Indian Patents. 259637:ANTIGEN BINDING PROTEINS DIRECTED AGAINST SCAVENGER RECEPTOR B1 THAT INHIBIT HCV REPLICATION

Title of Invention	ANTIGEN BINDING PROTEINS DIRECTED AGAINST SCAVENGER RECEPTOR B1 THAT INHIBIT HCV REPLICATION
Abstract	The present invention features antigen binding protein that bind to a SR-BI target region identified herein as a region involved in HCV E2 binding. Identified target regions are regions bound by a single-chain antibody of SEQ ID NOs:1,2, 3 or 4.

Full Text	TITLE OF THE INVENTION ANTIGEN BINDING PROTEINS DIRECTED AGAINST SCAVENGER' RECEPTOR Bl THAT INHIBIT HCV REPLICATION BACKGROUND OF THE INVENTION The references cited in the present application are not admitted to be prior art to the claimed invention. It is estimated that about 3% of the world's population is infected with the hepatitis C virus (HCV). (Wasley el al.. Semin. Liver Dis. 20:1-16, 2000.) HCV exposure results in an overt acute disease in a small percentage of cases, while in most instances the virus establishes a chronic infection causing liver inflammation and slowly progresses into liver failure and cirrhosis. (Slradere/ al.. JLAR J. 42:107-116, 2001.) Epidemiological surveys indicate an important role for HCV in the onset of hepatocellular carcinoma. (Strader^/o/., ILARJ. 42:107-116, 2001.) HCV can be classified into a number of distinct genotypes (1 to 6), and subtypes (a to c). The distribution of the genotypes and subtypes varies both geographically and between risk groups. (Robertson et al., Arch Virol. 743:2493-2503, 1998.) The HCV genome consists of a single strand RNA about 9.5 kb encoding a precursor polyprotein of about 3000 amino acids. (Choo ei al.. Science 244:362-364, 1989, Choo et al.. Science 244:359-362, 1989.) The HCV polyprotein contains the viral proteins in the order: C-El-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B. Cleavage of the preciusor polyprotein results in mature structural and non-structural viral proteins. (Neddermann et al.. Rial Chem 375:469-476, 1997.) As part of its infection cycle, HCV enters into a cell. Host cell LDL receptors and CD81 molecules have been identified as putative HCV receptors. The LDL receptor has been suggested to mediate* virus internalization via binding to LDL particles that are virus-associated. (Agnello et al., r'roc. Nail. Acml Sc, U.S.A. %: 12766-12771, 1999.) TheCHHl molecule has been suggested to bind HCV E2 based on recombinant envelope protein E2 from HCV genotype la. (Pileri et ai. Science 262:938-941, 1998.) HCV envelope glycoprotein L2 was found to bind human hepaioma cells independently l CDK1 The receptor responsible for E2 binding to human hepatic cells was identified as the human M .ivoij'cr icccplor class H type I (SR-BI). (Scarselh ,-t ,,l . The EM HO Journal 27:5017 5025. 2002.) SUMMARY OF THE INVENTION The present invention features antigen binding protein that bind to a SR-BI target region identified herein as a region involved in HCV E2 binding. Identified target regions are regions bound by a single-chain antibody of SEQ ID NOs: 1, 2, 3 or 4. Thus, a first aspect of the present invention features an isolated antigen binding protein . comprising a first variable region and a second variable region. The first and second variable regions bind one or more target regions selected from the group consisting of: the SEQ ID NO: 1 target region, the SEQ ID NO: 2 target region, the SEQ ID NO: 3 target region, and the SEQ ID NO: 4 target region. Reference to "isolated" indicates a different form than found in nature. The different form can be, for example, a different purity than found in nature and/or a structure that is not found in nature. A structure not found in nature includes recombinant structures where different regions are combined together, for example, humanized antibodies where one or more murine CDR is inserted onto a human framework scaffold, hybrid antibodies where one or more CDR from an antibody binding protein is inserted into a different framework scaffold, and antibodies derived from natural human sequences where genes coding light and heavy variable domains were randomly combined together. The isolated protein is preferably substantially free of serum proteins. A protein substantially free of serum proteins is present in an environment lacking most or all serum proteins. A "variable region" has the structure of an antibody variable region from a heavy or light chain. Antibody heavy and light chain variable regions contain three complementary determining regions interspaced onto a framework. The complementary determining regions are primarily responsible for recognizing a particular epitope. A target region defined with respect to SEQ ID NOs: 1, 2, 3 or 4, is an SR-BI region to which the corresponding single-chain antibody binds. For example, the SEQ ID NO: 1 target region is a region to which a polypeptide of SEQ ID NO: 1 binds. A protein binding to same target region as an identified target region competes with either SEQ ID NOs: 1, 2, 3 or 4 for binding to the identified target region. For example, a protein that competes with a polypeplide of SEQ ID NO: 1 for binding to SR-BI binds to the SEQ ID NO: 1 target region. Reference to "protein" or "polypeplide" indicates a contiguous amino acid sequence and does not provide a minimum or maximum size limitation. One or more amino acids present in the protein or polypcptide rnay contain a post-translational modification, such as glycosylation and disulfide bond formal ion. A preferred antigen binding protein is a monoclonal antibody. Reference to a "monoclonal antibody" indicates a collection of antibodies having the same, or substantially the same. complementary determining region, and binding specificity. The variation in the monoclonal antibodies is that which would occur if the