serial concentrations of (A) daclatasvir or (B) CLDN1- specific mAb or respective control reagents before incubation with HCVcc-J4 containing the NS5A inhibitor resistant mutations NS5A-Y93H (A and B) respectively in the presence of each compound. HCV infection was analyzed 72 hours post-infection by quantifying intracellular HCV RNA using RT-PCR. Means + SEM from at least three independent experiments performed in triplicate are shown.

Figure 18. (A) Treatment of chronic HCV infection using a SR-BI-specific mAb in vivo. Chimeric uPA-SCID mice were chronically infected with Jcl (genotype 2a/2a). Twenty-four to 50 days following inoculation and establishment of chronic infection, the animals received 500 μg control (white circles, dashed line; n=l) or SR- BI-specific QQ-2A10-A5 (black circles, full line; n=l) mAbs each week (arrows) for 4 weeks. The mice were followed-up until week 11 post- initiation of treatment as indicated. Serum viral load was quantified by the clinically licensed Abbott RealTime™ HCV assay. The horizontal dashed line indicates the threshold for HCV detection in mouse serum (LOQ: limit of quantification). The linear range of the assay is 12 IU/mL to 10⁸ IU/mL, the limit of quantification (LOQ) of HCV RNA is 12 IU/mL. Given a mouse serum dilution of 1/100 in PBS, LOQ is 1200 UI/mL, i.e. 5160 copies/mL. Viral load became undetectable in the SR-BI-specific mAb-treated mouse after the second antibody administration and remained undetectable for at least 5 weeks following the last antibody administration. (B) Absent detectable adverse effects of SR-BI-specific mAb on liver functions. Human albumin (huAlb) level in chronically Jcl-infected SR-BI-specific QQ-2A10-A5 (black circles, full line) or control mAb (white circles, dashed line) treated mice was quantified using ELISA (E80-129, Bethyl Laboratories). The species- specificity of the assay was confirmed by the absence of detectable albumin in serum samples of non-transplanted uPA-SCID mice. HuAlb levels did not change significantly during and after treatment period.

Figure 19. Synergy between anti-SR-BI and anti-CLDNl antibodies in inhibiting HCV infection. Huh7.5.1 cells were pre-incubated with increasing concentrations of anti-SR-BI mAbs: NK-8H5-E3 (A), QQ-4G9-A6 (B) or QQ-4A3- Al (C) and anti-CLDNl mAbs OM-7D3-B3, OM-8A9-A4 or OM-6E1-B5 in a checkerboard format and then infected with HCVcc (Luc-Jcl) for 4 hours at 37 °C in the presence of both compounds. HCVcc infection was analyzed by luciferase reporter gene expression. Effects of antibody combinations on HCVcc infection were evaluated using the method of Prichard and Shipman. Combination studies for each pair of compounds were performed in duplicate. The theoretical additive effect is calculated from the dose-response curves of individual compounds by the equation Z=X+Y(1-X) where X and Y represent the inhibition produced by the individual compounds and Z represents the effect produced by the combination of compounds. The theoretical additive surface is subtracted from the actual experimental surface, resulting in a horizontal surface that equals the zero plane when the combination is additive. A surface that lies higher than 20% above the zero plane indicates a synergistic effect of the combination and a surface lower than 20% below the zero plane indicates antagonism.

Definitions

Throughout the specification, several terms are employed that are defined in the following paragraphs.

As used herein, the term "subject" refers to a human or another mammal {e.g., primate, dog, cat, goat, horse, pig, mouse, rat, rabbit, and the like), that can be the host of Hepatitis C virus (HCV), but may or may not be infected with the virus, and/or may or may not suffer from a HCV-related disease. Non-human subjects may be transgenic or otherwise modified animals. In many embodiments of the present invention, the subject is a human being. In such embodiments, the subject is often referred to as an "individual" . The term "individual" does not denote a particular age, and thus encompasses newborns, children, teenagers, and adults. As used herein, the term "HCV refers to any major HCV genotype, subtype, isolate and/or quasispecies. HCV genotypes include, but are not limited to, genotypes 1, 2, 3, 4, 5, and 6; HCV subtypes include, but are not limited to, subtypes la, lb, 2a, 2b, 2c, 3a, 4a-f, 5a and 6a. The terms "afflicted with HCV or "infected with HCV" are used herein interchangeably. When used in reference to a subject, they refer to a subject that has at least one cell which is infected by HCV. The term "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. The terms "HCV-related disease" and "HCV -associated disease" are herein used interchangeably. They refer to any disease or disorder known or suspected to be associated with and/or caused, directly or indirectly, by HCV. HCV-related (or HCV- associated) diseases include, but are not limited to, a wide variety of liver diseases, such as subclinical carrier state of acute hepatitis, chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The term includes symptoms and side effects of any HCV infection, including latent, persistent and sub-clinical infections, whether or not the infection is clinically apparent.

The term "treatment" is used herein to characterize a method or process that is aimed at (1) delaying or preventing the onset of a disease or condition (e.g., HCV infection or HCV-related disease); (2) slowing down or stopping the progression, aggravation, or deterioration of the symptoms of the disease or condition; (3) bringing about amelioration of the symptoms of the disease or condition; or (4) curing the disease or condition. A treatment may be administered prior to the onset of the disease or condition, for a prophylactic or preventive action. Alternatively or additionally, a treatment may be administered after initiation of the disease or condition, for a therapeutic action.

A "pharmaceutical composition" is defined herein as comprising an effective amount of at least one antibody (or a fragment thereof) of the invention, and at least one pharmaceutically acceptable carrier or excipient. As used herein, the term "effective amount" refers to any amount of a compound, agent, antibody, or composition that is sufficient to fulfil its intended purpose(s), e.g., a desired biological or medicinal response in a cell, tissue, system or subject. For example, in certain embodiments of the present invention, the purpose(s) may be: to prevent HCV infection, to prevent the onset of a HCV-related disease, to slow down, alleviate or stop the progression, aggravation or deterioration of the symptoms of a HCV-related disease (e.g., chronic hepatitis C, cirrhosis, and the like); to bring about amelioration of the symptoms of the disease, or to cure the HCV- related disease.

The term "pharmaceutically acceptable carrier or excipient" refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredient(s) and which is not excessively toxic to the host at the concentration at which it is administered. The term includes solvents, dispersion, media, coatings, antibacterial and antifungal agents, isotonic agents, and adsorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art (see for example "Remington's Pharmaceutical Sciences", E.W. Martin, 18^th Ed., 1990, Mack Publishing Co.: Easton, PA, which is incorporated herein by reference in its entirety).

The term "antibody", as used herein, refers to any immunoglobulin (i.e., an intact immunoglobulin molecule, an active portion of an immunoglobulin molecule, etc.) that binds to a specific epitope. The term encompasses monoclonal antibodies and polyclonal antibodies. All derivatives and fragments thereof, which maintain specific binding ability, are also included in the term. The term also covers any protein having a binding domain, which is homologous or largely homologous to an immunoglobulin-binding domain. These proteins may be derived from natural sources, or partly or wholly synthetically produced.

The term "specific binding", when used in reference to an antibody, refers to an antibody binding to a predetermined antigen. Typically, the antibody binds with an affinity of at least 1 x 10 7 M -^"1 , and binds to the predetermined antigen with an affinity that is at least two-fold greater than the affinity for binding to a non-specific antigen (e.g., BSA, casein).

The term "human SR-BI or SR-BI" refers the scavenger receptor class B member 1, a protein having the sequence shown in NCBI Accession Number NP_005496.4, or any naturally occurring variants commonly found in HCV permissive human populations. The term "extracellular domain" or "ectodomain" of human SR-BI refers to the region of the SR-BI sequence that extends into the extracellular space (i.e., the space outside a cell). The term "N-terminal half of the extracellular domain of human SR-BI" refers to the region of the sequence as shown in NCBI Accession Number NP_005496.4.1 spanning from amino acid 38 to amino acid 398.

The terms "susceptible cell" and "HCV -susceptible cell" are used interchangeably. They refer to any cell that may be infected with HCV. Susceptible cells include, are not limited to, liver or hepatic cells, primary cells, hepatoma cells, CaCo2 cells, dendritic cells, placental cells, endometrial cells, lymph node cells, lymphoid cells (B and T cells), peripheral blood mononuclear cells, and monocytes/macrophages .

The term "preventing, inhibiting or blocking HCV infection" when used in reference to an inventive antibody or antibody-related molecule, means reducing the amount of HCV genetic information introduced into a susceptible cell or susceptible cell population as compared to the amount that would be introduced in the absence of the antibody or antibody-related molecule.

The term "isolated", as used herein in reference to a protein or polypeptide, means a protein or polypeptide, which by virtue of its origin or manipulation is separated from at least some of the components with which it is naturally associated or with which it is associated when initially obtained. By "isolated", it is alternatively or additionally meant that the protein or polypeptide of interest is produced or synthesized by the hand of man.

The terms "protein", "polypeptide", and "peptide" are used herein interchangeably, and refer to amino acid sequences of a variety of lengths, either in their neutral (uncharged) forms or as salts, and either unmodified or modified by glycosylation, side-chain oxidation, or phosphorylation. In certain embodiments, the amino acid sequence is a full-length native protein. In other embodiments, the amino acid sequence is a smaller fragment of the full-length protein. In still other embodiments, the amino acid sequence is modified by additional substituents attached to the amino acid side chains, such as glycosyl units, lipids, or inorganic ions such as phosphates, as well as modifications relating to chemical conversions of the chains such as oxidation of sulfydryl groups. Thus, the term "protein" (or its equivalent terms) is intended to include the amino acid sequence of the full-length native protein, or a fragment thereof, subject to those modifications that do not significantly change its specific properties. In particular, the term "protein" encompasses protein isoforms, i.e. , variants that are encoded by the same gene, but that differ in their pi or MW, or both. Such isoforms can differ in their amino acid sequence (e.g. , as a result of allelic variation, alternative splicing or limited proteolysis), or in the alternative, may arise from differential post-translational modification (e.g., glycosylation, acylation, phosphorylation) .

The term "analog", as used herein in reference to a protein, refers to a polypeptide that possesses a similar or identical function as the protein but need not necessarily comprise an amino acid sequence that is similar or identical to the amino acid sequence of the protein or a structure that is similar or identical to that of the protein. Preferably, in the context of the present invention, a protein analog has an amino acid sequence that is at least 30%, more preferably, at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical to the amino acid sequence of the protein.

The term "fragment" or the term "portion", as used herein in reference to a protein, refers to a polypeptide comprising an amino acid sequence of at least 5 consecutive amino acid residues (preferably, at least about: 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250 or more amino acid residues) of the amino acid sequence of a protein. The fragment of a protein may or may not possess a functional activity of the protein.

The term "biologically active", as used herein to characterize a protein variant, analog or fragment, refers to a molecule that shares sufficient amino acid sequence identity or homology with the protein to exhibit similar or identical properties to the protein. For, example, in many embodiments of the present invention, a biologically active fragment of an inventive antibody is a fragment that retains the ability of the antibody to bind to the extracellular domain of Claudin- 1.

The term "homologous" (or "homology"), as used herein, is synonymous with the term "identity" and refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both compared sequences is occupied by the same base or same amino acid residue, the respective molecules are then homologous at that position. The percentage of homology between two sequences corresponds to the number of matching or homologous positions shared by the two sequences divided by the number of positions compared and multiplied by 100. Generally, a comparison is made when two sequences are aligned to give maximum homology. Homologous amino acid sequences share identical or similar amino acid sequences. Similar residues are conservative substitutions for, or "allowed point mutations" of, corresponding amino acid residues in a reference sequence. "Conservative substitutions" of a residue in a reference sequence are substitutions that are physically or functionally similar to the corresponding reference residue, e.g. that have a similar size, shape, electric charge, chemical properties, including the ability to form covalent or hydrogen bonds, or the like. Particularly preferred conservative substitutions are those fulfilling the criteria defined for an "accepted point mutation" as described by Dayhoff et al. ("Atlas of Protein Sequence and Structure", 1978, Nat. Biomed. Res. Foundation, Washington, DC, Suppl. 3, 22: 354-352).

The terms "labeled' , "labeled with a detectable agent" and "labeled with a detectable moiety" are used herein interchangeably. These terms are used to specify that an entity {e.g., an antibody) can be visualized, for example, following binding to another entity {e.g., an antigen). Preferably, a detectable agent or moiety is selected such that it generates a signal which can be measured and whose intensity is related to the amount of bound entity. Methods for labeling proteins and polypeptides, including antibodies, are well-known in the art. Labeled polypeptides can be prepared by incorporation of or conjugation to a label, that is directly or indirectly detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means, or any other suitable means. Suitable detectable agents include, but are not limited to, various ligands, radionuclides, fluorescent dyes, chemiluminescent agents, microparticles, enzymes, colorimetric labels, magnetic labels, and haptens.

The terms "approximately" and "about", as used herein in reference to a number, generally include numbers that fall within a range of 10% in either direction of the number (greater than or less than the number) unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Detailed Description of Certain Preferred Embodiments

As mentioned above, the present invention provides monoclonal antibodies that prevent HCV infection and HCV spread by interfering with HCV-host cells interactions post-binding, and hybridoma cell lines which secrete such monoclonal antibodies.

I - Hybridomas and Anti-SR-BI Monoclonal Antibodies

The Applicants have used genetic immunization of rats and mice and screening methods to generate hybridoma cell lines which secrete monoclonal antibodies that efficiently inhibit HCV infection and HCV viral spread and which specifically bind to the extracellular domain of human SR-BI, and more particularly to the N-terminal half of the extracellular domain of human SR-BI (see the Examples section).

A. Hybridoma Cell Lines and Anti-SR-BI Monoclonal Antibodies

Accordingly, the present invention provides hybridoma cell lines which secrete monoclonal antibodies that specifically bind to the N-terminal half of the extracellular domain of human SR-BI. In particular, the present invention provides four of such hybridoma cell lines, generated by genetic immunization as described the examples (Lohrmann et ah, Curr. Drug Disc, 2003, October, 17-21). These hybridoma cell lines, which are called NK-8H5-E3, QQ-2A10-A5, QQ-4G9-A6 and QQ-4A3-A1, were deposited on August 1, 2012 at the CNCM (Collection Nationale de Culture de Microorganismes (Institut Pasteur, Paris, France) under Accession Numbers CNCM I- 4662, CNCM 1-4663, CNCM 1-4664, and CNCM 1-4665, respectively.

Also provided by the present invention are monoclonal antibodies secreted by any one of these hybridoma cell lines. Methods for the production and isolation of monoclonal antibodies from hybridoma cultures are well known in the art. Hybridoma cells are grown using standard methods, in suitable culture media such as, for example, D-MEM and RPMI-1640 medium. An anti-SR-BI monoclonal antibody can be recovered and purified from hybridoma cell cultures by protein A purification, ammonium sulphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, such as Protein A column, hydroxylapatite chromatography, lectin chromatography, or any suitable combination of these methods. High performance liquid chromatography (HPLC) can also be employed for purification.

Each of the anti-SR-BI monoclonal antibodies secreted by the hybridoma cell lines of the invention was determined to be an immunoglobulin of the rIgG2b heavy (H) chain and kappa light (L) chain isotype or of the mIgG2b heavy (H) chain and kappa light (L) chain isotype. However, monoclonal antibodies of the present invention more generally comprise any monoclonal antibody (or fragment thereof), that is secreted by an inventive hybridoma cell line (or a derivatized cell line), and that specifically binds to the extracellular domain of human SR-BI, and more specifically to the N-terminal half of the extracellular domain of human SR-BI. Without wishing to be bound by any theory, it is believed that binding of a monoclonal antibody to the extracellular domain of human SR-BI on a susceptible cell interferes with HCV-host cells interactions during post-binding steps, and thereby prevents, inhibits or blocks HCV from entering into the cell and from infecting the cell. Instead of using the hybridomas described herein as a source of the antibodies, the monoclonal antibodies may be prepared by any other suitable method known in the art. For example, an inventive anti-SR-BI monoclonal antibody may be prepared by recombinant DNA methods. These methods generally involve isolation of the genes encoding the desired antibody, transfer of the genes into a suitable vector, and bulk expression in a cell culture system. The genes or DNA encoding the desired monoclonal antibody may be readily isolated and sequenced using conventional procedures (e.g. , by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cell lines provided herein serve as a preferred source of such DNA. Suitable host cells for recombinant production of monoclonal antibodies include, but are not limited to, appropriate mammalian host cells, such as CHO, HeLa, or CV1. Suitable expression plasmids include, without limitation, pcDNA3.1 Zeo, pIND(SPl), pREP8 (all commercially available from Invitrogen, Carlsboad, CA, USA), and the like. The antibody genes may be expressed via viral or retroviral vectors, including MLV-based vectors, vaccinia virus-based vectors, and the like. Antibodies of the present invention may be expressed as single chain antibodies. Isolation and purification of recombinantly produced monoclonal antibodies may be performed as described above. B. Antibody Fragments

In certain embodiments, an inventive monoclonal antibody is used in its native form. In other embodiments, it may be truncated (e.g. , via enzymatic cleavage or other suitable method) to provide immunoglobulin fragments or portions, in particular, fragments or portions that are biological active. Biologically active fragments or portions of an inventive monoclonal antibody include fragments or portions that retain the ability of the monoclonal antibody to interfere with HCV-host cells interactions during post-binding steps, and/or to specifically bind to the N- terminal half of the extracellular domain of human SR-BI, and/or to inhibit or block HCV entry into susceptible cells, and/or to reduce or prevent HCV infection of susceptible cells. Biologically active fragments or portions of inventive monoclonal antibodies described herein are encompassed by the present invention.

A biologically active fragment or portion of an inventive monoclonal antibody may be an Fab fragment or portion, an F(ab')₂ fragment or portion, a variable domain, or one or more CDRs (complementary determining regions) of the antibody. Alternatively, a biologically active fragment or portion of an inventive monoclonal antibody may be derived from the carboxyl portion or terminus of the antibody protein and may comprise an Fc fragment, an Fd fragment or an Fv fragment.

Antibody fragments of the present invention may be produced by any suitable method known in the art including, but not limited to, enzymatic cleavage (e.g. , proteolytic digestion of intact antibodies) or by synthetic or recombinant techniques. F(ab')₂, Fab, Fv and ScFv (single chain Fv) antibody fragments can, for example, be expressed in and secreted from mammalian host cells or from E. coli. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. The various portions of antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques.

C. Fusion Proteins

Antibodies of the present invention (or fragments thereof) may be produced in a modified form, such as a fusion protein (i.e. , an immunoglobulin molecule or portion linked to a polypeptide entity). Preferably, fusion proteins of the invention retain the binding capability of the monoclonal antibody towards the N-terminal half of the extracellular domain of human SR-BI. A polypeptide entity to be fused to an inventive monoclonal antibody, or a fragment thereof, may be selected to confer any of a number of advantageous properties to the resulting fusion protein. For example, the polypeptide entity may be selected to provide increased expression of the recombinant fusion protein. Alternatively or additionally, the polypeptide entity may facilitate purification of the fusion protein by, for example, acting as a ligand in affinity purification. A proteolytic cleavage site may be added to the recombinant protein so that the desired sequence can ultimately be separated from the polypeptide entity after purification. The polypeptide entity may also be selected to confer an improved stability to the fusion protein, when stability is a goal. Examples of suitable polypeptide entities include, for example, polyhistidine tags, that allow for the easy purification of the resulting fusion protein on a nickel chelating column. Glutathione- S-transferase (GST), maltose B binding protein, or protein A are other examples of suitable polypeptide entities. Depending on the use intended, an antibody of the invention may be re- engineered so as to optimize stability, solubility, in vivo half-like, or ability to bind additional targets. Genetic engineering approaches as well as chemical modifications to accomplish any or all of these changes in properties are well known in the art. For example, the addition, removal, and/or modification of the constant regions of an antibody are known to play a particularly important role in the bioavailability, distribution, and half-life of therapeutically administered antibodies. The antibody class and subclass, determined by the Fc or constant region of the antibody (which mediates effector functions), when present, imparts important additional properties. Thus, anti-SR-BI monoclonal antibodies with reconfigured, redesigned, or otherwise altered constant domains are encompassed by the present invention.

Additional fusion proteins of the invention may be generated through the techniques of DNA shuffling well known in the art (see, for example, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458). DNA shuffling may be employed to modulate the activity of antibodies, or fragments thereof, for example, to obtain antibodies with higher affinity and lower dissociation rates. In such methods, polynucleotides encoding antibodies of the invention may be altered through random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. Alternatively, one or more portions of a polynucleotide encoding an inventive antibody may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc of one or more heterologous molecules.

Alternatively, an inventive antibody may be linked to another antibody, e.g., to produce a bispecific or a multispecific antibody. For example, an anti-SR-BI monoclonal antibody of the present invention, or a biologically active fragment thereof, may be linked to an antibody (or a fragment thereof) that specifically binds to another receptor of HCV on susceptible cells, such as CD81 and Claudin- 1. Methods for producing bispecific and multispecific antibodies are known in the art and include, for example, chemical synthesis involving cross-linking through reducible disulfide bonds or non-reducible thioether bonds, and recombinant methods.

D. Chimeric/Humanized of De-immunized Antibodies

Anti-SR-BI monoclonal antibodies of the present invention can also be "humanized": sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site-directed mutagenesis of individual residues or by grafting of entire regions or by chemical synthesis. Humanized antibodies can also be produced using recombinant methods. In the humanized form of the antibody, some, most or all of the amino acids outside the CDR regions 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 as long as they do not abrogate the ability of the resulting antibody to bind to the N-terminal half of the extracellular domain of human SR-BI. Suitable human "replacement" immunoglobulin molecules include IgGl, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgA, IgM, IgD or IgE molecules, and fragments thereof. Alternatively, the T-cell epitopes present in rodent antibodies can be modified by mutation (de-immunization) to generate non-immunogenic rodent antibodies that can be applied for therapeutic purposes in humans (see www.accurobio.com).

E. Antibody Conjugates

A monoclonal antibody of the invention, or a biologically active variant or fragment thereof, may be functionally linked {e.g. , by chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other molecular entities. Methods for the preparation of such modified antibodies (or conjugated antibodies) are known in the art. (see, for example, "Affinity Techniques. Enzyme Purification: Part B", Methods in Enzymol., 1974, Vol. 34, W.B. Jakoby and M. Wilneck (Eds.), Academic Press: New York, NY; and M. Wilchek and E.A. Bayer, Anal. Biochem., 1988, 171: 1-32). Preferably, molecular entities are attached at positions on the antibody molecule that do not interfere with the binding properties of the resulting conjugate, i.e., positions that do not participate in the specific binding of the antibody to the N-terminal half of the extracellular domain of human SR-BI.

In certain embodiments, the antibody molecule and molecular entity are covalently, directly linked to each other. The direct covalent binding can be through a linkage such as an amide, ester, carbon-carbon, disulfide, carbamate, ether, thioether, urea, amine or carbonate linkage. Covalent binding can be achieved by taking advantage of functional groups present on the antibody and the molecular entity. An activating agent, such as a carbodiimide, can be used to form a direct linkage. In other embodiments, the antibody molecule and the molecular entity are covalently linked to each other through a linker group. This can be accomplished by using any of a wide variety of stable bifunctional agents well known in the art, including homofunctional and heterofunctional linkers.

In certain embodiments, an antibody of the present invention (or a biologically active fragment thereof) is conjugated to a therapeutic moiety. Any of a wide variety of therapeutic moieties may be suitable for use in the practice of the present invention including, without limitation, cytotoxins (e.g., cytostatic or cytocidal agents), therapeutic agents, and radioactive metal ions (e.g., alpha-emitters and alpha-emitters attached to macrocyclic chelators such as DOTA). Cytotoxins or cytotoxc agents include any agent that is detrimental to cells. Examples include, but are not limited to, paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, thymidine kinase, endonuclease, RNAse, and puromycin and fragments, variants or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g. , daunorubicin and doxorubicin), antibiotics (e.g. , dactinomycin, bleomycin, mithramycin, and anthramycin), and anti-mitotic agents (e.g. , vincristine and vinblastine). The resulting antibody conjugates may find application in the treatment of liver cancer associated with HCV infection (see below).

Other therapeutic moieties include proteins or polypeptides possessing a desired biological activity. Such proteins include, but are not limited to, toxins (e.g. , abrin, ricin A, alpha toxin, pseudomonas exotoxin, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin); proteins such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; apoptotic agents (e.g. , TNF-cc, TNF-β) or, biological response modifiers (e.g. , lymphokines, interleukin-1 (IL- 1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), or other growth factors).

Alternatively or additionally, an antibody of the present invention (or a biologically active fragment thereof) may be conjugated to a detectable agent. Any of a wide variety of detectable agents can be used in the practice of the present invention,

3 125 131 including, without limitation, various ligands, radionuclides (e.g., H, I, I, and the like), fluorescent dyes (e.g. , fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthal-dehyde and fluorescamine), chemiluminescent agents (e.g. , luciferin, luciferase and aequorin), microparticles (such as, for example, quantum dots, nanocrystals, phosphors and the like), enzymes (such as, for example, those used in an ELISA, i.e. , horseradish peroxidase, beta- galactosidase, luciferase, alkaline phosphatase), colorimetric labels, magnetic labels, and biotin, dioxigenin or other haptens and proteins for which antisera or monoclonal antibodies are available. The resulting detectable antibodies may be used in diagnostic and/or prognostic methods (see below). Other molecular entities that can be conjugated to an antibody of the present invention (or a biologically active fragment thereof) include, but are not limited to, linear or branched hydrophilic polymeric groups, fatty acid groups, or fatty ester groups.

Thus, in addition to anti-SR-BI monoclonal antibodies secreted by the hybridoma cell lines described herein, and any biologically active variants or fragments thereof, the present invention also encompasses chimeric antibodies, humanized antibodies, and antibody-derived molecules comprising at least one complementary determining region (CDR) from either a heavy chain or light chain variable region of an inventive anti-SR-BI monoclonal antibody, including molecules such as Fab fragments, F(ab')₂ fragments, Fd fragments, Fabc fragments, Sc antibodies (single chain antibodies), diabodies, individual antibody light single chains, individual antibody heavy chains, chimeric fusions between antibody chains and other molecules, and antibody conjugates, such as antibodies conjugated to a diagnostic or therapeutic agent. All these antibodies and antibody-related molecules encompassed by the present invention retain the ability to specifically bind to the N-terminal half of the extracellular domain of human SR-BI.

F. Activity and Specificity of Inventive Monoclonal Antibodies and Related Molecules

Each of the inventive anti-SR-BI monoclonal antibodies described in the Examples was produced from a hybridoma cell line provided herein and was selected for its ability to inhibit HCVcc infection of Huh7.5.1 cells. As will be appreciated by those skilled in the art, the HCV infection inhibitory effect of other antibodies and antibody-related molecules of the invention may also be assessed using a HCVcc infection system. The inhibitory effect of antibodies and antibody-related molecules on HCV infection may, alternatively or additionally, be assessed using retroviral HCV pseudotyped particles (HCVpp) as known in the art. Preferably, an antibody or antibody-related molecule of the present invention will be shown to inhibit HCV infection of susceptible cells by HCVcc or HCVpp in a dose-dependent manner.

Other methods that can be used for testing the specificity of antibodies and reagents of the present invention include, but are not limited to, flow cytometry analysis, Western blot analysis, ELISA and inhibiting binding assays involving ligand/receptor binding by the antibody. These methods can be used for testing supernatants from hybridomas producing antibodies, for testing the activity of isolated/purified antibodies, and/or for testing the activity of modified antibodies (antibody-related molecules). Binding specificity testing may be performed using the antibody or antibody-related molecule against a panel of cells, e.g., human cells, including, without limitation, liver cell lines (such as, for example, Huh7, Hep3b or HepG2), embryonic kidney cells (293T), fibroblasts (HeLa), B cells, T cells (e.g., Molt-4, Sup-TI, 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 SkHepI, human peripheral blood cells and various fractionated subtypes thereof including lymphocytes and monocytes or other cell lines including CaCo2 cells. Flow cytometry analysis can reveal binding specificity of the antibody or antibody-related molecule for SR-BI on various cell types. Cells from non-human mammals may also be used in such assays.

Using such assays, IC₅₀ values may be determined for the antibodies and antibody-related molecules of the present invention. These values, which give an indication of the concentration of antibody or antibody-related molecule required for 50% inhibition of viral infectivity, provide meaningful and significant quantitative criteria and allow comparison of the infection inhibiting activity of different antibodies and antibody-related molecules.

II - Treatment or Prevention of HCV infection and HCV-associated Diseases A. Indications

Anti-SR-BI antibodies of the present invention may be used in therapeutic and prophylactic methods to treat and/or prevent HCV infection, or to treat and/or prevent a liver disease or a pathological condition affecting HCV- susceptible cells, such as liver cells, lymphoid cells, or monocytes/macrophages. An inventive anti-SR-BI antibody interferes with HCV-host cells interactions during post-binding steps by binding to the extracellular domain of SR-BI on a cell surface, thereby reducing, inhibiting, blocking or preventing HCV entry into the cell and/or HCV infection of the cell.

Methods of treatment of the present invention may be accomplished using an inventive antibody or a pharmaceutical composition comprising an inventive antibody (see below). These methods generally comprise administration of an effective amount of at least one inventive anti-SR-BI antibody, or a pharmaceutical composition thereof, to a subject in need thereof. Administration may be performed using any of the methods known to one skilled in the art. In particular, the antibody or composition may be administered by various routes including, but not limited to, aerosol, parenteral, oral or topical route. In general, an inventive antibody or composition will be administered in an effective amount, i.e. an amount that is sufficient to fulfill its intended purpose. The exact amount of antibody or pharmaceutical composition to be administered will vary from subject to subject, depending on the age, sex, weight and general health condition of the subject to be treated, the desired biological or medical response {e.g., prevention of HCV infection or treatment of HCV- associated liver disease), and the like. In many embodiments, an effective amount is one that inhibits or prevents HCV from entering into a subject's susceptible cells and/or infecting a subject's cells, so as to thereby prevent HCV infection, treat or prevent liver disease or another HCV- associated pathology in the subject. Antibodies and compositions of the present invention may be used in a variety of therapeutic or prophylactic methods. In particular, the present invention provides a method for treating or preventing a liver disease or pathology in a subject, which comprises administering to the subject an effective amount of an inventive antibody (or composition thereof) which inhibits HCV from entering or infecting the subject's cells, so as to thereby treat or prevent the liver disease or pathology in the subject. The liver disease or pathology may be inflammation of the liver, liver fibrosis, cirrhosis, and/or hepatocellular carcinoma {i.e., liver cancer) associated with HCV infection.

The present invention also provides a method for treating or preventing a HCV- associated disease or condition (including a liver disease) in a subject, which comprises administering to the subject an effective amount of an inventive antibody (or composition thereof) which inhibits HCV from entering or infecting the subject's cells, so as to thereby treat or prevent the HCV-associated disease or condition in the subject. In certain embodiments of the present invention, the antibody or composition is administered to a subject diagnosed with acute hepatitis C. In other embodiments of the invention, the antibody or composition is administered to a subject diagnosed with chronic hepatitis C. Administration of an inventive antibody or composition according to such methods may result in amelioration of at least one of the symptoms experienced by the individual including, but not limited to, symptoms of acute hepatitis C such as decreased appetite, fatigue, abdominal pain, jaundice, itching, and flu-like symptoms; symptoms of chronic hepatitis C such as fatigue, marked weight loss, flu-like symptoms, muscle pain, joint pain, intermittent low-grade fevers, itching, sleep disturbances, abdominal pain, appetite changes, nausea, diarrhea, dyspepsia, cognitive changes, depression, headaches, and mood swings; symptoms of cirrhosis such as ascites, bruising and bleeding tendency, bone pain, varices (especially in the stomach and esophagus), steatorrhea, jaundice and hepatic encephalopathy; and symptoms of extrahepatic manifestations associated with HCV such as thyroiditis, porphyria cutanea tarda, cryoglobulinemia, glomerulonephritis, sicca syndrome, thrombocytopenia, lichen planus, diabetes mellitus and B-cell lymphoproliferative disorders. Alternatively or additionally, administration of an inventive antibody or composition according to such methods may slow, reduce, stop or alleviate the progression of HCV infection or an HCV-associated disease, or reverse the progression to the point of eliminating the infection or disease. Administration of an inventive antibody or composition according to such methods may also result in a reduction of the number of viral infections, reduction of the number of infectious viral particles, and/or reduction in the number of virally infected cells.

The effects of a treatment according to the invention may be monitored using any of the assays known in the art for the diagnosis of HCV infection and/or liver disease. Such assays include, but are not limited to, serological blood tests, liver function tests to measure one or more of albumin, alanine transaminase (ALT), alkaline phosphatase (ALP), aspartate transaminase (AST), and gamma glutamyl transpeptidase (GGT), and molecular nucleic acid tests using different techniques such as polymerase chain reaction (PCR), transcription mediated amplification (TMA), or branched DNA (bDNA). Antibodies and compositions of the present invention may also be used in immunization therapies. Accordingly, the present invention provides a method of reducing the likelihood of susceptible cells of becoming infected with HCV as a result of contact with HCV. The method comprises contacting the susceptible cells with an effective amount of an inventive antibody or composition which inhibits HCV from entering or infecting the susceptible cells, so as to reduce the likelihood of the cells to become infected with HCV as a result of contact with HCV. The present invention also provides a method of reducing the likelihood of a subject's susceptible cells of becoming infected with HCV as a result of contact with HCV. In this method, contacting the susceptible cells with the inventive antibody or composition may be performed by administrating the antibody or composition to the subject.

Reducing the likelihood of susceptible cells or of a subject of becoming infected with HCV means decreasing the probability of susceptible cells or a subject to become infected with HCV as a result of contact with HCV. The decrease may be of any significant amount, e.g., at least a 2-fold decrease, more than a 2-fold decrease, at least a 10-fold decrease, more than a 10-fold decrease, at least a 100-fold decrease, or more than a 100-fold decrease. In certain embodiments, the subject is infected with HCV prior to administration of the inventive antibody or composition. In other embodiments, the subject is not infected with HCV prior to administration of the inventive antibody or composition. In yet other embodiments, the subject is not infected with, but has been exposed to, HCV. In certain embodiments, the subject may be infected with HIV or HBV. For example, the methods of the present invention may be used to reduce the likelihood of a subject's susceptible cells of becoming infected with HCV as a result of liver transplant. As already mentioned above, when a diseased liver is removed from a HCV-infected patient, serum viral levels plummet. However, after receiving a healthy liver transplant, virus levels rebound and can surpass pre-transplant levels within a few days (Powers et ah, Liver TranspL, 2006, 12: 207-216). Liver transplant patients may benefit from administration of an inventive antibody that binds to N- terminal half of the extracellular domain of human SR-BI on the surface of hepatocytes and thereby reduce, inhibit, block or prevent HCV entry into the cells. Administration may be performed prior to liver transplant, during liver transplant, and/or following liver transplant.

Other subjects that may benefit from administration of an inventive antibody or composition include, but are not limited to, babies born to HCV-infected mothers, in particular if the mother is also HIV-positive; health-care workers who have been in contact with HCV-contaminated blood or blood contaminated medical instruments; drug users who have been exposed to HCV by sharing equipments for injecting or otherwise administering drugs; and people who have been exposed to HCV through tattooing, ear/body piercing and acupuncture with poor infection control procedures.

Other subjects that may benefit from administration of an inventive antibody or composition include, but are not limited to, subjects that exhibit one or more factors that are known to increase the rate of HCV disease progression. Such factors include, in particular, age, gender (males generally exhibit more rapid disease progression than females), alcohol consumption, HIV co-infection (associated with a markedly increased rate of disease progression), and fatty liver.