antibodies were produced from the same construct(s). Monoclonal antibodies can be produced, for example, from a particular hybridoma and from a recombinant cell containing one or more recombinant genes encoding the antibody. The antibody may be encoded by more than one recombinant gene where, for example, one gene encodes the heavy chain and one gene encodes the light chain. Another aspect of the present invention describes a pharmaceutical composition. The composition contains an antigen binding protein and a pharmaceutically acceptable carrier. Another aspect of the present invention describes a nucleic acid containing a recombinant gene encoding an antigen binding protein. A recombinant gene contains recombinant nucleic acid encoding a polypeptide along with regulatory elements for proper transcription and processing (which may include translational and post translational elements). The recombinant gene can exist independent of a host genome or can be part of a host genome. A recombinant nucleic acid is nucleic acid that by virtue of its sequence and/or form does not occur in nature. Examples of recombinant nucleic acid include purified nucleic acid, two or more nucleic acid regions combined together providing a different nucleic acid than found in nature, and the absence of one or more nucleic acid regions (e.g., upstream or downstream regions) that are naturally associated with each other. Another aspect of the present invention describes a method of inhibiting HCV replication in cell. The method involves providing to the cell an effective amount of an antigen binding protein. Another aspect of the present invention describes a method of inhibiting HCV replication in a patient. The method involves administering to the patient an effective amount of an antigen binding protein. Another aspect of the present invention describes a recombinant cell comprising a recombinant nucleic acid encoding an antigen binding protein. Another aspect of the present invention describes a method of producing an antigen binding piotein. The method involves growing a cell comprising a recombinant gene encoding an antigen binding under conditions where the nucleotide sequence is expressed in the cell and isolating the antigen binding protein. Reference to "isolating" indicates separation of the protein from one or more cellular components. Preferably, the protein is substantially purified. Unless particular terms are mutually exclusive, reference to "or" indicates cither or both possibilities. Occasionally phrases such as "and/or" are used to highlight either or both possibilities. Reference to open-ended terms such as "comprises" allows for additional elements or steps. Occasionally phrases such as "one or more" are used with or without open-ended terms to highlight the possibility of additional elements or steps. Unless explicitly stated reference to terms such as "a" or "an" is not limited to one. For example, "a cell" does not exclude "cells". Occasionally phrases such as one or more are used to highlight the possible presence of a plurality. Other features and advantages of the present invention are apparent from the additional descriptions provided herein including the different examples. The provided examples illustrate different components and methodology useful in practicing the present invention. The examples do not limit the claimed invention. Based on the present disclosure the skilled artisan can identify and employ other components and methodology useful for practicing the present invention. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the structure of an IgG molecule. "VL" refers to a light chain variable region. "Vji" refers to a heavy chain variable region. "CL" refers to a light chain constant region. "CHi", "CH2" and "CH3" are heavy chain constant regions. Figure 2 illustrates the structure of a single-chain antibody. "VL" refers to a light chain variable region. "V\|-j" refers to a heavy chain variable region. Figures 3A and 3B provide an amino acid sequence alignment of single-chain antibodies of SEQ ID NOs: 1, 2, 3, and 4, and indicates the different complementary determining regions ("CDR"). framework regions ("FW") and linker. "SEQ 1" refers to SEQ ID NO: 1. "SEQ 2" refers to SEQ ID NO: 2. "SEQ 3" refers to SEQ ID NO: 3. "SEQ 4" refers to SEQ ID NO: 4. The CDR's can be longer than illustrated in the figure. The provided alignment eliminates common gaps in the CDR's. Figures 4A-4D illustrate nucleic acid sequences encoding SEQ DD NOs: 1, 2, 3 and 4. Figure 4A illustrates a nucleic acid sequence (SEQ ID NO: 5) encoding SEQ ID NO: 1. Figure 4B illustrates a nucleic acid sequence (SEQ ID NO: 6) encoding SEQ ID NO: 2. Figure 4C illustrates a nucleic acid sequence (SEQ ID NO: 7) encoding SEQ ID NO: 3. Figure 4D illustrates a nucleic acid sequence (SEQ ID NO: 8) encoding SEQ ID NO: 4. Figure 5 provides the amino acid sequence of SEQ ID NO: 9. Figure 6 provides results illustrating the ability of single-chain antibodies of SEQ ID NOs: 1.23, and 4 to inhibit E2 protein binding to the HepG2 cell line. The vertical axis represents the percentage ol E2 protein binding to HcpG2 cells, referring to cells not pre-incubated with single-chain antibodies. As a control, an unrelated single-chain antibody was used (D5). The experiment was performed in duplicate, the averages of the duplicate samples arc shown. Figure 7 provides results illustrating the inhibition of HCV infection of cultured human hepatocytes by the single-chain antibody of SEQ ID NO: 2 ("scFVCll"). Viral replication was measured on total RNA by quantitative PCR and expressed as number of HCV copies/350000 cells. The experiment was performed in triplicate wells and values are. shown with standard deviations. Single-chain antibody of SEQ ID NO: 2 was tested at two different