In certain embodiments, an inventive antibody or composition is administered alone according to a method of treatment of the present invention. In other embodiments, an inventive antibody or composition is administered in combination with at least one additional therapeutic agent. The inventive antibody or composition may be administered prior to administration of the therapeutic agent, concurrently with the therapeutic agent, and/or following administration of the therapeutic agent.

Therapeutic agents that may be administered in combination with an inventive antibody or composition may be selected among a large variety of biologically active compounds that are known to have a beneficial effect in the treatment or prevention of HCV infection, or a HCV-associated disease or condition. Such agents include, in particular, antiviral agents including, but not limited to, interferons (e.g., interferon- alpha, pegylated interferon- alpha), ribavirin, anti-HCV (monoclonal or polyclonal) antibodies, RNA polymerase inhibitors, protease inhibitors, IRES inhibitors, helicase inhibitors, antisense compounds, ribozymes, and any combination thereof.

B. Administration

An inventive antibody, (optionally after formulation with one or more appropriate pharmaceutically acceptable carriers or excipients), in a desired dosage can be administered to a subject in need thereof by any suitable route. Various delivery systems are known and can be used to administer antibodies of the present invention, including tablets, capsules, injectable solutions, encapsulation in liposomes, microparticles, microcapsules, etc. Methods of administration include, but are not limited to, dermal, intradermal, intramuscular, intraperitoneal, intralesional, intravenous, subcutaneous, intranasal, pulmonary, epidural, ocular, and oral routes. An inventive antibody or composition may be administered by any convenient or other appropriate route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g. , oral, mucosa, rectal and intestinal mucosa, etc). Administration can be systemic or local. Parenteral administration may be preferentially directed to the patient's liver, such as by catheterization to hepatic arteries or into a bile duct. As will be appreciated by those of ordinary skill in the art, in embodiments where an inventive antibody is administered in combination with an additional therapeutic agent, the antibody and therapeutic agent may be administered by the same route (e.g. , intravenously) or by different routes (e.g. , intravenously and orally).

C. Dosage

Administration of an inventive antibody (or composition) of the present invention will be in a dosage such that the amount delivered is effective for the intended purpose. The route of administration, formulation and dosage administered will depend upon the therapeutic effect desired, the severity of the HCV-related condition to be treated if already present, the presence of any infection, the age, sex, weight, and general health condition of the patient as well as upon the potency, bioavailability, and in vivo half-life of the antibody or composition used, the use (or not) of concomitant therapies, and other clinical factors. These factors are readily determinable by the attending physician in the course of the therapy. Alternatively or additionally, the dosage to be administered can be determined from studies using animal models (e.g., chimpanzee or mice). Adjusting the dose to achieve maximal efficacy based on these or other methods are well known in the art and are within the capabilities of trained physicians. As studies are conducted using the inventive monoclonal antibodies, further information will emerge regarding the appropriate dosage levels and duration of treatment.

A treatment according to the present invention may consist of a single dose or multiple doses. Thus, administration of an inventive antibody, or composition thereof, may be constant for a certain period of time or periodic and at specific intervals, e.g., hourly, daily, weekly (or at some other multiple day interval), monthly, yearly (e.g., in a time release form). 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 general, the amount of monoclonal antibody administered will preferably be in the range of about 1 ng/kg to about 100 mg/kg body weight of the subject, for example, between about 100 ng/kg and about 50 mg/kg body weight of the subject; or between about 1 g/kg and about 10 mg/kg body weight of the subject, or between about 100 g/kg and about 1 mg/kg body weight of the subject. III - Pharmaceutical Compositions

As mentioned above, anti-SR-BI antibodies (and related molecules) of the invention may be administered per se or as a pharmaceutical composition. Accordingly, the present invention provides pharmaceutical compositions comprising an effective amount of an inventive antibody described herein and at least one pharmaceutically acceptable carrier or excipient. In some embodiments, the composition further comprises one or more additional biologically active agents.

Inventive antibodies and pharmaceutical compositions may be administered in any amount and using any route of administration effective for achieving the desired prophylactic and/or therapeutic effect. The optimal pharmaceutical formulation can be varied depending upon the route of administration and desired dosage. Such formulations may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the administered active ingredient.

The pharmaceutical compositions of the present invention may be formulated in dosage unit form for ease of administration and uniformity of dosage. The expression "unit dosage form", as used herein, refers to a physically discrete unit of an inventive anti-SR-BI antibody for the patient to be treated. It will be understood, however, that the total daily dosage of the compositions will be decided by the attending physician within the scope of sound medical judgement.

A. Formulation

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents, and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 2,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S. P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solution or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or di-glycerides. Fatty acids such as oleic acid may also be used in the preparation of injectable formulations. Sterile liquid carriers are useful in sterile liquid form compositions for parenteral administration.

Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. Liquid pharmaceutical compositions which are sterile solutions or suspensions can be administered by, for example, intravenous, intramuscular, intraperitoneal or subcutaneous injection. Injection may be via single push or by gradual infusion. Where necessary or desired, the composition may include a local anesthetic to ease pain at the site of injection.

In order to prolong the effect of an active ingredient (here an inventive anti-SR- BI monoclonal antibody), it is often desirable to slow the absorption of the ingredient from subcutaneous or intramuscular injection. Delaying absorption of a parenterally administered active ingredient may be accomplished by dissolving or suspending the ingredient in an oil vehicle. Injectable depot forms are made by forming microencapsulated matrices of the active ingredient in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of active ingredient to polymer and the nature of the particular polymer employed, the rate of ingredient release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by entrapping the active ingredient in liposomes or microemulsions which are compatible with body tissues.

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, elixirs, and pressurized compositions. In addition to the anti-SR-BI monoclonal antibody, the liquid dosage form may contain inert diluents commonly used in the art such as, for example, water or other solvent, solubilising agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cotton seed, ground nut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, and fatty acid esters of sorbitan and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, suspending agents, preservatives, sweetening, flavouring, and perfuming agents, thickening agents, colors, viscosity regulators, stabilizes or osmo-regulators. Examples of suitable liquid carriers for oral administration include water (potentially containing additives as above, e.g. , cellulose derivatives, such as sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols such as glycols) and their derivatives, and oils (e.g. , fractionated coconut oil and arachis oil). For pressurized compositions, the liquid carrier can be halogenated hydrocarbon or other pharmaceutically acceptable propellant.

Solid dosage forms for oral administration include, for example, capsules, tablets, pills, powders, and granules. In such solid dosage forms, an inventive anti- SR-BI monoclonal antibody may be mixed with at least one inert, physiologically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and one or more of: (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannital, and silicic acid; (b) binders such as, for example, carboxymethylcellulose, alginates, gelatine, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants such as glycerol; (d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (e) solution retarding agents such as paraffin; absorption accelerators such as quaternary ammonium compounds; (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate; (h) absorbents such as kaolin and bentonite clay; and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulphate, and mixtures thereof. Other excipients suitable for solid formulations include surface modifying agents such as non-ionic and anionic surface modifying agents. Representative examples of surface modifying agents include, but are not limited to, poloxamer 188, benzalkonium chloride, calcium stearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, colloidal silicon dioxide, phosphates, sodium dodecylsulfate, magnesium aluminum silicate, and triethanolamine. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatine capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition such that they release the active ingredient(s) only, or preferably, in a certain part of the intestinal tract, optionally, in a delaying manner. Examples of embedding compositions which can be used include polymeric substances and waxes.

In certain embodiments, it may be desirable to administer an inventive composition locally to an area in need of treatment (e.g. , the liver). This may be achieved, for example, and not by way of limitation, by local infusion during surgery (e.g., liver transplant), topical application, by injection, by means of a catheter, by means of suppository, or by means of a skin patch or stent or other implant.

For topical administration, the composition is preferably formulated as a gel, an ointment, a lotion, or a cream which can include carriers such as water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, or mineral oil. Other topical carriers include liquid petroleum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylenemonolaurat (5%) in water, or sodium lauryl sulphate (5%) in water. Other materials such as antioxidants, humectants, viscosity stabilizers, and similar agents may be added as necessary.

In addition, in certain instances, it is expected that the inventive compositions may be disposed within transdermal devices placed upon, in, or under the skin. Such devices include patches, implants, and injections which release the active ingredient by either passive or active release mechanisms. Transdermal administrations include all administration across the surface of the body and the inner linings of bodily passage including epithelial and mucosal tissues. Such administrations may be carried out using the present compositions in lotions, creams, foams, patches, suspensions, solutions, and suppositories (rectal and vaginal).

Transdermal administration may be accomplished through the use of a transdermal patch containing an active ingredient (i.e. , an inventive anti-SR-BI monoclonal antibody) and a carrier that is non-toxic to the skin, and allows the delivery of the ingredient for systemic absorption into the bloodstream via the skin. The carrier may take any number of forms such as creams and ointments, pastes, gels, and occlusive devices. The creams and ointments may be viscous liquid or semisolid emulsions of either the oil-in- water or water-in-oil type. Pastes comprised of absorptive powders dispersed in petroleum or hydrophilic petroleum containing the active ingredient may be suitable. A variety of occlusive devices may be used to release the active ingredient into the bloodstream such as a semi-permeable membrane covering a reservoir containing the active ingredient with or without a carrier, or a matrix containing the active ingredient.

Suppository formulations may be made from traditional materials, including cocoa butter, with or without the addition of waxes to alter the suppository' s melting point, and glycerine. Water soluble suppository bases, such as polyethylene glycols of various molecular weights, may also be used. When a pharmaceutical composition of the present invention is used as

"vaccine" to prevent HCV- susceptible cells to become infected with HCV, the pharmaceutical composition may further comprise vaccine carriers known in the art such as, for example, thyroglobulin, albumin, tetanus toxoid, and polyamino acids such as polymers of D-lysine and D-glutamate. The vaccine may also include any of a variety of well known adjuvants such as, for example, incomplete Freund' s adjuvant, alum, aluminium phosphate, aluminium hydroxide, monophosphoryl lipid A (MPL, GlaxoSmithKline), a saponin, CpG oligonucleotides, montanide, vitamin A 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. Materials and methods for producing various formulations are known in the art and may be adapted for practicing the subject invention. Suitable formulations for the delivery of antibodies can be found, for example, in "Remington's Pharmaceutical Sciences", E.W. Martin, 18^th Ed., 1990, Mack Publishing Co.: Easton, PA.

B. Additional Biologically Active Agents

In certain embodiments, an inventive anti-SR-BI monoclonal antibody is the only active ingredient in a pharmaceutical composition of the present invention. In other embodiments, the pharmaceutical composition further comprises one or more biologically active agents. Examples of suitable biologically active agents include, but are not limited to, vaccine adjuvants and therapeutic agents such as anti-viral agents (as described above), anti-inflammatory agents, immunomodulatory agents, analgesics, antimicrobial agents, antibacterial agents, antibiotics, antioxidants, antiseptic agents, and combinations thereof.

In such pharmaceutical compositions, the anti-SR-BI monoclonal antibody and additional therapeutic agent(s) may be combined in one or more preparations for simultaneous, separate or sequential administration of the anti-SR-BI antibody and therapeutic agent(s). More specifically, an inventive composition may be formulated in such a way that the antibody and therapeutic agent(s) can be administered together or independently from each other. For example, an anti-SR-BI antibody and a therapeutic agent can be formulated together in a single composition. Alternatively, they may be maintained {e.g., in different compositions and/or containers) and administered separately.

In certain embodiments, the at least one anti-SR-BI monoclonal antibody and at one active ingredient act in synergy to inhibit HCV infection. Accordingly, the present invention also provides synergistic combinations.

C. Synergistic Combinations comprising an Inventive anti-SR-BI Monoclonal Antibody

Thus, the present invention provides a combination comprising at least one anti- SR-BI monoclonal antibody according to the invention and at least one additional agent for use in the treatment or prevention of HCV infection, wherein the at least one anti-SR-BI monoclonal antibody and at least one additional agent act in synergy to inhibit HCV infection. In certain embodiments, the at least one anti-SR-BI monoclonal antibody according to the invention decreases the IC₅₀ for the inhibition of HCV infection by the additional agent by a factor of at least 10 fold or at least 15 fold, preferably at least 20 fold or at least 25 fold, more preferably at least 30 fold or 40 fold, and even more preferably 50 fold or more than 50 fold. In other words, in the presence of the anti- SR-BI monoclonal antibody, the concentration of the additional agent necessary to obtain a 50% inhibition of HCV entry is at least 10 times or at least 15 times, preferably at least 20 times or at least 25 times, more preferably at least 30 times or at least 40 times, and even more preferably 50 times lower or more than 50 times lower than the concentration of the additional agent that would be necessary to obtain the same HCV entry inhibition in the absence of anti-SR-BI monoclonal antibody.

In other embodiments, the additional agent decreases the IC₅₀ for the inhibition of HCV infection by the at least one anti-SR-BI monoclonal antibody according to the invention by a factor of at least 5 times or 10 fold or at least 15 fold, preferably at least 20 fold or at least 25 fold, more preferably at least 30 fold or 40 fold, and even more preferably 50 fold or more than 50 fold. In other words, in the presence of the additional agent, the concentration of anti-SR-BI monoclonal antibody necessary to obtain a 50% inhibition of HCV entry is at least 5 times or at least 10 times or at least 15 times, preferably at least 20 times or at least 25 times, more preferably at least 30 times or at least 40 times, and even more preferably 50 times lower or more than 50 times lower than the concentration of anti-SR-BI monoclonal antibody that would be necessary to obtain the same HCV entry inhibition in the absence of additional agent.

In certain embodiments, a combination of the present invention is characterized by a combination index (CI) that is lower than 1 (which is defined as a marked synergy). A combination of the present invention is preferably characterized by a CI lower than 1, preferably lower than 0.50 or lower than 0.25, more preferably lower than 0.15, and even more preferably lower than 0.10.

In particular, in certain embodiments, the at least one additional agent is an anti- HCV envelope antibody. The at least one anti-HCV envelope antibody is preferably selected from anti-El antibodies, anti-E2 antibodies, anti-HCV IgGs from individuals chronically or previously infected with HCV and mixtures thereof. The Applicants have shown that such combinations act in synergy to inhibit HCV entry into susceptible cells.

In other embodiments, the one additional agent is a protein kinase inhibitor. As used herein, the term "protein kinase inhibitor" refers to any molecule that specifically blocks the action of one or more protein kinases. Protein kinase inhibitors are subdivided by the amino acids whose phosphorylation is inhibited. Most kinases act on both serine and threonine, the tyrosine kinases act on tyrosine, and a number (dual- specificity) kinases act on all three. As used herein, the term "serine/threonine kinase inhibitor" refers to a molecule that specifically blocks the action of one or more serine and/or threonine kinases. As used herein, the term "tyrosine kinase inhibitor" refers to a molecule that specifically blocks the action of one or more tyrosine kinases.

As mentioned above, the present Applicants have shown that erlotinib and dasatinib act in synergy with an anti-SRBI antibody of the invention to inhibit HCV infection. Both erlotinib and dasatinib are tyrosine kinase inhibitors. Therefore, in certain preferred embodiments, the at least one protein kinase inhibitor present in a combination according to the invention is a tyrosine kinase inhibitor.

Tyrosine kinase inhibitors are generally used in cancer therapy. Indeed, research indicates that mutations which make tyrosine kinases constantly active can be a contributing factor in the development of cancerous cells. So, when a tyrosine kinase inhibitor is administered, the cell communication and reproduction is reduced, and cancerous cell growth can be lowered to the point of stopping growth. However, the research team of the laboratory of the Applicants has recently demonstrated that blocking the activity of HCV entry cofactors, EGFR (epidermal growth factor receptor) and EphA2 (ephrin type-A receptor A), by the approved tyrosine kinase inhibitors, erlotinib and dasatinib, broadly impaired infection by all major HCV genotypes and viral escape variants in vitro and in the human liver-chimeric Alb- uPA/SCID mouse model (Lupberger et al, Nature Medicine, 2011, 17: 589-595). They showed that erlotinib and dasatinib interfere with CD81-CLDN1 co-receptor interactions and with glycoprotein-dependant viral fusion. These results suggest that tyrosine kinase inhibitors that act on these HCV entry cofactors may represent a promising class of novel antivirals that target the first step of the viral life cycle. Thus, in certain embodiments, the at least one protein kinase inhibitor present in a combination of the invention is a tyrosine kinase inhibitor that acts on the epidermal growth factor receptor (EGFR). The target protein EGFR is also sometimes referred to as Herl or ErbB-1. Examples of tyrosine kinase inhibitors that act on EGFR include, but are not limited to, erlotinib, gefitinib, vandetanib, and lapatinib.

Thus, in certain embodiments, the at least one tyrosine kinase inhibitor that acts on EGFR is erlotinib. Erlotinib is marketed under the tradename TARCEVA^® by Genentech and OSI pharmaceuticals in the United States and by Roche elsewhere. Erlotinib binds in a reversible fashion to the adenosine triphosphate (ATP) binding site of the epidermal growth factor receptor. For the signal to be transmitted, two members of the EGFR family need to come together to form a homodimer. These then use the molecule of ATP to autophosphorylate each other, which causes a conformational change in their intracellular structure, exposing a further binding site for binding proteins that cause a signal cascade to the nucleus. By inhibiting the ATP, autophosphorylation is not possible and the signal is stopped. The FDA (U.S. Food and Drug Administration) has approved the use of erolotinib for the treatment of locally advanced or metastatic non- small cell lung cancer that has failed at least one prior chemotherapy regimen.

In certain embodiments, the at least one tyrosine kinase inhibitor that acts on EGFR is gefitinib. Gefitinib (tradename IRESSA^®) is marketed by AstraZeneca and Teva. In Europe, gefitinib is indicated in advanced non-small cell lung cancer in all lines of treatment for patients harboring EGFR mutations.

In certain embodiments, the at least one tyrosine kinase inhibitor that acts on EGFR is vandetanib. Vandetanib, also known as ZD6474 is being developed by AstraZeneca. It is an antagonist of the epidermal growth factor receptor (EGFR) and the vascular endothelial growth factor receptor (VEGFR). In April 2011, Vandetanib became the first drug to be approved by the FDA for the treatment of late-stage (metastatic) medullatory thyroid cancer in adult patients who are ineligible for surgery. In certain embodiments, the at least one tyrosine kinase inhibitor that acts on

EGFR is lapatinib. Lapatinib, in the form of lapatinib ditosylate (tradenames TYKERB^® in the U.S. and TYVERB^® in Europe) is marketed by GlaxoSmithKline. Lapatinib is a dual tyrosine kinase inhibitor, which inhibits the tyrosine kinase activity associated with EGFR and HER2/neu (Human EGFR type 2). In February 2010, lapatinib received accelerated approval as front-line therapy in triple positive breast cancer. Other tyrosine kinase inhibitors that act on EGFR and that are suitable for use in the present invention include molecules that are currently under development for human use, including, but not limited to, neratinib (also known as HKI-272, being developed by Pfizer), which is under investigation for the treatment of breast cancer and other solid tumors; and afatinib (also known as BIBW 2992, being developed by Boehringer Ingelheim), which is a candidate drug against non-small cell lung carcinoma, both of which are dual inhibitors of EGFR and Her2.

Other examples of tyrosine kinase inhibitors that act on EGFR include anti- EGFR antibodies, such as Cetuximab, Panitumumab, Matuzumab, Zalutumumab, Nimotuzumab, Necitumumab, and the like. In other embodiments, the at least one protein kinase inhibitor present in a combination of the invention is a tyrosine kinase inhibitor that acts on the ephrin type- A receptor 2 (EphA2). Examples of such tyrosine kinase inhibitors include, for example, dasatinib.

Dasatinib (BMS-354825) is sold under the tradename SPRYCEL^® by Bristol- Myers Squibb. Dasatinib is a multi-targeted kinase inhibitor mainly developed for Bcr-Abl and Src family kinases, but which also inhibits multiple Eph kinases, including EphA2. Dasatinib is approved for use in patients with chronic myelogenous leukemia (CML) after imatinib treatment, and Philadelphia chromosome-positive acute lymphoblastic leukemia. It is being evaluated for use in numerous other cancers, including advanced prostate cancer.

Other examples of tyrosine kinase inhibitors that act on EphA2 include anti- EphA2 antibodies, such as those developed by Medlmmune Inc.

In other embodiments, the at least one protein kinase inhibitor present in a combination of the present invention is an inhibitor that acts on a receptor tyrosine kinase (RTK) other than EGFR and EphA2, for example an anti-RTK monoclonal antibody. Examples of such anti-RTK monoclonal antibodies include, but are not limited to, anti-VEGF antibodies such as Bevacizumab and Ranibizumab; anti-Erb2 antibodies such as Trastuzumab; anti-HER2/neu antibodies such as Trastuzumab, Ertimaxomab, and Pertuzumab; anti-VEGFR2 antibodies such as Ramucirumab and Alacizumab pegol; anti-VEGF-A antibodies such as Ranibizumab and Bevacizumab; anti-PDGF-R antibodies such as Olaratumab; and anti-IGF-1 receptor antibodies such as Figitumumab; Robatumumal and Cixutumumab.

The invention also provides a combination comprising at least one anti-SR-BI monoclonal antibody according to the invention and at least one direct acting antiviral (DAA) for use in the treatment or prevention of HCV infection, wherein the at least one anti-SR-BI monoclonal antibody and at least one DAA in synergy to inhibit HCV infection. The terms "direct acting antiviral', "direct acting antiviral agent", "DAA", "specifically targeted antiviral therapy for hepatitis C" and "STAT-C" are used herein interchangeably. They refer to molecules that interfere with specific steps of the lifecycle of HCV and are thus useful in the prevention or treatment of HCV infection.

A major effort of the pharmaceutical industry is being focused on the development of direct- acting antiviral agents. Direct acting antivirals have been shown to increase the efficacy of the standard of care in randomized clinical trials (for a review, see Hofmann et al., Nat. Rev. Gastroenterol. Hepatol.; 2011, 8: 257-264). However, along with these encouraging results, significant treatment-related adverse events including rash, gastrointestinal side-effects, and anemia as well as the emergence of HCV resistance have been reported (Poordad et al., New Engl. J. Med., 2011, 364: 1195-1206; Hezode et al, New Engl. J. Med., 2009, 360: 1839-1850; Pawlotsky et al, Hepatology, 2011, 53: 1742-1751). As mentioned above, the present Applicants have shown that an anti-SRBI antibody of the invention in combination with a direct acting antiviral act in high synergy to inhibit HCV infection. The efficient inhibition observed for these combinations suggest that they may represent a new approach for interferon- or ribavirin-free treatment strategies. Furthermore, such combinations may overcome antiviral resistance as they result in a greater decrease of the viral load in shorter periods of time, thereby limiting the frequency of appearance of resistant variants. A direct acting antiviral agent suitable for use in a combination of the present invention may exert its effects by any mechanism that interferes with one or more specific steps of the lifecycle of HCV. For example, VX-950 (also known as telaprevir), ITMN-191 (also known as danoprevir), telaprevir, boceprevir, danoprevir and TMC-435 used by the present Applicants are HCV protease inhibitors, mericitabine (also known as RG7128) and GS-7977 are HCV polymerase inhibitors, and daclatasvir (also known as BMS-790052) is an NS5A inhibitor. Thus, in certain preferred embodiments, the at least one direct acting antiviral present in a combination according to the invention is a HCV protease inhibitor or a HCV polymerase inhibitor or an NS5A inhibitor.

Protease inhibitors suitable for use in the context of the present invention include NS3/4A protease inhibitors. Examples of NS3/4A protease inhibitors that can be present in a combination of the present invention include, but are not limited to, VX-950 (also known as telaprevir), ITMN-191 (also known as danoprevir), boceprevir, BMS-650032, VX-985, BI 201335, and TMC435. In certain preferred embodiments, the at least one direct acting antiviral present in a combination according to the invention is NS3/4A protease inhibitor telaprevir, boceprevir, danoprevir or TMC-435.

Telaprevir (also known as VX-950), marketed under the tradename INCIVEK^®, was co-developed by Vertex Pharmaceuticals, Inc. and Johnson & Johnson. In May 2011, the FDA approved telaprevir for the treatment of patients with genotype 1 chronic hepatitis C. ΓΓΜΝ-191 (also known as R7227 or danoprevir) was being co- developed by Roche and InterMune Inc., but is now fully owned by Roche. Boceprevir (initially developed by Schering-Plough, and then by Merck and marketed under the tradename VICTRELIS^®) was approved by the FDA for the treatment of hepatitic C genotype 1 in May 2011. BMS-650032 is being developed by Bristol- Myers-Squibb. VX-985 is a NS3/4A protease inhibitor being developed by Vertex Pharmaceuticals, Inc. BI 201335 is being developed by Boehringer Ingelheim and is now in Phase III clinical trials in the United States. TMC435, a NS3/4A protease inhibitor being developed by Medivir/Tibotec/Johnson & Johnson, is also in Phase III clinical trials. Other examples of NS3/4A protease inhibitors that can be present in a combination according to the invention include, but are not limited to, NS3/4A protease inhibitors that are currently in phase II clinical trials such as GS 9256 and GS 9451 (being developed by Gilead), MK-7009 (also known as vaniprevir, being developed by Merck), ACH-1625 (being developed by Achillion), and ABT-450 (being developed by Abbott/Enanta); NS3/4A protease inhibitors that are currently in phase I clinical trials such as BMS-791325 (being developed by Bristol-Myers Squibb), VX-985 and VX-500 (being developed by Vertex pharmaceuticals), and PHX1766 (being developed by Phenomix); and NS3/4A protease inhibitors that are currently in preclinical trials such as VX-813 (being developed by Vertex), AVL-181 and AVL-192 (being developed by Avila Therapeutics), and ACH-2684 (being developed by Achillion).

Polymerase inhibitors suitable for use in the context of the present invention include NS5B polymerase inhibitors. The NS5B, an RNA-dependent RNA polymerase (RdRp) enzyme, is a highly conserved structure across all hepatitis C genotypes. It is therefore, an ideal target for drug therapy. There are two classes of polymerase inhibitors: nucleoside/nucleotide analogues and non-nucleoside RdRp inhibitors. Nucleoside inhibitors target the catalytic sites of the enzyme and act as chain terminators. Non-nucleoside inhibitors are allosteric inhibitors. In certain embodiments, the at least one direct acting antiviral present in a combination according to the invention is HCV NS5B polymerase inhibitor, mericitabine or GS- 7977.

Mericitabine (also known as RG7128 or RO5024048), is a prodrug of PSI-6130, an oral cytidine nucleoside analogue. It is being developed by Roche and Pharmasset. Mericitabine has shown in vitro activity against all of the most common HCV genotypes.

GS-7977 (also known as PSI-7977) is being developed by Gilead Sciences. It is currently in Phase III clinical trials. It is being studied as a treatment to be used in combination with ribavirin. GS-78977 is a prodrug that is metabolized to the active antiviral agent 2'-deoxy-2'-a-fluoro-P-C-methyluridine-5'-monophosphate.

Other examples of NS5B polymerase inhibitors that can be present in a combination according to the invention include, but are not limited to, nucleoside/nucleotide polymerase inhibitors that are currently in Phase II clinical trials such as IDX184 (being developed by Idenix) and PS 1-7977 (being developed by Pharmasset); non-nucleoside polymerase inhibitors that are currently in Phase II clinical trials such as VX-222 (initially developed by ViroChem, now owned by Vertex); PF-868554 (being developed by Pfizer); ABT-072 and ABT-333 (being developed by Abbott), GS 9190 (being developed by Gilead) and ANA598 (also known as setrobuvir, being developed by Anadys); nucleoside/nucleotide polymerase inhibitors that are currently in Phase I clinical trials such as BI 207127 (being developed by Boehringer Ingelheim), MK-0608 (being developed by Isis/Merck), TMC649128 (being developed by Medivir/Tibotec), RG7348 (being developed by Roche/Ligand (Metabasis)), PSI-938 (being developed by Pharmasset), and INX-189 (being developed by Inhibitex); and non-nucleoside polymerase inhibitors that are currently in Phase I clinical trials such as VCH-759 (initially developed by ViroChem Pharma, now owned by Vertex), IDX375 (being developed by Idenix), and A-837093 (being developed by Abbott).

NS5A inhibitors suitable for use in the context of the present invention include, in particular in particular daclatasvir (also known as BMS-790052), which was developed by Bristol-Myers-Squibb.

The invention also provides a combination comprising at least one anti-SR-BI monoclonal antibody according to the invention and at least one interferon for use in the treatment or prevention of HCV infection, wherein the at least one anti-SR-BI monoclonal antibody and at least one interferon in synergy to inhibit HCV infection. The terms "interferon" and ^ίΊFN^,, are used herein interchangeably. They refer to any interferon or interferon derivative {e.g., pegylated interferon) that can be used in the prevention or treatment of HCV infection and/or in the prevention or treatment of HCV-related diseases, in particular cirrhosis and liver cancer.

Interferons are a family of cytokines produced by eukaryotic cells in response to viral infection and other antigenic stimuli, which display broad-spectrum antiviral, antiproliferative and immunomodulatory effects. Recombinant forms of interferons have been widely applied in the treatment of various conditions and diseases, such as viral infections {e.g., HCV, HBV and HIV), inflammatory disorders and diseases (e.g. , multiple sclerosis, arthritis, cystic fibrosis), and tumors (e.g. , liver cancer, lymphomas, myelomas, etc .).

In certain embodiments, the at least one interferon molecule present in a combination according to the invention is selected from the group consisting of IFN- a, IFN-β, IFN-CO, IFN-γ, IFN-λ, analogs thereof and derivatives thereof.

As used herein, the terms "interferon" and "IFN" more specifically refer to a peptide or protein having an amino acid substantially identical (e.g. , et least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or even 100% identical) to all or a portion of the sequence of an interferon (e.g., a human interferon), such as IFN- a, IFN-β, IFN-CO, IFN-γ, and IFN-λ that are known in the art. Interferons suitable for use in the present invention include, but are not limited to, natural human interferons produced using human cells, recombinant human interferons produced from mammalian cells, E-co/i-produced recombinant human interferons, synthetic versions of human interferons and equivalents thereof. Other suitable interferons include consensus interferons which are a type of synthetic interferons having an amino acid sequence that is a rough average of the sequence of all the known human IFN subtypes (for example, all the known IFN-a subtypes, or all the known IFN-β subtypes, or all the known IFN-CO subtypes, or all the known IFN-γ subtypes, or all the known IFN-λ subtypes. In certain embodiments, interferons present in combinations according to the invention have been approved for human use. In other embodiments, interferons present in combinations according to the present are undergoing human clinical trials.

The terms "interferon" and "IFN" also include interferon derivatives, i.e., molecules of interferon (as described above) that have been modified or transformed. A suitable transformation may be any modification that imparts a desirable property to the interferon molecule. Examples of desirable properties include, but are not limited to, prolongation of in vivo half-life, improvement of therapeutic efficacy, decrease of dosing frequency, increase of solubility/water solubility, increase of resistance against proteolysis, facilitation of controlled release, and the like. As mentioned above, pegylated interferons have been produced (e.g. , pegylated IFN-a) and are currently used to treat hepatitis. Pegylated interferons exhibit longer half-lifes, which allows for less frequent administration of the drug. Pegylating an interferon molecule involves covalently binding the interferon to polyethylene glycol (PEG), an inert, nontoxic and biodegradable organic polymer. Therefore, in certain embodiments, the at least one interferon present in a combination according to the invention is a pegylated interferon. Interferons have also been produced as fusion proteins with human albumin (e.g. , albumin-IFN-Cc). The albumin-fusion platform takes advantage of the long half-life of human albumin to provide a treatment that allows the dosing frequency of IFN to be reduced in individuals with chronic hepatitis C. Therefore, in certain embodiments, the at least one interferon present in a combination according to the invention is an albumin-interferon fusion protein. The terms "alpha interferon" , "interferon-alpha" , "interferon- of and "/FN- of are used herein interchangeably and refer to the family of highly homologous species- specific proteins (i.e. , glycoproteins) that are known in the art and inhibit viral replication and cellular proliferation, and modulate immune response. Typical IFN-a molecules suitable for use in the present invention include, but are not limited to, recombinant IFN-CC-2b (such as INTRON-A^® interferon available from Schering Corporation); recombinant IFN-CC-2a (such as ROFERON^® interferon available from Hoffman-La Roche); recombinant IFN-CC-2C (such as BEROFOR^® alpha 2 interferon available from Boehringer Ingelheim Pharmaceutical, Inc); IFN-a-nl, a purified blend of natural alpha interferons (such as SUMERIFERON^® available from Sumitomo, Japan or WELLFERON^® interferon alpha-nl (INS) available from Glaxo-Wellcome Ltd); IFN-a-n3, a mixture of natural alpha interferons (such as ALFERON^® made by Interferon Sciences); human leukocyte interferon-a obtained from the leukocyte fraction of human blood following induction with Sendai virus (such as MULTIFERON^®, available from Swedish Orphan Biovitrium, which contains several naturally occurring IFN-a subtypes); a consensus IFN-a (such as INFERGEN^®, interferon alfacon- 1, available from Three Rivers Pharmaceuticals, LLC, and those described in U.S. Pat. Nos. 4,897,471); and equivalents thereof.

Other suitable interferon alpha molecules include IFN-a derivatives, including, but not limited to, pegylated IFN-a-2a (such as PEGASYS^® available from Hoffman- La Roche); pegylated IFN-a-2b (such as PEGINTRON^® available from Schering Corporation); albumin IFN-a-2b also known as albinterferon (such as ALBUFERON^® available from Human Genome Sciences), and equivalents thereof. The terms "beta interferon", "interferon-beta" , "interferon- β' and "ΙΙ Ν-β^' are used herein interchangeably and refer to the family of highly homologous species- specific proteins (i.e., glycoproteins) that are known in the art and have the ability to induce resistance to viral antigens. Typical IFN-β molecules suitable for use in the present invention include, but are not limited to, recombinant ΙΡΝ-β-la (such as, REBIF^® available from Pfizer or AVONEX^® available from Biogen Idex), recombinant ΙΡΝ-β-lb (such as BETAFERON^®/BETASERON^® available from Bayer HealthCare or EXTAVIA^®, the generic form of BETAFERON, available from Novartis, or ZIFERON^®, an interferon-β lb biosimilar, available from Zistdaru Danesh Ltd), IFN-β molecules described in U.S. Pat. Nos. 4,820,638 and 5,795,779) and, equivalents thereof.

Other suitable interferon beta molecules include IFN-β derivatives, including, but not limited to, pegylated INF-β (such as TRK-560 being developed by Toray Industries, Inc.), pegylated ΙΡΝ-β-la (such as BUBO 17 being developed by Biogen Idee); pegylated ΙΡΝ-β-lb (such as NU100 and NU400 being developed by Nuron Biotech); albumin- IFN-β fusion proteins such as those described in U.S. Pat. No. 7,572,437, and equivalents thereof.

The terms "omega interferon" , "interferon-omega" , "interferon- (ά' and "IFN-

(ά' are used herein interchangeably and refer to the family of highly homologous species-specific proteins (i.e., glycoproteins) that are known in the art and have the ability to inhibit viral replication and cellular proliferation and modulate immune response. Typical IFN-CO molecules suitable for use in the present invention include, but are not limited to, IFN-CO described in European patent No. EP0 170 204, ITCA being developed by Intarcia Therapeutics, Inc., and equivalents thereof. Other suitable interferon omega molecules include IFN-CO derivatives, including, but not limited to, pegylated INF-CO that can be obtained using a method described in U.S. Pat. Nos. 5,612,460; 5,711,944; 5,951,974 or 5,951,974; albumin-IFN-CO fusion proteins such as those described in U.S. Pat. No. 7,572,437, and equivalents thereof.