concentrations: 25 and 5 jig/ml. As a control, an unrelated single-chain antibody was used (FV) at the concentration of 25 U-g/ml. As a positive control of inhibition of infection, a HCV replicase inhibitor was used ("Pol inhibitor"). Figure 8 provides results illustrating the ability of IgG4 molecules containing SEQ ID NO: 2 or SEQ ID NO: 4 variable regions to inhibit E2 protein binding to the CHO7s cell line. An unrelated IgG was used as a negative control. DETAILED DESCRIPTION OF THE INVENTION The present application identifies particular SR-BI regions that can be targeted to inhibit HCV E2 binding to a cell. The examples provided below illustrate that ability of single-chain antibodies of SEQ ID NOs: 1-4 to inhibit HCV E2 binding, the ability of IgG molecules containing SEQ ID NOs: 2 or 4 variable region to inhibit HCV E2 binding, and the ability of SEQ ID NO: 2 to inhibit HCV replication. Inhibiting HCV E2 binding can have research tool and therapeutic applications. Research tool applications include using the binding protein as a tool to study HCV binding and replication, and to identify additional binding protein that bind to the same region. Therapeutic applications include using those compounds having appropriate pharmacological properties such as efficacy and lack of unacceptable toxicity to treat, or inhibit onset of, HCV in a patient. The target SR-B1 is a glycoprotein containing a large extracellular loop anchored to the plasma membrane at both the amino and carboxyl termini by transmcmbrane domains. (Krieger Journal of Clinical Investigation 108:193-791, 2001.) SR-BI is highly expressed in the liver hepatocytes and sleroidogenic tissues, and mediates the selective cellular uptake of cholesterol and phospholipids. (Acton ct al.. Science 271:518-520, 1996, Urban et al., J. Biol. Chem. 275:33409-33415, 2000.) SEQ ID NO: 9 provides the amino acid sequence of SR-BI used to obtain single-chain antibodies of SEQ ID NOs 1,2, 3, and 4. SEQ ID NO: 9 can be used a frame of reference for antigen binding protein. i. Antigen Bitjding_Proiein Antigen binding proteins contain antibody variable regions providing for specific binding to an cpitopc. The antibody variable region can be present in, for example, a complete antibody. an antibody fragment, and a recombinant derivative of an antibody or antibody fragment. Figures 1 and 2 provide some examples of different types of antigen binding proteins. Figure 1 illustrates a complete IgG molecule and different antibody regions. An IgG molecule contains four polypeptide chains: two longer length heavy chains and two shorter light chains. Heavy and light chains each contain a constant region and a variable region. Within the variable regions are hypervariable regions responsible for antigen specificity. (See. for example. Breitling el al., Recombinant Antibodies, John Wiley & Sons, Inc. and Spektrum Akademischer Veriag, 1999; and Lewin, Genes W, Oxford University Press and Cell Press, 1990.) The two heavy chain carboxyl regions are constant regions joined by disulfide binding to produce an Fc region. The Fc region is important for providing biological activity such as complement and macrophage activation. Each of the two heavy chain polypeptides making up the Fc region extend into different Fab regions through a hinge region. In higher vertebrates there are two classes of light chains and five classes of heavy chains. The light chains are either K or X. The heavy chains define the antibody class and are either a, 6, c, y, or u. For example, IgG has a y heavy chain. Subclasses also exist for different types of heavy chains such as y\, 72. Yi> and -,4. Heavy chains impart a distinctive conformation to hinge and tail regions. {Lewin, Genes FV, Oxford University Press and Cell Press, 1990.) Subclasses can be further characterized. For example, IgG2 subtypes can be further ' • divided into lgG2a and IgG2b- (Hahn G.S. (1982) Antibody Structure, Function and Active Sites. In Physiology of Immunoglobulins: Diagnostic and Clinical Aspects. S.E. Ritzmann (ed) Alan Liss Inc., New York; and Turner M.W. (1983) Immunoglobulins. In Immunology in Medicine. A Comprehensive Guide to Clinical Immunology. 2" Edition. E.J. Holborow & W.G. Reeves (eds.) Grune & Stratton, London.) Antibody fragments containing an antibody variable region include Fv, Fab, and Fat>2 regions. Each Fab region contains a light chain made up ot a variable region and a constant region, and a heavy chain region containing a variable region and a constant region. A light chain is joined to a heavy chain by disulfide bonding through constant regions. The light and heavy chain variable regions of a Fab region provide for an Fv region that participates in antigen binding. The antibody variable region can also be part of protein containing variable regions such as single chain antibody and a minibody. A single chain antibody contains a light and a heavy variable region joined together by a linker. (Sec Figure 2.) The linker can be, for example, about 5 to 16 amino acids. A minibody is a single chain-CH3 fusion protein that self assembles into a bivalent dimcr of about 80 kDa. Specificity of the variable region is determined by three hypervariable regions (also referred to as complementarity determining regions), that are interposed between more conserved flanking regions (also referred to as framework regions) Amino acids associated with framework regions and complementarity determining regions can be numbered and aligned as described by Rabat e/ al. Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991. H SEQ ID NOs: 1-4 Targeted Regions , The SR-B1 regions bound by SEQ ID NOs: 1,2.3 and/or 4 provide target regions for inhibiting HCV E2 binding. Single-chain antibodies of SEQ ID NOs: 1-4 do not necessarily bind to different regions. SEQ ID NOs: 1 and 4 should recognize the same epitope. SEQ ID NOs: 1 and 4 have a very high homology with one different amino acid in CDR3 and a few minor changes in the framework regions. Antigen binding protein described herein binds to a SR-B1 target region involved in HCV E2 binding. While inhibition of HCV E2 binding is expected to occur by interacting with a site directly involved with HCV E2 binding, a region involved in HCV E2 binding can include regions not directly interacting with HCV E2 binding. For example, the targeted region may be involved in providing a proper conformation for a different region which directly binds to HCV E2. Single-chain antibodies of SEQ ID NOs: 1-4 are examples of antigen binding proteins that can be used to inhibit HCV E2 binding. SEQ ID NOs: 1-4 can also be used to design additional antigen-binding proteins that bind to a targeted region. The design of additional binding protein can be performed, for example, using techniques involving deriviiizing SEQ ID NOs: 1-4, using of the sequence information provided in SEQ ID NOs: 1-4, or employing SEQ ED NOs: i-4 as tool to experimentally identify protein that bind to the same region. SEQ ID NOs: 1-4 provide variable region sequences and complementary determining region sequences that can be incorporated into an antigen binding protein. Figures 3A and 3B provide the ammo acid sequence of SEQ ID NOs: 1-4 and indicate the location of different complementary determining regions, frame work regions, and a linker region. The ability of antigen binding protein to inhibit HCV E2 binding and HCV replication can k' evaluated using methods such as those described in the Examples Infra An antigen binding protein inhibiting HCV E2 binding can be used as a starting construct to obtain additional antigen binding protein. li-A. Single-Chain Antibody Modification Single-chain antibodies of a known sequence, such as SEQ ID NOs: 1,2, 3 or 4. can be dcnvaii/cd to enhance stability and to enhance antigen binding. Factors effecting stability include cxposuie of hydiophobic residues that arc hidden at the interlace of a whole Ig molecule at the constant n mierl;icc; hydrophohic icj-ion exposure on the Fv surface leading to intennolccular interaction; and hydrophilic residues in the interior of the Fv beta sheet or at the normally interface between VHand VL. (Chowdhury el at.. Engineering scFvs for Improved Stability, p. 237-254 in Recombinant Antibodies for Cancer Therapy Methods and Protocols, (Eds. Welschof and Krauss) Humana Press, Totowa, New Jersey, 2003.) Stability can be enhanced by substituting problematic residues impacting on stability. Buried hydrophobic residue and exposed hydrophobic residues are potentially problematic. Techniques for enhancing single chain antibody stability taking into account problematic residue are well known in art. (Chowdhury et at.. Engineering scFvs for Improved Stability, p; 237-254 in Recombinant Antibodies for Cancer Therapy Methods and Protocols, (Eds. Welschof and Krauss) Humana Press, Totowa, New Jersey, 2003.) Single chain antigen affinity can be enhanced using techniques such as site directed mutagenesis and chain shuffling. Site directed mulagenesis can be performed to substitute one or more complementary determining region amino acids and then identifying antibodies with higher affinity. (Azzazy et ai. Clinical Biochemistry 35:425-445, 2002.) Chain shuffling can be preformed to provide new combinations of variable regions binding to an antigen. Chain shuffling can be performed by combining a single chain antibody variable region (e.g., V,,) with a repertoire of different variable region (e.g., Vu's) to produce a single chain antibody library. The resulting library contains a variable region known to be specific for the antigen and a random variable region. The library can be panned against the antigen to identify single-chain antibodies binding to the antigen with enhanced affinity. Il.B. Antigen Binding Protein Construction Based on Variable Region Information Variable regions and complementary determining regions from single-chain antibodies of SEQ ID NOs: 1-4 can be incorporated into antigen binding proteins. Techniques for incorporating a variable region into an antibody or an antibody fragment are well known in the art. (E.g., Azzazy ft al.. Clinical Biochemistry J5:425-445, 2002, Persic et al.. Gene 757:9-18, 1997.) An example of such a technique is as follows. 1) Separately amplify the Fv domains using PCR primers specific for the VH and VL regions, the primers can include additional nuclcotidcs tor introducing unique restriction sites, for providing splice sites, and encoding additional ammo acids; 2) Incorporate the amplified variable encoding regions into mammalian expression cassettes. Vj-j encoding nucleic acid can be inserted into a plasmid containing a cassette for expressing a fuiiiuin heavy ( between the leader sequence and the constant region sequence of the antibody. The intron should " contain unique restriction sites suitable for cloning the amplified FV domains; and 3) IgG production can be achieved by co-transfecting the VH and VL expression vector in 293-EBNA. Numerous variations of the outlined procedure can be performed to incorporate a variable region into an antibody or an antibody fragment. Such variations include, for example, using a vector encoding different types of antibody light and heavy chains or fragments thereof, using a single vector, and using different types of host cells. Technique for grafting complementary determining regions into an antibody or antibody fragment are also well known in art. Such techniques are generally described with reference to humanizing murine antibodies by grafting murine variable regions onto a human antibody framework and, if needed making further modifications. (E.g., O'Brien