The terms "gamma interferon", ' interferon -gamma" , "interferon- and "IFN-f are used herein interchangeably and refer to the family of highly homologous species-specific proteins (i.e., glycoproteins) that are known in the art and have the ability to induce resistance to certain viral antigens. Typical IFN-γ molecules suitable for use in the present invention include, but are not limited to, IFN-γ described in U.S. Pat. Nos. 4,727,138, 4,762,791, 4,845,196, 4,929,554, 5,574,137, and 5,690,925; interferon gamma lb (such as ACTEVIMUNE^® available from InterMune, Inc.), and equivalents thereof.

Other suitable interferon omega molecules include IFN-γ derivatives, including, but not limited to, pegylated INF-γ that can be obtained using a method described in U.S. Pat. Nos. 5,612,460; 5,711,944; 5,951,974 or 5,951,974 albumin- IFN-CO fusion proteins such as those described in U.S. Pat. No. 7,572,437, and equivalents thereof. The terms "lambda interferon", "interferon-lambda", "interferon- " and

"IFN-λ" are used herein interchangeably and refer to the family of highly homologous species-specific proteins (i.e., glycoproteins) that are known in the art and have antiviral properties. Typical IFN-λ molecules suitable for use in the present invention include, but are not limited to, IFN-λΙ, ΙΡΝ-λ2 and ΙΡΝ-λ3 molecules described in international patent applications number WO02/086087, WO2004/037995 and WO/2005/023862 and equivalents thereof.

Other suitable interferon omega molecules include IFN-γ derivatives including, but not limited to, pegylated ΙΡΝ-λ-la (such as BMS-914143 being developed by Bristol-Myers Squibb), albumin- IFN-CO fusion proteins such as those described in U.S. Pat. No. 7,572,437, and equivalents thereof.

The terms "interferon" and "/ N' also include interferon-like molecules, i.e., molecules that have functional and/or structural features exhibited by or similar to known interferons or interferon analogs, such as those described above.

Finally, the invention provides a combination comprising at least one anti-SR- BI monoclonal antibody according to the invention and the host-targeting agent, alisporivir. Alisporivir (also known as Debia 0.25, DEB025 or UNIL-025) is a cyclophilin A inhibitor. It is under development by Debiopharm for Japan and by Novartis for the rest of the world since February 2010. It is being researched for potential use in the treatment of hepatitis C (Flisiak et ah, Hepatology, 2009, 49: 1460-1468), and also investigated for Duchenne muscular dystrophy (Reutenauer et al, Br. J. Pharmacol., 2008, 155: 574-584). The synergistic combinations of the present invention may be used in the treatment of HCV infection or a HCV-related disease in a subject; or for the control of chronic HCV infection in a subject; or for the prevention of HCV re-infection and recurrence in a liver transplantation patient, as described above for the anti-SR-BI monoclonal antibodies of the invention.

In addition, in certain embodiments, the HCV infection or HCV-related disease to be treated by a combination according the invention is caused by a Hepatitis C virus that is resistant to a direct acting antiviral and/or that is transmitted by cell-cell transmission. The invention also provides pharmaceutical compositions comprising an effective amount of at least one combination of the invention and at least one pharmaceutically acceptable carrier or excipient, as described above.

D. Pharmaceutical Packs of Kits

In another aspect, the present invention provides a pharmaceutical pack or kit comprising one or more containers (e.g. , vials, ampoules, test tubes, flasks or bottles) containing one or more ingredients of an inventive pharmaceutical composition, allowing administration of an anti-SR-BI monoclonal antibody of the present invention.

Different ingredients of a pharmaceutical pack or kit may be supplied in a solid (e.g. , lyophilized) or liquid form. Each ingredient will generally be suitable as aliquoted in its respective container or provided in a concentrated form. Pharmaceutical packs or kits may include media for the reconstitution of lyophilized ingredients. Individual containers of the kits will preferably be maintained in close confinement for commercial sale. In certain embodiments, a pharmaceutical pack or kit includes one or more additional therapeutic agent(s) (e.g., one or more anti-viral agents, as described above). Optionally associated with the container(s) can be a notice or package insert in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. The notice of package insert may contain instructions for use of a pharmaceutical composition according to methods of treatment disclosed herein.

An identifier, e.g. , a bar code, radio frequency, ID tags, etc., may be present in or on the kit. The identifier can be used, for example, to uniquely identify the kit for purposes of quality control, inventory control, tracking movement between workstations, etc.

IV - Non-Therapeutic Uses of Anti-SR-BI Monoclonal Antibodies

Antibodies of the present invention, e.g. , an anti-SR-BI monoclonal antibody produced by a hybridoma cells line provided herein, may be employed in a variety of non-therapeutic applications, such as purification and screening methods.

A. Purification Methods

Thus, antibodies of the invention may be used as affinity purification agents. In this application, an inventive antibody is immobilized on a solid phase such as Sephadex resin or filter paper, using methods well known in the art. The immobilized antibody is contacted with a sample containing human SR-BI (or a fragment thereof) to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the SR-BI protein, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent which will release the SR-BI protein from the antibody. B. Screening Methods

Anti-SR-BI monoclonal antibodies of the present invention may also be used in drug screening methods based on competitive binding assays. Such methods may involve the steps of allowing competitive binding between a test compound (e.g. , a test antibody) in a sample and a known amount of an inventive anti-SR-BI monoclonal antibody, for binding to cells to which the inventive antibody binds, and measuring the amount of the known monoclonal antibody bound. The inventive monoclonal antibody is appropriately labeled, for example, with an enzymatic, chemiluminescent, or fluorescent label. Examples

The following examples describe some of the preferred modes of making and practicing the present invention. However, it should be understood that the examples are for illustrative purposes only and are not meant to limit the scope of the invention. Furthermore, unless the description in an Example is presented in the past tense, the text, like the rest of the specification, is not intended to suggest that experiments were actually performed or data are actually obtained.

Some of the results reported below have been described in an article (M. Zahid et al, Hepatology, 2013, 57: 492-504) and in two manuscripts entitled "Targeting Hepatitis C Virus Cell-cell Transmission Prevents Viral Resistance to Direct- Acting Antivirals and Results in Viral Clearance" by F. Xiao et al. and "Synergy of entry inhibitors with direct-acting antivirals uncovers novel combinations for prevention and treatment of hepatitis C" by Fofana et al. These manuscripts were submitted to Hepatology in June 2013 and August 2013, respectively. Example 1 : The Post-binding Activity of SR-BI

mediates HCV Cell- Cell Transmission

Materials and Methods

Cells. HEK293T, Chinese hamster ovary (CHO), Buffalo Rat Liver (BRL3A), Huh7, Huh7.5-GFP and Huh7.5.1 cells were cultured as described (Krieger et al., Hepatology, 2010, 51: 1144-1157; Pestka et al, Proc. Natl. Acad. Sci. USA, 2007, 104: 6025-6030; Dreux et al, PLoS Pathog., 2009, 5:el00031; Witteveldt et al, J. Gen. Virol., 2009, 90: 48-58). Primary human hepatocytes were isolated as previously described (Krieger et al, Hepatology, 2010, 51: 1144-1157). CHO and BRL3A cells expressing SR-BI were produced as described (Zeisel et al, Hepatology, 2007, 46: 1722-1731; Dreux et al, PLoS Pathog., 2009, 5:el00031).

Antibodies. Polyclonal (Zeisel et al, Hepatology, 2007, 46: 1722-1731) and monoclonal antibodies (mAbs) directed against the extracellular loop of SR-BI were raised by genetic immunization of Wistar rats and Balb/c mice as described (Zeisel et al, Hepatology, 2007, 46: 1722-1731) according to proprietary technology (Aldevron GmbH, Freiburg, Germany). Anti-SR-BI mAbs were purified using protein G and selected by flow cytometry for their ability to bind to human SR-BI (Zeisel et al, Hepatology, 2007, 46: 1722-1731). To determine the affinity of the anti-SR-BI mAbs for human SR-BI, Huh7.5.1 cells were incubated with increasing concentrations of mAbs and binding was assessed using flow cytometry. Kd values were determined as half- saturating concentrations of the mAbs using SigmaPlot. Anti-CD81 (JS-81), anti-SR-BI (CLA-1) and phycoerythrin (PE)-conjugated anti-mouse antibodies were from BD Biosciences. Anti-His and FITC-conjugated anti-His antibodies were from Qiagen and rabbit anti-actin (AA20-30) antibodies from Sigma- Aldrich. Anti-El (IGH526, Innogenetics), anti-E2 (IGH461, Innogenetics; AP33, Genentech; CBH23, a kind gift from S. K. H. Foung) and patient-derived anti-HCV IgG have been described (Haberstroh et al, Gastroenterology, 2008, 135: 1719-1728; Fofana et al, Gastroenterology, 2010, 39: 953-964; Fofana et al, Gastroenterology, 2012, 143: 223- 233).

Cell Culture-derived HCV (HCVcc) and Pseudoparticle Production and Infection. Production of luciferase reporter HCVcc, HCVpp, MLVpp and VSV-Gpp, infection and kinetic experiments have been described (Zeisel et al, Hepatology, 2007, 46: 1722-1731; Harris et al, J. Virol., 2008, 82: 5007-5020; Pestka et al, Proc. Natl. Acad. Sci. USA, 2007, 104: 6025-6030; Fofana et al, Gastroenterology, 2010, 39: 953-964; Bartosch et al, J. Exp. Med., 2003, 197: 633-642). HCVpp of genotypes 1-6 have been described (Lupberger et al, Nature Medicine, 2011, 17: 589- 595; Fofana et al, Gastroenterology, 2012, 143: 223-233). Chimeric HCVcc of genotypes 1-4 have been described (Koutsoudakis et al, J. Virol., 2006, 80: 5308- 5320). Unless otherwise stated, HCVcc experiments were performed using Luc-Jcl and infection was analyzed in cell lysates by quantification of luciferase activity (Koutsoudakis et al, J. Virol., 2006, 80: 5308-5320). For combination experiments, each antibody was tested individually or in combination with a second antibody. Huh7.5.1 cells were pre-incubated with anti-SR-BI or control mAb for 1 hour and then incubated for 4 hours at 37 °C with HCVcc or HCVpp (P02VJ) (pre-incubated for 1 hour with or without anti-envelope antibodies). Synergy was assessed using the combination index and the method of Prichard and Shipman (Zhu et al, J. Infect. Dis., 2012, 205: 656-662; Prichard et al, Antiviral Res., 1990, 14: 181-205). Cell viability was assessed using a MTT test (Lupberger et al, Nature Medicine, 2011, 17: 589- 595). Cellular Binding of Envelope Glycoprotein E2. Recombinant His-tagged soluble E2 (sE2) was produced as described (Dreux et al, PLoS Pathog., 2009, 5:el00031). Huh7.5.1 cells were pre-incubated with control or anti-SR-BI serum (1:50), anti-SR-BI or control mAbs (20 μg/mL) for 1 hour at room temperature (RT) and then incubated with sE2 for 1 hour at RT. Binding of sE2 was revealed using flow cytometry as described (Krieger et al, Hepatology, 2010, 51: 1144-1157; Dreux et al, PLoS Pathog., 2009, 5:el00031).

HCVcc Binding. Huh7.5.1 cells were pre-incubated with heparin (100 μg/mL), control or anti-SR-BI serum (1:50), anti-SR-BI or control mAbs (20 μg/mL) for 1 hour at 37°C prior to incubation with HCVcc as described (Krieger et al, Hepatology, 2010, 51: 1144-1157; Dreux et al, PLoS Pathog., 2009, 5:el00031). Non-bound HCVcc were removed by washing of cells with PBS and cell bound HCV RNA was then quantified by RT-PCR (Krieger et al, Hepatology, 2010, 51: 1144-1157; Dreux et al, PLoS Pathog., 2009, 5:el00031). HCV Cell-to-cell Transmission. HCV cell-to-cell transmission was assessed as described (Lupberger et al., Nature Medicine, 2011, 17: 589-595; Witteveldt et al., J. Gen. Virol., 2009, 90: 48-58). Producer Huh7.5.1 cells were electroporated with Jcl RNA (Pietschmann et al., Proc. Natl. Acad. Sci USA, 2006, 103: 7408-7413) and cultured with naive target Huh7.5-GFP cells in the presence or absence of anti-SR-BI or control mAbs. An HCV E2-neutralizing antibody (AP33, 25 μg/mL) was added to block cell-free transmission (Witteveldt et al, J. Gen. Virol., 2009, 90: 48-58). After 24 hours of co-culture, cells were fixed with paraformaldehyde, stained with an NS5A-specific antibody and analyzed by flow cytometry (Lupberger et al., Nature Medicine, 2011, 17: 589-595; Witteveldt et al, J. Gen. Virol., 2009, 90: 48-58). Immunofluorescence. Cell spread was assessed by visualizing Jcl -infected

Huh7.5.1 cells by immunoflorescence using anti-NS5A and anti-E2 (CBH23) antibody as described (Lupberger et al, Nature Medicine, 2011, 17: 589-595).

HDL Binding. HDL was labeled using Amersham Cy5 Mono-Reactive Dye Pack (GE Healthcare). Unbound Cy5 was removed by applying labeled HDL on illustra MicroSpin G-25 Columns (GE Healthcare). Blocking of Cy5-HDL binding with indicated reagents was performed for 1 hour at RT prior to Cy5-HDL binding for 1 hour at 4°C on 10⁶ target cells. Lipid Transfer Assays. Selective HDL-CE uptake and lipid efflux assays were performed as described (Dreux et ah, PLoS Pathog., 2009, 5:el00031; Le Goff et ah, J. Lipid Res., 2006, 47: 51-58). HDL-CE uptake was assessed in the presence or absence of anti-SR-BI mAbs (20 μg/mL) and ³H-CE-labelled HDL (60 μg protein) for 5 hours at 37°C. Selective uptake was calculated from the known specific radioactivity of radiolabeled HDL-CE and is denoted in μg HDL-CE^g cell protein. For lipid efflux assay, Huh7 cells were labeled with H-cholesterol (1 μθ/ι ί) and incubated at 37°C for 48 hours as described (Dreux et ah, PLoS Pathog., 2009, 5:el00031; de la Llera Moya et al, Arterioscler. Thromb. 1994, 14: 1056-1065). Cells were incubated with anti-SR-BI mAbs (20 μg/mL) for 1 hour prior to incubation with unlabeled HDL for 4 hours. Fractional cholesterol efflux was calculated as the amount of label obtained in the medium divided by the total in each well (radioactivity in the medium + radioactivity in the cells) regained after lipid extraction from cells. Statistical Analysis. Unless otherwise stated, data are presented as means + SD of three independent experiments. Statistical analyses were performed using Student's t test and Mann-Whitney test with a P value of <0.01 being considered statistically significant.

Results

Production of SR-BI-specific Monoclonal Antibodies Interfering with the

Post-Binding Steps of Viral Entry. To further explore the role of HCV-SR-BI interaction during HCV infection, the present Applicants have generated five rat and three mouse monoclonal antibodies (mAbs) directed against the human SR-BI (hSR- BI) ectodomain (Table 1). These antibodies bound to endogenous SR-BI on human hepatoma Huh7.5.1 cells and primary human hepatocytes (PHH) but did not bind to mouse SR-BI (mSR-BI) expressed on rat BRL cells (Figure 1(A)-(B), Figure 2). Three rats (QQ-4A3-A1, QQ-2A10-A5 and QQ-4G9-A6) and one mouse mAb (NK- 8H5-E3) markedly inhibited HCVcc infection in a dose-dependent manner with 50% inhibitory concentrations (IC₅₀) between 0.2 and 8 μg/mL (Figure 1(C)-(D), Table 1). The apparent K_d (Kaapp) corresponding to the half- saturating concentrations for binding to Huh7.5.1 cells ranged from 0.5 to 7.4 nM demonstrating that these antibodies recognize SR-BI with high affinity (Table 1). It is noteworthy that there seems to be a correlation between the antibody affinity and inhibitory capacity with the low affinity antibodies unable to block HCV infection.