et al., Humanization of Monoclonal Antibodies by CDR Grafting, p 81-100, From Meiliods in Molecular Biology Vol 207: Recombinant antibodies for Cancer Therapy: Methods and Protocols (Eds Welschof and Krauss) Humana Press, Totowa. New Jersey, 2003.) In different embodiments the antigen binding protein is a complete antibody, an antibody fragment, or a recombinant derivative of an antibody or antibody fragment, wherein; a) the first variable region is a Vfo region comprising a first CDR comprising amino acids 31 -35 of SEQ ID NO: 1, a second CDR comprising amino acids 50-66 of SEQ ID NO: I, and a third CDR comprising amino acids 99-108 of SEQ ID NO: 1; and the second variable region is a Vj region comprising a first CDR comprising amino acids 158-170 of SEQ ID NO: 1, a second CDR comprising amino acids 186-192 of SEQ ID NO: l,and a third CDR comprising amino acids 225-235 of SEQ ID NO: 1, b) the first variable region is a V^ region comprising a first CDR comprising amino acids 31-37 of SEQ ID NO: 2. a second CDR comprising amino acids 52-67 of SEQ ID NO: 2, and a third CDR comprising arnino acids 100-114 of SEQ ID NO: 2; and the second variable region is a V\| region comprising a first CDR comprising ainino acids 164-176 of SEQ ID NO: 2, a second CDR comprising amino acids 192-198 of SEQ ID NO: 2, and a third CDR comprising amino acids 231-241 of SEQ ID NO: 2; c) the first variable region is a region comprising a first CDR comprising amino acids 31-35 of SEQ ID NO. 3, a second CDR comprising amino acids 50-66 of SEQ ID NO: 3. and a third (.T)R comprising ainino acids 99-108 of SEQ ID NO: 3; and the second variable region is a Vj region comprising a first CDR comprising amino acids 158-170 of SEQ ID NO: 3, a second CDR comprising aminu acids -186-192 of SEQ ID NO: 3, and a third CDR comprising amino acids 225-235 of SEQ ID NO: 3, d) the first variable region is a Vn region comprising a first CDR comprising amino acids 31-35 of SEQ ID NO: 4, a second CDR comprising amino acids 50-66 of SEQ ID NO: 4, and a third CDR comprising amino acids 99-108 of SEQ ID NO: 4; and the second variable region is a Vj region comprising a first CDR comprising amino acids 158-170 of SEQ ID NO: 4, a second CDR comprising amino acids 186-192 of SEQ ID NO: 4, and a third CDR comprising amino acids 225-235 of SEQ ID NO: 4; e) the first variable region consists of amino acids 1-119 of SEQ ED NO: 1 and the second variable region consists of amino acids 136-245 of SEQ ID NO: 1; f) the first variable region consists of amino acids 1-125 of SEQ ID NO: 2 and the second variable region consists of amino acids 142-251 of SEQ ID NO: 2; g) the first variable region consists of amino acids 1 -119 of SEQ ID NO: 3 and the second variable region consists of amino acids 136-245 of SEQ ID NO: 3; or h) the first variable region consists of amino acids 1-119 of SEQ ID NO: 4 and the second variable region consists of amino acids 136-245 of SEQ ID NO: 4. II.C. Further Identification of Antigen Binding Proteins Single-chain antibodies SEQ ID NOs: 1, 2, 3 or 4 can be used to identify additional antigen binding proteins binding to a targeted region. Identification can be performed using different techniques such as screening for antigen binding proteins that compete with SEQ ED NOs: 1, 2, 3 or 4 binding to SR-B1. mapping theepitope recognized by single-chain antibodies SEQ ED NOs: 1, 2, 3 or 4, and utilizing the epitope itself to select for additional antigen binding protein. Antigen binding proteins for use in a competition assay can be generated using SR-B1 as an antigen Techniques for generating antigen binding protein such as a single-chain antibody, an antibody, or an antibody fragment are well known in the art. Examples of such techniques include the use of phage display technology, identification and humanization of rodent antibodies, and generation of human antibodies using a XenoMouse or Trans-Chromo mouse. (E.g.. Azzazy ct al.. Clinical biochemistry 35:425-445, 2CK)2, Bcrger et al.. Am. J. Mcd. Sci 324(1): [4-40, 2002.) J1J. Protein Production Antigen binding protein are preferably produced using recombinant nucleic acid techniques or through the use of a hybridoma. Recombinant nucleic acid techniques involve constructing a nucleic acid template for protein synthesis. A hybridoma is an immortalized cell line producing the antigen binding protein. Kecninbinunt nucleic acid encoding an antigen binding protein can IK- expressed in a host eel! that in effect serves as a factory for the encoded protein. The recombinant nucleic acid can provide a recombinant gene encoding the antigen binding protein that exists autonomously from a host cell genome or as part of the host cell genome. A recombinant gene contains nucleic acid encoding a protein along with regulatory elements for protein expression. Generally, the regulatory elements that are present in a recombinant gene include a transcriptional promoter, a ribosome binding site, a terminator, and an optionally present operator. A preferred element for processing in eukaryotic cells is a polyadenylation signal. Antibody associated introns may also be present. Examples of expression cassettes for antibody or antibody fragment production are well known in art. (E.g.. Persic et ai. Gene 757:9-18, 1997, Boel et ai. J. Immunol. Methods 239:153-166, 2000, Liang et ai, J. Immunol. Methods 247:119-130, 2001.) Expression of a recombinant gene in a cell is facilitated using an expression vector. Preferably, an expression vector, in addition to a recombinant gene, also contains an origin of replication for autonomous replication in a host cell, a selectable marker, a limited number of useful restriction enzyme sites, and a potential for high copy number. Examples of expression vectors for antibody and antibody fragment production are well known in art. (E.g.. Persic et ai. Gene 757:9-18, 1997, Boel et ai. J. Immunol. Methods 239:153-166, 2000, Liangetai. J. Immunol. Methods 247:119-130, 2001.) If desired, nucleic acid encoding an antibody may be integrated into the host chromosome using techniques well known in the art. (See, Ausubel, Current Protocols in Molecular Biology. John Wiley, 1987-1998, Marks et ai, International Application Number WO 95/17516, International Publication Date June 29, 1995.) A variety of different cell lines can be used for recombinant antigen binding protein expression, including those from prokaryotic organisms (e.g.. E. coli. Bacillus, and Streptomyces) and from Eukaryotic (e.g.. yeast, Baculovirus. and mammalian). (Breitling et al.. Recombinant Antibodies, John Wiley & Sons, Inc. and Spektrum Akademischer Verlag, 1999.) Preferred hosts for recombinant antigen binding protein expression are mammalian cells able to produce antigen binding protein with proper post translational modifications. Post translational modifications include disulfidc bond formation and glycosylation. Another type of post translational modification is signal pcptide cleavage. Proper glycosylation can be important for antibody function. (Yoo et ai. Journal of Immunologival Methods 267:1-20, 2002.) Naturally occurring antibodies contain at least one N-linked carbohydrate attached to a heavy chain (Id.) Additional N-linked carbohydrates and O-linked carbohydrates may be present and may be important for antibody function. (Id.) Different types of mammalian host cells can be used to provide for efficient posl-translational mcxlifications. Examples of such host cells include Chinese hamster ovary (Cho), HeLa, C6, PC 12. and myeloma cells. (Yoo «.•/ ai. Journal of Immunological Methods 267:1-20, 2002. Persic el ai. Gene /«7:9-l8, 1997.) A hybridoma is an immortalized antibody producing cell line. A hybridoma can be produced using techniques such as those described in Ausubel Current Protocols in Molecular Biology, John Wiley, 1987-1998, Harlow el a\.. Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, and Kohler et al. Nature 256, 495-497, 1975. IV. Combination Treatment Antigen binding protein that bind to suitable SR-BI sites can be used to inhibit HCV and treat HCV patients by itself, or in combination with one or more olher anti-HGV agents. Currently approved anti-HCV agents are interferon alpha, and interferon alpha in combination with ribovarin. Different forms of interferon alpha, such as recombinant interferon and peglyated interferons, can used to treat HCV infections. (De Francesco et al., Antiviral Research 58:1-16, 2003, Walker et al., Antiviral Chemistry & Chemotherapy 14:1-21, 2003.) A variety of different anti-HCV agents are in different phases of clinical developments. The different anti-HCV agents being developed include agents directed against different HCV targets. Examples of different HCV targets include HCV polymerase and HCV NS3-NS4A protease. (De Francesco et al.. Antiviral Research 55:1-16, 2003, Walker et al.. Antiviral Chemistry & Chemotherapy 14:1-21,2003.) V. Administration Guidelines for pharmaceutical administration in general are provided in, for example, Remington's Pharmaceutical Sciences 20h Edition, Ed. Gennaro, Mack Publishing, 2000; and Modem Pharmaceutics 2nd Edition, Eds. Banker and Rhodes, Marcel Dekker, Inc., 1990. Pharmaceutically acceptable carriers facilitate storage or administration of an antigen binding protein. Substances used to stabilize protein solution formulations include carbohydrates, amino acids, and buffering salts. (Middaugh et al.. Handbook of Experimental Pharmacology 757:33-58, 1999.) Antigen binding proteins can be administered by different routes such as subcutaneous, intramuscular, or mucosal. Subcutaneous and intramuscular administration can be performed using, for example, needles or jet-injectors Mucosal delivery, such as nasal delivery, can involve using enhancers or mucoadhcsivcs to produce a longer retention lime at adsorption sites. (Middaugh et al.. Handbook of Experimental Pharmacology 737:33-58. 1999.) Suitable dosing regimens are preferably determined taking into account factors well known in the art including age, weight, sex and medical condition of the patient; the route of administration; the desired effect, and the particular compound employed. It is expected that a dose would consist of the range of 1.0 ug to 1.0 mg total protein, in different embodiments of the present invention the range is 0.01 mg to 1.0 ing and 0.1 nig to'1.0 nig. VI. Examples Examples are provided below further illustrating different features of the present invention. The examples also illustrate useful methodology for practicing the invention. These examples do not limit the claimed invention. Example 1: Experimental Procedure to Obtain SR-B1-Specific Phage Antibodies The CAT (Cambridge antibody technology) phage library CS was screened for single-chain antibodies binding to SR-B 1. The library provides single-chain antibodies containing a variable portion of heavy chain and light chain antibodies exposed on the surface of a filamentous phage as a fusion to the N terminus of the pUl protein. The VH region is joined to the VL region by a linker of Ser and Gly. To select phage displaying antibodies specifically binding to the SR-B 1 receptor, whole cells were utilized for phage enrichment. The library (10" phage) was pre-incubated with 101 CHO cells for 1 hour at room temperature, followed by centrifugation. The unbound phage present in the supernatant were recovered and incubated for 1 hour with CHO cells stably expressing the human SR-B1 receptor (SEQ ID NO: 9). Cells were then washed several limes with PBS and resuspended in eluiion buffer (triethylamina, 100 mM) for 25 minutes, followed by adjustment of the pH with Tris.HCl. Recovered phage were amplified by infecting TGI cells and subjected to other two rounds of selection as described above. Following the third round of selection, 3 x 10s phage were recovered. A sample of these (144 phage) was tested in a cell based EL1SA, using SR-B 1 expressing CHO cells, in parallel with CHO ceils as negative control. Among these 11 phage clones were scored as SR-B1- specific. Clones were subsequently tested for the ability to HCV E2 binding. Example 2: Inhibition of HCV E2 Clones encoding SEQ ID NOs: 1, 2, 3 and 4 produced outside the phage context were able to inhihit the binding of the HCV E2 protein to HepG2 cells HepG2 is a human hepatoma cell line. Cells were detached and washed in phosphate buffered saline (PBS), 0.2% BSA, 10 mM Hopes (washing buffer). 