Table 1. Monoclonal antibodies directed against human SR-BI. Isotype, binding affinity to Huh7.5.1 cells (Kd_app) as well as inhibition of HCVcc infection (IC₅₀) and inhibition of lipid transfer of anti-SR-BI mAbs are shown. Huh7.5.1 cells were incubated with increasing concentrations of mAbs and Kd values were determined as half- saturating concentrations of the mAbs. IC₅₀ was determined after incubation of Huh7.5.1 cells with serial dilutions of anti-SR-BI mAbs for 1 hour at room temperature before infection with HCVcc. The results represent means of three independent experiments performed in triplicate. Lipid uptake and efflux were assessed in Huh7 cells as described above in the presence of anti-SR-BI mAbs (20 μg/mL). The results are expressed as % inhibition of lipid transfer relative to cells incubated in the absence of antibody and represent means + SD of three independent experiments. N. d. : not determined

Inhibition of Inhibition of HDL-CE cholesterol

Kdg_pp IC50

mAb Isotype HCVcc uptake efflux (mean

Huh7.5.1 (nM)

(Mg/mL) (mean % ± % ± SD)

SD)

QQ-4A3-A1 rat lgG2b 1 .0 0.7 44.1 8 ± 1 .42 40.97 ± 0.92

QQ-2A10-A5 rat lgG2b 0.5 0.2 47.64 ± 1 .2 40 ± 1 .01

QQ-4G9-A6 rat lgG2b 0.5 1 .0 44.64 ± 1 .57 39.02 ± 1 .14

PS-6A7-C4 rat lgG2b n. d. n. d. 10.24 ± 1 .52 -2.52 ± 1 .25

PS-7B1 1 -E3 rat lgG2b n. d. n. d. 1 1 .73 ± 2.1 5.04 ± 0.83 mouse

NK-8H5-E3 7.4 8.0 56.28 ± 0.8 44.74 ± 0.55 lgG2b

NK-6B10-E6 mouse lgG1 n. d. n. d. 1 .28 ± 1 .69 18.41 ± 0.81

NK-6G8-B5 mouse lgG1 n. d. n. d. 5.64 ± 1 .04 13.08 ± 0.77

The Applicants then aimed at characterizing the viral entry steps targeted by these anti-SR-BI mAbs. They first assessed the ability of these mAbs to interfere with viral binding. To reflect the complex interaction between HCV and hSR-BI during viral binding, they studied the effect of anti-SR-BI mAbs on HCVcc binding to Huh7.5.1 cells at 4°C. Incubation of Huh7.5.1 cells with anti-SR-BI mAbs prior to and during HCVcc binding did not inhibit virus particle binding (Figure 2A). These data suggest that, in contrast to previously described anti-SR-BI mAbs, these novel anti-SR-BI mAbs do not inhibit HCV binding but interfere with HCV entry during post-binding steps. To characterize the potential post-binding steps targeted by these anti-SR-BI mAbs, the Applicants assessed HCVcc entry kinetics into Huh7.5.1 cells in the presence of anti-SR-BI mAbs inhibiting HCV infection (QQ-4A3-A1, QQ-2A10-A5 and QQ-4G9-A6 and NK-8H5-E3) added at different time-points during or after viral binding (Figure 2(B)). This assays was performed side-by-side with an anti-CD81 mAb inhibiting HCV post-binding (Zeisel et al, Hepatology, 2007, 46: 1722-1731; Krieger et al, Hepatology, 2010, 51: 1144-1157; Koutsoudakis et al, J. Virol., 2006, 80: 5308-5320) and proteinase K (Schwartz et al, J. Virol., 2009, 83: 12407-12414) to remove HCV from the cell surface. HCVcc binding to Huh7.5 cells was performed for 1 hour at 4°C in the presence or absence of compounds. Subsequently, unbound virus was washed away, cells were shifted to 37 °C to allow HCVcc entry and compounds were added every 20 minutes for up to 120 minutes after viral binding. These kinetic experiments indicate that anti-SR-BI mAbs inhibited HCVcc infection when added immediately after viral binding as well as 20 to 30 min after initiation of viral entry (Figure 2(C)) demonstrating that QQ-4A3-A1, QQ-2A10-A5 and QQ-4G9- A6 and NK-8H5-E3 indeed target post-binding steps of the HCV entry process. This timeframe is comparable to the kinetics of resistance of internalized virus to proteinase K (Figure 2(C)) indicating that these post-binding steps precede completion of virus internalization. Taken together, these data indicate that a post-binding function of SR-BI is essential for initiation of HCV infection. In contrast to previous anti-SR-BI mAbs inhibiting HCV binding (Catanes et al, J. Virol., 2010, 84: 34-43) as well as polyclonal anti-SR-BI antibodies and small molecules interfering with both viral binding and post-binding (Zeisel et al, Hepatology, 2007, 46: 1722-1731; Syder et al, J. Hepatol., 2011, 54: 48-55; Dreux et al, PLoS Pathog., 2009, 5:el00031), these antibodies are the first molecules exclusively targeting the post-binding function of SR-BI and thus represent a unique tool to more thoroughly assess the relevance of this function for HCV infection.

A Post-binding Function of SR-BI is Essential for Cell-to-cell Transmission and Viral Spread. HCV disseminates via direct cell-to-cell transmission (Witteveldt et al, J. Gen. Virol., 2009, 90: 48-58; Brimacombe et al, J. Virol., 2011, 85: 596- 605). To assess the role of SR-BI post-binding function in viral dissemination, the Applicants first investigated the ability of the anti-SR-BI mAbs to interfere with neutralizing antibody-resistant viral spread by studying direct HCV cell-to-cell transmission in the presence of anti-SR-BI mAbs QQ-2A10-A5 and QQ-4G9-A6. Viral "producer" cells containing replicating HCV Jcl (Pi) are co-cultured with GFP- expressing "target" cells (T) in the presence of E2-neutralizing mAb (AP33, 25 μg/mL) to prevent cell-free HCV transmission (Witteveldt et ah, J. Gen. Virol., 2009, 90: 48-58), AP33 reduces cell-free transmission by >90 and infectivity of producer cell supernatants is minimal at the time of co-culture; viral transmission thus occurs predominantly by cell-to-cell transmission (Lupberger et ah, Nature Medicine, 2011, 17: 589-595). HCV cell-to-cell transmission was assessed by quantifying HCV- infected, GFP-positive target cells (Ti) by flow cytometry (Lupberger et ah, Nature Medicine, 2011, 17: 589-595). Both anti-SR-BI mAbs (10 μg/mL) efficiently blocked HCV cell-to-cell transmission (Figure 3(A), Figure 4(A)-(B)) indicating that these antibodies may prevent viral spread in vitro. As these anti-SR-BI mAbs do not block HCV-SR-BI binding (Figure 2(A)) but inhibit HCV entry during post-binding steps (Figure 2C), these data suggest that a SR-BI post-binding function plays an important role during HCV cell-to-cell transmission.

To ascertain the importance of the SR-BI post-binding function in this process, the Applicants performed additional cell-to-cell transmission assays using mSR-BI, which is unable to bind E2. Cells lacking SR-BI and robustly replicating HCV, that would be an ideal model cell to study cell-to-cell transmission by mSR-BI in the absence of hSR-BI, have not been described. However, hSR-BI has been reported to be a limiting factor for HCV spread in Huh7-derived cells as overexpression of hSR- BI increases cell-to-cell transmission (Brimacombe et ah, J. Virol., 2011, 85: 596- 605). The Applicants thus used Huh7.5 cells or Huh7.5 cells overexpressing either mSR-BI, which is unable to bind E2, or hSR-BI, which is able to bind E2, as target cells. Cell-to-cell transmission was enhanced in Huh7.5 cells overexpressing either hSR-BI (2.04 + 0.03 fold overexpression) or mSR-BI (1.92 + 0.19 fold overexpression) as compared to parental cells (Figure 3(B)). These data indicate that E2-SR-BI binding is not mandatory for viral dissemination and confirm the crucial role of SR-BI post-binding function in this process. To assess whether anti-SR-BI mAbs may prevent viral dissemination when added post-infection, the Applicants performed a long-term analysis of HCVcc infection by culturing Luc-Jcl infected Huh7.5.1 cells in the presence or absence of control or anti-SR-BI mAbs QQ-4G9-A6 and NK-8H5-E3 as previously described (Lupberger et al, Nature Medicine, 2011, 17: 589-595). When added 48 hours after infection and maintained in the cell culture medium throughout the experiment, these anti-SR-BI mAbs efficiently inhibited HCV spread over 2 weeks in a dose-dependent manner without affecting cell viability (Figure 3(C)-(D), Figure 4(C)). The Applicants also assessed Jcl spread in Huh7.5.1 cells by NS5A- and E2-staining of infected cells as described (Lupberger et ah, Nature Medicine, 2011, 17: 589-595). While 74.5 + 2.3% and 70.0 + 3.2% of cells incubated with control rat or mouse mAbs stained positive for NS5A and E2, respectively, incubation with anti-SR-BI mAbs QQ-4G9-A6 and NK-8H5-E3 markedly reduced the number of NS5A-positive (14.2 + 3.4%) and E2-positive (16.7 + 2.6%) cells (Figure 3(E)-(F)). Taken together, these data indicate that a post-binding function of SR-BI is required for HCV cell-to- cell transmission and spread.

SR-BI Determinants Relevant for HCV Post-Binding Steps May be Linked to the Lipid Transfer Function of the Entry Factor. The SR-BI ectodomain has been demonstrated to be important for both HDL binding and CE uptake but the determinants involved in these processes have not been precisely defined yet. To assess whether anti-SR-BI mAbs inhibiting HCV post-binding steps affect HDL binding to SR-BI, the Applicants studied Cy5-labeled HDL binding to hSR-BI in the presence or absence of anti-SR-BI mAbs. In contrast to polyclonal anti-SR-BI serum which inhibited Cy5-labeled HDL binding, none of the anti-SR-BI mAbs markedly interfered with HDL-SR-BI binding at concentrations inhibiting HCV infection by up to 90% (Figure 5(A)).

Furthermore, the Applicants investigated the effect of these mAbs on CE uptake and cholesterol efflux. While PS-6A7-C4, PS-7B11-E3, NK-6B10-E6 and NK-6G8- B5 had no effect on lipid transfer, QQ-4A3-A1, QQ-2A10-A5, QQ-4G9-A6 and NK- 8H5-E3 partially reduced both CE uptake and cholesterol efflux at concentrations inhibiting HCV infection by up to 90% (Figure 5(B)-(C)). These data indicate that the anti-SR-BI mAbs inhibiting HCVcc infection also partially inhibit SR-BI mediated lipid transfer (Table 1). The Applicants also assessed the ability of the anti-SR-BI mAbs to bind to SR-BI mutants reported to modulate lipid transfer (Dreux et ah, PLoS Pathog., 2009, 5:el00031). Taken together, these data suggest that SR-BI determinants involved in HCV post-binding events do not mediate HDL binding but may contribute to lipid transfer, in line with the reported link between the SR-BI lipid transfer function and HCV infection (Bartosch et al, J. Virol, 2005, 79: 8217-8229; Dreux et al, PLoS Pathog., 2009, 5:el00031).

Synergy Between Antibodies Targeting SR-BI Post-Binding Function and Neutralizing Antibodies on Inhibition of HCV Infection. To assess the clinical relevance of blocking SR-BI post-binding function to inhibit HCV infection, the Applicants determined the effect of anti-SR-BI mAbs on entry into Huh7.5.1 cells of HCVpp of major genotypes and highly infectious HCV strains selected during liver transplantation (P02VJ). All anti-SR-BI mAbs inhibiting HCVcc genotype 2a infection (QQ-4A3-A1, QQ-2A10-A5, QQ-4G9-A6 and NK-8H5-E3) also inhibited infection of HCVpp of all major genotypes whereas VSV-Gpp entry was unaffected (Figure 6, Figure 7).

Moreover, entry of patient-derived HCVpp P02VJ into both Huh7.5.1 cells and PHH (primary human hepatocytes) was also efficiently inhibited by these anti-SR-BI mAbs (Figure 8). Given that combining compounds targeting both viral and host factors represents a promising future approach to prevent and treat HCV infection, the Applicants next determined whether the combination of anti-SR-BI mAb NK-8H5-E3 or QQ-2A10-A5 and anti-HCV envelope antibodies results in an additive or synergistic effect on inhibiting HCV infection. Combination of anti-SR-BI and anti- HCV envelope antibodies resulted in a synergistic effect on inhibition of HCVpp P02VJ entry and HCVcc infection as assessed using the combination index (combination index of 0.06-0.67) and the method of Prichard and Shipman (Zhu et al., J. Infect. Dis., 2012, 205: 656-662; Prichard et al., Antiviral Res., 1990, 14: 181-205). These combinations reduced the IC₅₀ of anti-SR-BI mAb by up to 100-fold (Figure 8(A)-(D)). The marked synergy may be explained by the fact that the E2- and SR-BI- specific antibodies target highly complementary steps during HCV entry. Taken together, these data indicate that interfering with SR-BI post-binding function may hold promise for the design of novel antiviral strategies targeting HCV entry factors.

Protein Determinants Relevant for HCV Post-Binding Steps Lie Within the N-Terminal Half of the Human SR-BI Ectodomain. To map the protein determinants important for SR-BI post-binding function during HCV entry, the Applicants first performed cross-competition studies in order to determine whether these antibodies recognize overlapping or distinct epitopes. Labeled anti-SR-BI mAb NK-8H5-E3 was incubated with Huh7.5.1 cells in the presence of increasing concentrations of unlabeled anti-SR-BI mAbs. Cross-competition experiments with labeled versions of QQ-4A3-A1, QQ-2A10-A5 and QQ-4G9-A6 demonstrated that each of these mAbs reduced binding of unlabeled rat mAbs but not mouse mAb (Figure 9(A)-(C)). Moreover, in contrast to unlabeled mouse NK-8H5-E3, none of the three unlabeled rat mAbs (QQ-4A3-A1, QQ-2A10-A5 and QQ-4G9-A6) reduced binding of NK-8H5-E3 to Huh7.5.1 cells, comparable to control isotype mAb (Figure 9(D)). The mutual cross competition between the three rat mAbs suggests that they recognize overlapping or closely related epitopes on SR-BI while the mouse mAb recognizes a distinct epitope.

To further define the epitopes targeted by these antibodies, the Applicants investigated their ability to bind to human-mouse SR-BI chimeras, where part of the mouse SR-BI ectodomain was replaced by the corresponding human sequence. While the HHH and MMM SR-BI constructs refer to the wild- type human (H) and mouse (M) SR-BI molecules, respectively, the human/mouse SR-BI chimeras were denominated according to the origin of either SR-BI region, e.g., HMM bears region 1 from human SR-BI and regions 2 and 3 from murine SR-BI (Figure 10(A)). The overall homology between human and mouse SR-BI is 80% (54 aa difference). There is a total of 31, 14 and 9 different aa within the first, second and third region of the SR-BI human/mouse chimeras, respectively. The three rat anti-SR-BI mAbs QQ- 4A3-A1, QQ-2A10-A5, QQ-4G9-A6 bind to HMM SR-BI, i.e. aa 38-215, with high affinity and also to MHM, i. e. 216-398, to a lesser extent while the mouse mAb NK- 8H5-E3 only recognizes HMM SR-BI with high affinity (Figure 10(B)). These data suggest that the epitope targeted by NK-8H5-E3 lies in the N-terminal half of the human SR-BI ectodomain, between aa 38 and aa 215, while the epitope(s) targeted by QQ-4A3-A1, QQ-2A10-A5, QQ-4G9-A6 probably lie more downstream within the SR-BI ectodomain. To further map residues within SR-BI contributing to antibody binding we used previously described SR-BI point mutants (Dreux et al., PLoS Pathol., 2009, 5: el000310). Interestingly, point mutation Q420R and double mutation Q402R-E418R within human SR-BI abolished binding of the four anti-SR- BI mAbs inhibiting HCV infection (Figure 10(E)). These data suggest that aa 402, 418 and 420 may be part of the epitope of these antibodies or that these mutations may induce conformational changes within this epitopes.

Finally, to further characterize the nature of the epitopes targeted by the present panel of anti-SR-BI mAbs, the Applicants assessed the ability of the anti-SR-BI mAbs to bind to human SR-BI using SDS-PAGE and Western blot. Staining of SR-BI by anti-SR-BI mAb PS-6A7-C4, NK-6B10-E6 and NK-6G8-B5 suggest that the epitopes interacting with these antibodies, that do not inhibit HCV infection, probably include linear domains (Figure 11). In contrast, none of the antibodies inhibiting HCV infection interacted with linear SR-BI in Western blot experiments suggesting that the antibodies inhibiting HCV infection likely recognize predominantly conformational epitopes (Figure 11). Taken together, these data indicate that anti-SR-BI mAbs inhibiting HCVcc infection recognize conformational epitopes within the N-terminal half of the SR-BI ectodomain. Moreover, these data suggest that the N-terminal ectodomain of SR-BI contains protein determinants relevant for the SR-BI post- binding function in HCV entry.

Discussion

The Applicants have generated novel anti-SR-BI mAbs specifically inhibiting HCV entry during post-binding steps that enabled them for the first time, using endogenous SR-BI, to explore and validate the hypothesis that SR-BI has a multifunctional role during HCV entry and to elucidate the functional role of SR-BI post-binding activity for HCV infection. The present data demonstrate that the HCV post-binding function of hSR-BI can indeed be dissociated from its E2-binding function. Moreover, the Applicants demonstrated that the post-binding activity of SR- BI is of key relevance for cell-free HCV infection as well as cell-to-cell transmission. SR-BI mediates uptake of HDL-CE in a two-step process including HDL binding and subsequent transfer of CE into the cell without internalization of HDL. At the same time, SR-BI also participates in HCV binding and entry into target cells. SR-BI is able to directly bind E2 and virus-associated lipoproteins but additional function(s) of SR-BI have been reported to be at play during HCV infection (Bartosch et al, J. Virol., 2005, 79: 8217-8229; Zeisel et al, Hepatology, 2007, 46: 1722-1731; Dreux et al, PLoS Pathog., 2009, 5:el00031). The results from this study highlight the importance of a SR-BI post-binding function for HCV entry and further extend the relevance of this function for HCV cell-to-cell transmission.

The molecular mechanisms underlying HCV cell-to-cell transmission are only partially understood. A recent study showed that SR-BI contributes to this process (Brimacombe et al, J. Virol., 2011, 85: 596-605) and that E2-SR-BI interaction and/or SR-BI-mediated lipid transfer likely takes place during HCV dissemination as antibodies and small molecule inhibitors targeting both SR-BI binding and lipid transfer reduce HCV cell-to-cell transmission (Meuleman et al, Hepatology, 2012, 55: 364-372; Syder et al, J. Hepatol., 2011, 54: 48-55). However, which SR-BI functions are relevant for this process remained to be determined. Taking advantage of the novel mAbs uniquely inhibiting SR-BI post-binding activity required for HCV entry, the Applicants demonstrated that an E2 binding-independent post-binding function is involved in neutralizing antibody-resistant cell-to-cell transmission. E2- independent SR-BI function in HCV dissemination is in line with the observation that cell-to-cell transmission is largely insensitive to E2-specific antiviral mAbs (Brimacombe et al., J. Virol., 2011, 85: 596-605). Given that mSR-BI does not bind sE2 but mediates HCV entry and promotes cell-to-cell transmission, the post-binding function of SR-BI seems to be essential for HCV infection and dissemination while the binding function may be dispensable. Furthermore, since HVR1 -deleted HCV is less sensitive to inhibition by anti-SR-BI mAbs, HVR1-SR-BI interaction may play an important role during post-binding steps of HCV entry.

Previous studies using small molecule inhibitors indicated a role for SR-BI lipid transfer function in HCV infection and HDL-mediated entry enhancement (Bartosch et al, J. Exp. Med., 2003, 197: 633-642; Voisset et al, J. Biol. Chem., 2005, 280: 7793-7799; Dreux et al, PLoS Pathog., 2009, 5:el00031). As inhibition of cell-free HCV entry and cell-to-cell transmission by the novel anti-SR-BI mAbs was associated with interference with lipid transfer, the present data suggest that the SR-BI lipid transfer function may be relevant for both initiation of HCV infection and viral dissemination. Noteworthy, the novel anti-SR-BI mAbs are the first anti-SR-BI mAbs that do not inhibit HDL binding to SR-BI. These data suggest that HCV entry and dissemination can be inhibited without blocking HDL-SR-BI binding. The further characterization of the SR-BI post-binding function will allow to determine whether the SR-BI-mediated post-binding steps of HCV entry and dissemination are directly linked to its lipid transfer function.

Using SR-BI chimeras, the Applicants demonstrated that the determinants relevant for HCV post-binding steps lie within N-terminal half of the human SR-BI ectodomain (Figures 9, 10 and 11). Amino acids 70 to 87 and residue E210 of SR-BI are required for E2 binding while distinct protein regions are involved in HDL binding (Catanese et al, J. Virol., 2010, 84: 34-43; Guo et al, J. Lipid Res., 2011, 52: 2272- 2278). Although the SR-BI determinants involved in HDL binding and CE uptake have not been precisely defined yet, a recent study reported that amino acid C323 is critical for these processes (Guo et al., J. Lipid Res., 2011, 52: 2272-2278). Given that the novel anti-SR-BI mAbs do not interfere with E2 and HDL binding, amino acids 70-87 and residues E210 and C323 are most likely not part of the targeted epitope(s). Interestingly, the amino acid associated with cholesterol homeostasis (Vergeer et al., N. Engl. J. Med., 2011, 364: 136-145) probably also lies outside these epitope(s). The further characterization of the(se) epitope(s) may allow to more thoroughly determine the regions of SR-BI relevant for its post-binding function during initiation of HCV infection and spread.

Finally, the present data suggest that the SR-BI post-binding function is a highly relevant target for antivirals. Therapeutic options for a large proportion of HCV- infected patients are still limited by drug resistance and adverse effects (Pawlotsky, Hepatology, 2011, 53: 1742-1751). Furthermore, a strategy for prevention of HCV liver graft infection is absent. Antivirals targeting essential host factors required for the HCV life cycle are attractive since they may increase the genetic barrier for antiviral resistance (Lupberger et al., Nature Medicine, 2011, 17: 589-595; Zeisel et al., J. Hepatol., 2011, 54: 566-576). Indeed, the present data demonstrate a marked synergy on the inhibition of HCV entry when combining antibodies directed against the viral envelope and SR-BI. These results suggest that combining molecules directed against the virus and host entry factors is a promising strategy for prevention of HCV infection such as liver graft infection. The potent effect on cell-to-cell transmission and viral spread also opens a perspective of SR-BI-based entry inhibitors for treatment of chronic infection. Small molecules and mAbs targeting SR-BI and interfering with HCV infection have previously been described (Bartosch et al, J. Exp. Med., 2003, 197: 633-642; Syder et al, J. Hepatol., 2011, 54: 48-55; Catanese et al, J. Virol., 2007, 81: 8063- 8071). A human anti-SR-BI mAb has been reported to inhibit HDL binding, to interfere with cholesterol efflux and to decrease HCVcc entry during attachment steps without having a relevant impact on SR-BI mediated post-binding steps (Catanese et al, J. Virol., 2010, 84: 34-43; Catanese et al, J. Virol., 2007, 81: 8063-8071). A codon-optimized version of this mAb has been demonstrated to prevent HCV spread in vivo (Meuleman et al, Hepatology, 2012, 55: 364-372) underscoring the potential of SR-BI as an antiviral target. The mAbs generated in the present study are highly novel in their function as they do not interfere with HCV-SR-BI binding but inhibit HCV entry during post-binding steps of cell-free infection and cell-to-cell transmission. Furthermore, in contrast to previously described anti-SR-BI mAbs (Catanese et al, J. Virol., 2007, 81: 8063-8071), these mAbs do not hinder HDL-SR- BI binding and only partially inhibit lipid transfer at concentrations significantly inhibiting HCV infection. Given their novel mechanism of action and their potential differential toxicity profile, QQ-4A3-A1, QQ-2A10-A5, QQ-4G9-A6 and NK-8H5- E3 define a novel class of anti-SR-BI mAbs for prevention and treatment of HCV infection. Example 2: Synergistic Effects of Combinations Comprising an anti-SR-BI mAb

Materials and Methods

Cell Lines. Cultures of Huh7.5.1 (Zhong et al, Proc. Natl. Acad. Sci. USA, 2005, 102: 9294-2929) and HEK293T (Pestka et al, Proc. Natl. Acad. Sci. USA, 2007, 104: 6025-6030) cells, which have previously been described, were used in this study.

Monoclonal Antibodies and Inhibitors. Anti-SR-BI (NK-8H5-E3, QQ-4G9- A6 and QQ-A43-A1) mAbs were used in this series of experiments. Erlotinib and dasatinib were obtained from IC laboratories. The Cyclophilin A inhibitor (alisporivir), protease inhibitors (telaprevir, boceprevir, danoprevir and TMC-435), NS5A inhibitor (daclatasvir) and polymerase inhibitors (mericitabine and GS-7977 (formally known as PSI-7977)) were synthesized by Acme Bioscience, Inc. Anti- CLDN1 mAbs (OM-7D3-B3, OM-8A9-A4 and OM-6E1-B5) were used. Analysis of Antiviral Activity of Compounds and Combinations on HCV Infection. The in vitro antiviral activity of each compound was tested individually and in combination with a second compound using the HCVcc Huh7.5.1 cell culture described (Lupberger et al, Nature Medicine, 2011, 17: 589-595; Zhong et al., Proc. Natl. Acad. Sci. USA, 2005, 102: 9294-9299; Koutsoudakis et al., J. Virol., 2006, 80: 5308-5320). Production of HCVcc (Luc-Jcl; TCID₅₀ approximately 10⁴/ml) has been described (Koutsoudakis et al., J. Virol., 2006, 80: 5308-5320). For combination of the entry inhibitor (anti-SRBI mAb) with IFN-a, DAAs (telaprevir, boceprevir, danoprevir, TMC-435, daclatasvir, mericitabine and GS-7977) or alisporivir, Huh7.5.1 cells (culture in 96-well-plates) were pre-incubated with IFN-a, DAAs or alisporivir and the anti-SR-BI mAb for 1 hour at 37 °C before incubation for 4 hours at 37 °C with HCVcc in the presence of both compounds. For combination of entry inhibitors with entry inhibitors (i.e., the anti-SR-BI mAb with erlotinib or with dasatinib or with an anti-CLDNl mAb), Huh7.5.1 cells were pre-incubated with both entry inhibitiors or control reagent for 1 hour at 37°C. The mix was removed and Huh7.5.1 cells were incubated for 4 hours at 37°C with HCVcc in the presence of both compounds. HCVcc infection was analyzed two days later by lucif erase reporter gene expression as previously described (Krieger et al., Hepatology, 2010, 54: 1144-1157; Fofana et al., Gastroenterology, 2010, 139: 953-964; Koutsoudakis et al., J. Virol., 2006, 80: 5308-5320).

Analysis of Synergy. Synergy was assessed by two independent methods: the combination index (Fofana et al., Gastroenterology, 210, 139: 953-964; Koutsoudakis et al, J. Virol., 2006, 80: 5308-5320) and the method of Prichard and Shipman (Zhao et al, Clin. Cancer Res., 2004, 10: 7994-8004; Prichard et al, Antiviral Res., 1990, 14: 181-205). A CI of less than 0.9 indicates synergy; a CI equal to 0.9-1.1 indicates additivity; and a CI of more than 1.1 indicates antagonism (Zhao et al, Clin. Cancer Res., 2004, 10: 7994-8004; Zhu et al, J. Infect. Dis., 2012, 205: 656-662). The method of Prichard and Shipman was applied as described (Prichard et al. , Antiviral Res., 1990, 14: 181-205). Surface amplitudes > 20% above the zero plane indicate a synergistic effect, while surface amplitudes < 20% below the zero plane indicate antagonism. The validity of the assay and methods were confirmed by comparative analyses of combinations showing a non- synergistic effect. Toxicity Assays. Huh7.5.1 cells and primary human hepatocytes isolated and cultured as described were incubated with the compounds for 48 hours (Krieger et ah, Hepatology, 2010, 51: 1144-1157). Cytotoxic effects were analysed by the ability to metabolize 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as described (Lupberger et al, Nat. Med., 2011, 17: 589-595). An anti-Fas antibody (10 g/m) was used as a positive control.

Results

The results are presented on Figures 12-15 and Tables 2-5.

Anti-SR-BI mAb potentiates the antiviral activity of interferon-alpha in a synergistic manner. Since IFN-a is the key component of standard-of-care, the Applicants investigated whether the anti-SR-BI mAb could potentiate the antiviral activity of IFN-a by investigating the effect of combining the anti-SR-BI mAb with IFN-a2a or IFN-a2b on HCVcc infection. The antiviral effect of each molecule was tested alone or in combination to determine the combination index (CI) (Figure 12(A- B)). Combination of IFN-a2a or IFN-a2b with a sub-ICso concentration of SR-BI- specific mAb - which exerts only minimal inhibitory effect on HCV infection - resulted in a synergistic activity in inhibition of HCVcc infection (CIs of 0.30 and 0.56, respectively) Figure 12(A-B) and Table 2). In contrast, combination of IFN-a2a or IFN-a2b with sorafenib, a different kinase inhibitor, resulted in an antagonistic effect (CI of 1.32+0.02 and 1.34+0.07, respectively), demonstrating that the observed synergies are specific for the combinations and not related to technical issues of the model system.

Table 2. Synergy of the anti-SR-BI mAb and IFN-a2a or IFN-a2b on inhibition of HCV infection. The experiments were performed as described above. Means + SD from at least three independent experiments performed in triplicate are shown. ICso of anti-SR-BI, 1.3+0.4 μg/ml.

IC50 IC50 (Ul/ml) for

Compound 1 Compound 2 CI

(Ul/ml) combination

IFN-a2a 0.3±0.1 6 anti-SR-BI 0.08±0.04 0.30±0.03

IFN-a2b 0.2±0.05 anti-SR-BI 0.08±0.04 0.56±0.03 Synergy of Anti-SR-BI mAb and direct acting antivirals (DAAs) uncovers novel combinations for interferon-free regimens. A major effort of the pharmaceutical industry and current clinical research is the further improvement of IFN-based therapies using DAAs and the development of IFN-free combinations based on the combination of DAAs with or without ribavirin. Addressing these future concepts, the present Applicants have studied the combined antiviral effect of the anti- SR-BI mAb with the clinically licensed protease inhibitors, such as telaprevir (McHutchison et al, N. Engl. J. Med., 2010, 362: 1292-1303) and boceprevir (Poordad et al., N. Engl. J. Med., 2011, 364: 1195-1206), as well as second-generation protease inhibitors in late stage development, such as TMC-435 and danoprevir (Gane et al., Hepatology, 2011; 54: 76A - abstract 34) in the HCVcc model system.

Combination of a clinical licensed protease inhibitor, telaprevir or boceprevir and of a sub-ICso concentration of the ant-SR-BI mAb (which exerts only minimal inhibitory effect on HCV infection) resulted in CIs of 0.02 to 0.49, indicating synergy (see Figure 12(C) and Table 3). In contrast, combination of two protease inhibitors (telaprevir and boceprevir) resulted in an additive activity confirming the validity of the assay (Figure 12(C)).

Table 3. Synergy of entry inhibitors and direct acting antivirals on inhibition of HCV infection. Huh7.5.1 cells were pre-incubated with serial concentrations of DAAs (protease inhibitors: telaprevir, boceprevir, TMC-435 or danaorevir; NS5A inhibitor: daclatasvir; or polymerase inhibitors: mericitabine or GS-7977) and 0.01 g/ml anti- SRBI mAb or isotype control mAb for 1 hour at 37°C. Means + SD of at least three independent experiments performed in triplicate are shown. IC₅₀ of anti-SR-BI mAb, 1.3+0.4 ug/ml.

IC50 IC₅₀ (μΜ or nM¹)

Compound 1 Compound 2 CI

(μΜ or nM¹) for combination

telaprevir 0.15±0.06 anti-SRBI 0.001 ±0.0001 0.0210.01 boceprevir 0.14±0.02 anti-SRBI 0.02±0.002 0.1510.01

TMC-435 0.013±0.001 anti-SRBI 0.006±0.0007 0.4910.06 danoprevir 0.006±0.003 anti-SRBI 0.0007±0.001 0.1510.02 daclatasvir 0.012±0.003 anti-SRBI 0.000210.0004 0.0310.004 mericitabine 0.12±0.03 anti-SRBI 0.01910.007 0.1710.06

GS-7977 0.12±0.03 anti-SRBI 0.00310.0007 0.1510.04

Telaprevir, boceprevir, danoprevir, TMC-435, mercitabine, PSI-7977: μΜ ; daclatasvir : nM Second-generation protease inhibitors have been demonstrated to have a higher genetic barrier for resistance. However, single amino acid substitutions are able to confer drug resistance in vivo. Importanly, it has been demonstrated that several telaprevir- and boceprevir-resistance mutations confer cross-resistance to these second-generation protease inhibitors (Sarrazin et ah, J. Hepatol., 2012, 56(1): S88- 100). Combination of a second-generation protease inhibitor, TMC-435 or danoprevir, and the anti-SR-BI mAb resulted in a synergistic activity (CIs of 0.49 and 0.15, respectively - see Figure 12(C) and Table 3), demonstrating the relevance of adding an entry-inhibitor as a concept to improve antiviral efficacy. A number of novel DDAs have reached early- to late-stage clinical development, including NS5A inhibitors and polymerase inhibitors. The first NS5A inhibitor, daclatasvir (Gao et ah, Nature, 2010, 465: 96-100), has been shown to have potent antivial activity against HCV genotype 1 in monotherapy. However, its genetic barrier to resistance is low and resistant variants developed rapidly without improsing a loss of in vivo viral fitness (Gao et ah, Nature, 2010, 465: 96-100). A marked synergy was observed for the combination of daclatasvir with all the anti-SR-BI mAb (CI of 0.03, see Figure 12(C) and Table 3). The combination of daclatasvir and the anti-SR-BI mAb decreased its IC₅₀ up to 60 fold (Figure 13(A)).

Finally, the Applicants investigated the synergy between the anti-SR-BI mAb and the polymerase inhibitor GS-7977. GS-7977 is currenlty in clinical development and has been suggested as having the potential to become the conerstone of an efficacious, all-oral combination regimen for many patients with chronic HCV infection (Zeisel et ah, Front Biosci., 2009, 14: 3274-3285; Zeisel et ah, J. Hepatol., 2011, 54: 566-576). Thus, the inventors investigated whether the anti-SR-BI mAb potentiates the antiviral activity of GS-7977. The combination of GS-7977 with the anti-SR-BI mAb resulted in a synergistic activity, with a CI of 0.15 (Figure 12(C) and Table 3); and decreased its IC₅₀ from 0.021 μΜ to 0.003 μΜ (Figure 14(A)). These data clearly demonstrate the potential of combining GS-7977 with entry inhibitors to improve antiviral activity. Similar results were obtained for the combination of another polymerase inhibitor, mericitabine (Gane et ah, Lancet, 2010, 376: 1467-1475) and the anti-SR-BI mAb (CI of 0.17 - see Figure 12(C) and Table 3). To further confirm the synergistic effect over a broad range of concentrations of both compounds, the inventors performed combinations testing a full checker-board of compounds dose-response curves using the method of Prichard and Shipman (Prichard et ah, Antiviral Res., 1990, 14: 181-205). Noteworthy, in particular low doses of both compounds results in an antiviral activity above the expected value (Figure 13(B)). There results also suggest an opportunity to reduce the doses of both compounds of the combination - a key requirement for improvement of future antiviral treatment.

Noteworthy, none of all the DAA-entry inhibitor combinations tested in the present study resulted in detectable toxicity in primary human hepatocytes (Table 4).

The results obtained suggest an opportunity to reduce the doses of both compounds - a key requirement for improvement of future antiviral treatment. Therefore, the present Applicants have demonstrated that the addition of a sub-ICso concentration of the anti-SR-BI mAb is sufficient to markedly decrease the IC₅₀ concentration of the different DAAs currently evaluated in IFN-free regimens, without displaying any toxic effects in vitro. These data demonstrate the proof-of-concept that entry inhibitors and DAAs are highly synergistic and define novel antiviral combinations for further preclinical and clinical development in IFN-free regimens.

Table 4. Absence of toxicity of combinations of compounds in primary human hepatocytes (PHH). Cytotoxic effects on PHH using the highest concentrations of each compound used in combination (DAAs, 10 μΜ; anti-SR-BI mAb, 10 μg/ml) were assessed by analyzing the ability to metabolize MTT). Anti-Fas antibody (10 μg/ml) was used as a positive control of toxicity. Toxicity analyses of the most efficient combinations are shown. Data are presented as relative cell viability compared to PHH cultured in the absence of compounds or solvent (=100%). Means + SD from one representative experiment performed in triplicate is shown.

Relative cell

Compound 1 Concentration Compound 2 Concentration

viability (%) daclatasvir 1 0 μΜ anti-SRB I 1 0 μς/ιτι Ι 1 01 ±2

GS-7977 1 0 μΜ anti-SR-B I 1 0 μς/ιτι Ι 1 1 0±7 anti-Fas 1 0 μς/ιη Ι 1 6±2

Combination of anti-SR-BI mAb and host-targeting agents. It is still not clear whether DAA-based therapies will be effective in difficult-to-treat populations such as patients with co-morbitity, co-medication or immunosuppression and patients undergoing liver transplantation. Combinations of host-targeting agents (HTAs) represent a promising alternative to DAAs for IFN-sparring regimens, allowing increasing the genetic barrier for resistance.