4xl()3 cells were allowed to bind to different concentrations (0.5-5-20-40 jag/ml) of single chain antibodies or an unrelated single chain control D5 at room temperature for 30 minutes. Cells were then incubalcd for I hour at room temperature with recombinant soluble E2. Binding was revealed by anti-E2 rat rnAb 6/la (Pafel et al. J. Gen. Viral. 57:2873-2883, 2000) and a secondary unti rat PE-conjugalcd mAb. Fluorescence associated lo the cells was measured by FACS analysis. As shown in Figure 6, an(i-SRBl single-chain antibodies of SEQ ID NOs: 1, 2, 3, and 4 inhibited E2 protein binding to the HepG2 cell line. The vertical axis represents the percentage of E2 protein binding to HepG2 cells, referring to cells not pre-incubated with antibodies. The experiment was performed in duplicate, the averages of the duplicate samples are shown. Example 3 Inhibition of HCV Infection Using The Single-Chain Antibody of SEO ID NO: 2 The ability of single-chain antibodies able to inhibit HCV E2 binding to also inhibit HCV replication was illustrated using the single-chain antibody of SEQ ID NO: 2. Isolated human hepatocytes from surgical liver resection were seeded in 24 well microplates at the density of 3xl05 cells/well. Cells were allowed to attach and recover 24 hours and then medium was replaced with fresh one containing different concentrations of ami-SRBl single-chain antibody SEQ ID NO: 2 (25 and 5 u,g/ml) or a control unrelated single chain antibody FV at the highest concentration (25 jig/ml). Hepatocytes were preincubated 1 hour at 37°C with the indicated amounts of single-chain antibodies, then medium was replaced with fresh one containing the same amounts of single-chain antibodies and a fixed amount (100 uT) of an infectious human serum from a HCV chronic patient. Cells were incubated 18 hours with the virus to allow infection, then washed and incubated for four days. Total RNA was extracted and viral replication was measured by quantitative RT-PCR. Typically, 104 to JO5 copies of genomes/well are detected after four days from infection. To be sure thai the measured viral RNA derived from active replication, a small molecule inhibitor of the viral replicasc was included as a positive control. As shown in Figure 7, anti-SRBl single chain of SEQ ID NO: 2 (scFVCll) can block HCV infection of cultured human hepatocytes. Example 4: 1^0production from Single-Chain Antibodies lpCi4 molecules were produced containing SEQ ID NO: 2 or SEQ ID NO: 4 variable regions SEQ ID NOs: 2 or 4 variable regions were separately amplified by PCR using primers specific for the Vjj ami the VL regions. The primers contained additional nuclcotidcs for the introduction of unique restriction sites, and bases representing splice sites or coding for additional amino acids. The amplified products were introduced into two separate mammalian expression vectors: Vj \ w;is inserted in the pEU8.2 containing a cassette for the expression of human gamma 4 heavy chain, while Vj was introduced into the vector pEU 4.2 expressing the constant region of the human lambda lij'hi chain Both veclois carry an intron between the leader sequence and the constant icgion sequentf of the antibody. The intron contains unique restriction sites suitable for cloning the amplified vaiiable domains IgG production was achieved by co-transfecting the Vj-j and VL expression vectors into 293-EBNA cells (Invitrogen), using Lipofectamine 2000 reagent (Invitrogcn) and collecting the supernatant for up to eight days. IgG's were purified from culture medium using Hi-Trap protein A columns, Amersham, following manufacturers instruction. Example 5: Inhibition of HCV E2 Protein Binding Using Anti-SRB I IeG Cho7s cells stably expressing the human SR-B1 receptor (Scarselli el ai, Tlie EMBO Journal 2/f/9j:5017-5025, 2002) were used to evaluate the ability of anti-SRBl IgG containing the SEQ ID NO: 2 or 4 variable regions to inhibit HCV E2 binding. Cells were detached and washed in phosphate buffered saline (PBS), 0.2% BSA. 10 mM Hepes (washing buffer). 4 x 105 cells were allowed to bind to different concentrations (60-12-2.4 ng/ml) of antibodies at room temperature for 30 minutes. Cells were then incubated for 1 hour at room temperature with recombinam soluble E2 (carrying a His-Tag). Binding was revealed by an anti-penta His biotin conjugate and streptavidin-R-PE. Fluorescence associated to the cells was measured by FACS analysts. As shown in Figure 8, anti-SRBl IgG4 SEQ ID NOs: 2 and 4 inhibit E2 protein binding to the Cho7s cell line. Unrelated IgGs were used as a negative control Other embodiments are within the following claims. While several embodiments have been shown and described, various modifications may be made without departing from the spirit and scope of ihe present invention WHAT IS CLAIMED IS: 1. An isolated antigen binding protein comprising a first variable region and a second variable region, wherein said first and said second variable regions bind one or more target regions selected from the group consisting of the SEQ ID NO: 1 target region, the SEQ ID NO: 2 target region, the SEQ ID NO: 3 target region, and the SEQ ID NO: 4 target region. 