The most advanced HTA is the cyclophilin A inhibitor alisporivir (Flisiak et ah, Hepatology, 2009, 49: 1460-1468). Therefore, the Applicants investigated the effects of a combination of alisporivir and the anti-SR-BI rriAb, and observed a marked synergistic activity (CI of 0.16 - see Figure 15(A) and Table 5). In contrast, combination of two receptor- specific mAbs, such as anti-CLDNl and anti-CD81 mAbs, resulted only in an additive activity with a CI of 0.98+0.06 (Figure 15(A)). Interestingly, combination of the anti-SR-BI mAb and a protein kinase inhibitor, erlotinib or dasatinib, also resulted in a synergistic effect on HCVcc infection, with CIs of 0.18 in both cases (Figure 15(B) and Table 5). Combination of an anti-SR-BI mAb and of an anti-CLDNl mAb also resulted in a synergistic effect (see Figure 19).

Table 5. Synergy of host-targeting agents on the inhibition of HCV infection. Huh7.5.1 cells were pre-incubated with serial concentrations of alisporivir and 0.01 μg/ml of anti-SRBI mAb or isotype control mAb or 0.1 μΜ PKIs (erlotinib or dasatinib) or with serial diluation of anti-SRBI mAb or isotype control mAbs for 1 hour at 37°C and 0.1 μΜ PKIs (erlotinib or dasatinib). Means + SD from at least three independent experiments performed in triplicate are shown. IC₅₀ of anti-SR-BI mAb 1.3+0.4

erlotinib, 0.5+0.06 μΜ; dasatinib, 0.4+0.3 μΜ.

IC50 IC50 (μΜ)

Compound 1 Compound 2 CI

(μΜ) for combination alisporivir 2.2±0.5 anti-SRBI 0.31 ±0.02 0.16±0.01 erlotinib 0.002±0.001 0.18±0.007 anti-SRBI 1 .3±0.4

dasatinib 0.015±0.007 0.18±0.01 anti-CLDN1 0.18±0.03 anti-CD81 0.056±0.002 0.98±0.06

Example 3: Effects of Monoclonal Anti-SR-BI Antibodies on Viruses Resistant to Direct-Acting Antivirals (DAAs)

Antiviral resistance remains a major challenge for treatment of chronic HCV infections. The functional role of viral dissemination for emergence and maintenance of antiviral resistance is largely unknown. HCV is transmitted via cell-free diffusion but also uses direct cell-cell transfer to infect neighboring cells (Meredith et ah, J. Hepatol, 2013, 53: 1074-1080; Timpe et al, Hepatology, 2008, 47: 17-24). While cell-free entry is most relevant for initiation of HCV infection, HCV neutralizing antibody-resistance cell-cell transmission is thought to play an important role in viral persistence (Zeisel et al, J. Hepatol., 2013, 58: 375-384). The majority of host factors involved in cell-free entry (including SR-BI) also contribute to cell-cell transmission. However, in contrast to the majority of monoclonal antibodies targeting the virus, host-targeting entry inhibitors (HTEIs) potently inhibit HCV cell-cell transmission (Zeisel et al, J. Hepatol., 2013, 58: 375-384; Lupberger et al, Nature Med., 2011, 17: 589-595; Sainz et al, Nature Med., 2012, 18: 281-285; Zahid et al, Hepatology, 2013, 57: 492-504). In the present study, the inventors aimed to assess the role of cell-cell transmission in antiviral resistance using HCV infection as a model, and explore cell-cell transmission as a target to prevent and treat resistance to direct-acting antivirals.

Materials and Methods

Cells. Origin and culture of Huh7 and Huh7.5-GFP have been described in

Example 2.

Monoclonal Antibodies and Inhibitors. Anti-SR-BI (NK-8H5-E3) mAb was used in this study. Erlotinib was obtained from IC laboratories. Anti-E2 mAb (AP33, Genetech) and human anti-HCV IgG have been described (Witteveldt et al, J. Gen. Virol., 2009, 90: 48-58; Fofana et al, Gastroenterology, 2012, 143: 223-233). Mouse IgG was purchased from BD. NS 5 -A- specific mAb was obtained from Virostat. Inhibitors of HCV protease (telaprevir, boceprevir and simeprevir) and HCV NS5A (daclatasvir) were synthesized by Acme Bioscience, Inc..

HCVcc Production, Infection and Cell-Free Inhibition. Drug-resistant individual or combined mutations were introduced in the NS3 region of the Luc-Jcl (genotype 2a/2a) and/or Jcl plasmid (Koutsoudakis et al, J. Virol., 2006, 80: 5308- 5320; Piestchmann et al, Proc. Natl. Acad. Sci. USA, 2006, 103: 7408-7413; Wakita et al, Nature Med., 2005, 11: 791-796) using the QuikChange II XL site-directed mutagenesis kit (Strategene) as previously described (Zhu et al, J. Infect. Dis., 2012, 205: 656-662). A one-step PCR mutagenesis was performed. Mutations A156S or L36M and R155K were confirmed by DNA sequence analysis (GATC Biotech) for the desired mutation and for exclusion of unexpected residue changes in the NS3 encoding sequences. HCVcc J4 (genotype 2a/lb) and HCVcc J4 NS5A-Y93H (Y2065H) have been described (Scheel et al., Gastroenterology, 2011, 140: 1032- 1042). HCVcc (TCIDso approximately 10³/ml to 10⁴/ml) were produced as previously described (Lupberger et al, Nature Med., 2011, 17: 589-595). Huh7.5.1 cells were pre-incubated with serial concentrations of protease inhibitors (telaprevir, boceprevir), NS5A inhibitor (daclatasvir), anti-SR-BI monoclonal antibody or control reagents for 1 hour at 37°C before incubation for 4 hours at 37°C with wild-type or chimeric HCVcc. Viral infection was analysed by assessing the intracellular luciferase activity (Lupberger et al, Nature Med., 2011, 17: 589-595; Fofana et al, Gastroenterology, 2010, 139: 953-964) or intracellular HCV RNA levels as previous described (Lupberger et al, Nature Med., 2011, 17: 589-595; Fofana et al, Gastroenterology, 2010, 139: 953-964; Zeisel et al, Hepatology, 2007, 46: 1722-1731) Absent HCV RNA quantification by RT-PCR was confirmed using Abbott RealTime HCV assay (LOD 48 IU/ml). Statistical Analysis. Unless otherwise stated, results are expressed as means + standard deviation (SD) from at least 3 independent experiments performed in triplicate. Statistical analyses were performed using Student t test.

Results and Discussion

Functional Characterization of DAA-resistant Viruses and their Sensitivity to Monoclonal Anti-SR-BI antibody. To functionally characterize DAA-resistant viruses in cell-free and cell-cell transmission model systems, the Applicants generated DAA-resistant viruses and assessed their infectivity in state-of-the-art infection models. They first used classical Huh7.5.1 cell infection assays and characterized the ability of DAA-resistant viruses to infect hepatoma cells and their sensitivity to DAAs and HTEIs. Thereto, they introduced individually in the backbone of cell culture- derived HCV (HCVcc) Luc-Jcl (genotype 2a/2a) two mutations at positions 155 and 156 of the HCV NS3 protein (Sarrazin et al, J. Hepatol., 2012, 56(Suppl. 1): S88-100; Hiraga et al, Hepatology, 2011, 54: 781-788) known to induce cross-resistance in vitro and in vivo to protease inhibitors (teleprevir and boceprevir). Introduction of mutations A156S and R155K into Luc-Jcl increased the IC₅₀S of telaprevir and boceprevir up to 10-fold, respectively, demonstrating that these DAA-resistant viruses are indeed able to escape cell-free inhibition by DAAs (Fig. 16(A)-(B)). In contrast, the SR-BI- specific monoclonal antibody potently inhibited infection of DAA-resistant viruses as shown by similar levels of inhibition for both wild-type and protease- resistant viruses (Fig. 16(C)).

Mutation Y2065H (NS5A-Y93H) confers resistance to daclatasvir, an HCV HS5A inhibitor, among recombinant HCV variants with NS5A from genotypes 1-7 (30). As shown in Fig. 2A, the inventors have demonstrated that mutation NS5A- Y93H in HCVcc J4 (genotype 2a/lb) increased the IC₅₀ of daclatasvir up to 10-fold. In contrast, the IC₅₀ of CLDN1 -specific mAb to J4 NS5A-Y93H remained unchanged (Fig. 2B). These data indicate that DAA-resistant viruses robustly infect hepatoma cells but are efficiently inhibited by HTEIs in cell-free assays without exhibiting cross-resistance. These data suggest that HTEIs are effective in preventing infection of DAA-resistance variants.

The ability of a virus to spread within a host is a key determinant of its persistence and virulence. While cell-free entry is important for initiation of infection by virions entering the liver through the bloodstream, HCV dissemination within the liver and establishment of chronic HCV infection may mainly occur by direct cell-cell transmission between adjacent hepatocytes (Timpe et al, Hepatology, 2008, 47: 17- 24). Functional results obtained in cell culture and animal models demonstrated strong evidence that cell-cell transmission plays a relevant role in dissemination of several viruses including HBV, HIV, herpes simplex virus (HSV), measles virus or human T-lymphotropic virus type 1 (HTLV-1) (Sattentau et al, Nat. Rev. Microbiol., 2008, 6: 815-826; Petersen et al, Nat. Biotechnol., 2008, 26: 335-341). Interestingly, HIV cell-cell transmission has been reported to be more efficient than cell-free infection and less sensitive to inhibition by antiretroviral drugs (Sigal et al, Nature, 2011, 477: 95-98). For HCV, all DAAs currently in development induce emergence of generally fit resistant variants in vitro after multiple passages (Rong et al, Sci. Transl. Med., 2011, 2: 30-32) and DAA-resistant variants may become in vivo the dominant strain, subsequently leading to viral breakthrough and therapy failure (Kuntzen et al, Hepatology, 2008, 48: 31769-1778). Although differences in the ability of diverse genotypes to spread via cell-free and cell-cell transmission have been observed (Meredith et al, J. Hepatol., 2013, 53: 1074-1080), cell-cell transmission appears to serve as an important route of transmission for most genotypes (Meredith et al., J. Hepatol., 2013, 53: 1074-1080; Brimacombe et al., J. Virol. 2011, 85: 596-605).

In this study, the Applicants have demonstrated for the first time that DAA- resistant HCV strains disseminate using preferentially cell-cell transmission (data not shown) and that effective blockade of cell-cell transmission using the anti-SR-BI monoclonal antibody prevents viral resistance resulting in rapid, efficient and sustained virus elimination. This has major consequences for the development and prevention of DAA-resistance. HTEIs act on cellular targets and thus may impose a higher genetic barrier for resistance than DAAs. Since drug-resistant variants pre- exist within the patient's quasispecies before treatment (Pawlotsky et al., Sci Transl. Med., 2012, 4: 140-142), blocking the spread of DAA-resistant variants will be of major importance for preventing viral breakthrough caused by DAA-resistant variants.

Example 4: In vivo Effects of Monoclonal Anti-SR-BI Antibodies

To investigate the effect of the anti-SR-BI mAb (QQ-2A10-A5) on chronic infection, the Applicants utilized mice persistently infected with HCVcc Jcl (genotype 2a/2a). HCV Jcl -infected mice, persistently infected for 24 to 50 days, received 4 weekly antibody doses of SR-BI-specific or control mAb. The SR-BI- specific mAb-treated mouse showed a rapid decline in viral load with undetectable HCV RNA after 2 injections (Fig. 18(A)) that were sustained for at least 6 weeks. Human albumin levels remained stable during and post antibody administration and were higher in the SR-BI mAb-treated mouse than in the control mAb-treated mouse following antibody-treatment (Fig. 18(B)), indicating the presence of viable and functional hepatocytes following mAb treatment and exclude that the antiviral activity was due to a toxic effect on hepatocytes.

Other Embodiments

Other embodiments of the invention will be apparent to those skilled in the art from a consideration of the specification or practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope of the invention being indicated by the following claims.

Claims

claimed is:

A hybridoma cell line deposited at the CNCM (Collection Nationale de Culture de Microorganismes, Institut Pasteur, Paris, France) on August 1, 2012 under an Accession Number selected from the group consisting of CNCM I- 4662, CNCM 1-4663, CNCM 1-4664 and CNCM 1-4665.

An anti-SR-BI monoclonal antibody secreted by a hybridoma cell line deposited according to claim 1.

The anti-SR-BI monoclonal antibody or biologically active fragment thereof according to claim 2, wherein the monoclonal antibody is humanized, de- immunized or chimeric.

The anti-SR-BI monoclonal antibody or biologically active fragment thereof according to claim 2, wherein the monoclonal antibody is humanized, de- immunized or chimeric, and comprises complementary determining regions (CDRs), or portions thereof, derived from the secreted monoclonal antibody.

A molecule comprising an anti-SR-BI monoclonal antibody or biologically active fragment thereof according to any one of claims 2-4, wherein, optionally, the monoclonal antibody, or fragment thereof, is attached to a detectable moiety or a therapeutic agent.

The anti-SR-BI monoclonal antibody or biologically active fragment thereof according to any one of claims 2-4 or the molecule according to claim 5 for use in the prevention of Hepatitis C virus (HCV) infection of a cell.

The anti-SR-BI monoclonal antibody or biologically active fragment thereof according to any one of claims 2-4 or the molecule according to claim 5 for use in the prevention or treatment of a HCV infection or a HCV-related disease in a subject.

The anti-SR-BI monoclonal antibody or biologically active fragment thereof according to any one of claims 2-4 or the molecule according to claim 5 for use in the prevention of HCV recurrence in a liver transplantation patient. The anti-SR-BI monoclonal antibody or biologically active fragment thereof according to any one of claims 2-4 or the molecule according to claim 5 for the use according to any one of claims 6 to 8, wherein HCV infection is due to HCV of a genotype selected from the group consisting of genotype 1, genotype 2, genotype 3, genotype 4, genotype 5 and genotype 6.

The anti-SR-BI monoclonal antibody or biologically active fragment thereof according to any one of claims 2-4 or the molecule according to claim 5 for the use according to claim 9, wherein HCV infection is due to HCV of a subtype selected from the group consisting of subtype la, subtype lb, subtype 2a, subtype 2b, subtype 2c, subtype 3a, subtype 4a-f, subtype 5a, and subtype 6a.

The anti-SR-BI monoclonal antibody or biologically active fragment thereof according to any one of claims 2-4 or the molecule according to claim 5 for the use according to any one of claims 6 to 8, wherein HCV infection is due to a HCV resistant to at least one direct-acting antiviral.

The anti-SR-BI monoclonal antibody or biologically active fragment thereof according to any one of claims 2-4 or the molecule according to claim 5 for the use according to claim 11, wherein the at least one direct-acting antiviral is a protease inhibitor.

The anti-SR-BI monoclonal antibody or biologically active fragment thereof according to any one of claims 2-4 or the molecule according to claim 5 for the use according to claim 12, wherein the at least one protease inhibitor is boceprevir or telaprevir.

A pharmaceutical composition comprising an effective amount of an anti-SR- BI monoclonal antibody or biologically active fragment thereof according to any one of claims 2-4 or a molecule according to claim 5, and at least one pharmaceutically acceptable carrier or excipient.

The pharmaceutical composition according to claim 14 adapted for use in combination with at least one anti-viral agent.

The pharmaceutical composition according to claim 14 further comprising at least one anti- viral agent. The pharmaceutical composition according to claim 15 or claim 16, wherein the anti-viral agent is selected from the group consisting of interferons, rabivirin, anti-hepatitis C virus monoclonal antibodies, anti-hepatitis C virus envelope monoclonal antibodies, anti-hepatitis C virus polyclonal antibodies, RNA polymerase inhibitors, protease inhibitors, IRES inhibitors, helicase inhibitors, antisense compounds, ribozymes, and any combination thereof.

A pharmaceutical kit comprising a monoclonal antibody or biologically active fragment thereof according to any one of claims 2-4 or the molecule according to claim 5

A combination of at least one anti-SR-BI monoclonal antibody according to any one of claims 2-4 and:

at least one anti-HCV envelope antibody selected from anti-El antibodies, anti-E2 antibodies, and anti-HCV IgGs from individuals chronically or previously infected with HCV, or

at least one protein kinase inhibitor selected from erlotinib and dasatinib, or

at least one direct acting antiviral selected from telaprevir, boceprevir, danoprevir, TMC-435, daclatasvir, mericitabine, and GS-7977; or

at least one interferon selected from IFNcc-2a and IFNcc-2b, or at least one host- targeting agent such as alisporivir or an anti-CLDNl monoclonal antibody,

wherein the anti-SR-BI monoclonal antibody and anti-HCV envelope antibody, or the anti-SR-BI monoclonal antibody and protein kinase inhibitor, or the anti-SR-BI monoclonal antibody and direct acting antiviral, or the anti- SR-BI monoclonal antibody and host-targeting agent act in synergy to inhibit HCV infection.

The combination according to claim 19 for use in the treatment of HCV infection or a HCV-related disease in a subject; or for the control of chronic HCV infection in a subject; or for the prevention of HCV re-infection and recurrence in a liver transplantation patient.

The combination according to claim 19 for the use according to claim 20, wherein the HCV infection, chronic HCV infection or HCV re-infection is due to HCV of a genotype selected from the group consisting of genotype 1, genotype 2, genotype 3, genotype 4, genotype 5 and genotype 6.

22. The combination according to claim 19 for the use according to claim 21, wherein the HCV infection, chronic HCV infection or HCV re-infection is due to HCV of a subtype selected from the group consisting of subtype la, subtype lb, subtype 2a, subtype 2b, subtype 2c, subtype 3a, subtype 4a-f, subtype 5a, and subtype 6a.

23. The combination according to claim 19 for the use according to claim 20, wherein the HCV infection, chronic HCV infection or HCV re-infection is due to a HCV resistant to at least one direct- acting antiviral.

24. The combination according to claim 19 for the use according to claim 23, wherein the at least one direct-acting antiviral is a protease inhibitor.

25. The combination according to claim 19 for the use according to claim 24, wherein the at least one protease inhibitor is boceprevir or telaprevir.