2. The antigen binding protein of claim 1, wherein either: a) said first variable region is a Vj, region comprising a first CDR comprising amino acids 31-35 of SEQ ID NO: I, a second CDR comprising amino acids 50-66 of SEQ ID NO: 1. and a third CDR comprising amino acids 99-108 of SEQ ID NO: 1; and said second variable region is a Vj region comprising a first CDR comprising amino acids 158-170 of SEQ ED NO: I, a second CDR comprising amino acids 186-192 of SEQ ID NO: 1, and a third CDR comprising amino acids 225-235 of SEQ ID NO: 1; b) said first variable region is a Vfo region comprising a first CDR comprising amino acids 31 -37 of SEQ ID NO: 2, a second CDR comprising amino acids 52-67 of SEQ ED NO: 2, and a third CDR comprising the amino acids 100-114 of SEQ ID NO: 2; and said second variable region is a V\| region comprising a first CDR comprising amino acids 164-176 of SEQ ID NO: 2, a second CDR comprising amino acids 192-198 of SEQ ID NO: 2, and a third CDR comprising amino acids 231-241 of SEQ ID NO: 2; c) said first variable region is a Vf, region comprising a first CDR comprising amino acids 31 -35 of SEQ ID NO: 3, a second CDR comprising amino acids 50-66 of SEQ ID NO: 3, and a third CDR comprising amino acids 99-108 of SEQ ID NO: 3; and said second variable region is a V\| region comprising a first CDK comprising amino acids 158-170 of SEQ ID NO: 3, a second CDR comprising amino acids 186-192 of SEQ ID NO. 3, and a third CDR comprising amino acids 225-235 of SEQ ID NO. 3; or d) said first variable icgion is a Vf, region comprising a first CDR comprising amino acids 31 -35 of SI:Q ID NO: 4, a second CDR comprising amino acids 50-66 of SEQ ID NO: 4, and a third CDR comprising amirio acids 99-108 ot SEQ ID NO: 4; and said second variable region is a V\| region comprising a first CDR comprising amino acids 158-170 of SEQ ID NO: 4, a second CDR comprising amino acids 186-192 of SEQ ID NO: 4, and a third CDR comprising amino acids 225-235 of SI-Q IJ) NO: 4. 3. The binding protein of claim 2, wherein said binding protein is an antibody 01 a fragment thereof 1 4. The binding protein of claim 3, wherein either: a) said first variable region consists of amino acids 1-119 of SEQ ID NO: 1 and said second variable region consists of amino acids 136-245 of SEQ ID NO: 1; b) said first variable region consists of amino acids 1-125 of SEQ ID NO: 2 and said second variable region consists of amino acids 142-251 of SEQ ID NO: 2, c) said third Vfo region consists of amino acids 1-119 of SEQ ID NO: 3 and said third Vj region consists of amino acids 136-245 of SEQ ED NO: 3; or d) said fourth Vfo region consists of amino acids 1-119 of SEQ ID NO: 4 and said fourth V\| region consists of amino acids 136-245 of SEQ ID NO: 4. 5. The binding protein of claim 4, wherein said binding protein is an antibody. 6. The binding protein of claim 5, wherein said antibody is a monoclonal antibody. 7. The binding protein of claim 3, wherein said binding protein is a single chain antibody wherein said Vh region is joined to said V\| region by an amino acid linker about 5 to 16 amino acids in length. 8. The binding protein of claim 7, wherein either: a) said first variable region consists of amino acids 1-119 of SEQ ED NO: 1 and said second variable region consists of amino acids 136-245 of SEQ ID NO: 1; b) said first variable region consists of amino acids 1-125 of SEQ ID NO: 2 and said second variable region consists of amino acids 142-251 of SEQ ID NO: 2; c) said first variable region consists of amino acids 1-119 of SEQ ID NO: 3 and said second variable region consists of amino acids 136-245 of SEQ ID NO: 3; or d) said first variable region consists of amino acids 1-119 of SEQ ID NO: 4 and said second variable region consists of amino acids 136-245 of SEQ ID NO: 4. 9. A nucleic acid comprising a recombinant gene encoding the antigen binding protein ol any one of claims 1 -X. 10. A pharmaceutical composition comprising the binding protein of any one of claims 1-8 and a pharmaceulically acceptable carrier 11. A method of inhibiting HCV replication in cell comprising the step of providing to said cell an effective amount of the binding protein of any one of claims 1-8. 12. A method of treating HCV in a patient comprising the step of administering to said patient an effective amount of the binding protein of any one of claims 1-8. 13. The method of claim 12, wherein said patient is a human infected with HCV. 14. A recombinant cell comprising the nucleic acid of claim 9, wherein said nucleotide sequence is expressed in said cell. 15. A method of producing an antigen binding protein comprising the step of growing the recombinant cell of claim 14 under conditions wherein said nucleotide sequence is expressed in said cell and isolating said antigen binding protein. 16. The Invention substantially such as herein before described.

Full Text

TITLE OF THE INVENTION
ANTIGEN BINDING PROTEINS DIRECTED AGAINST SCAVENGER' RECEPTOR Bl THAT INHIBIT HCV REPLICATION
BACKGROUND OF THE INVENTION
The references cited in the present application are not admitted to be prior art to the claimed invention.
It is estimated that about 3% of the world's population is infected with the hepatitis C virus (HCV). (Wasley el al.. Semin. Liver Dis. 20:1-16, 2000.) HCV exposure results in an overt acute disease in a small percentage of cases, while in most instances the virus establishes a chronic infection causing liver inflammation and slowly progresses into liver failure and cirrhosis. (Slradere/ al.. JLAR J. 42:107-116, 2001.) Epidemiological surveys indicate an important role for HCV in the onset of hepatocellular carcinoma. (Strader^/o/., ILARJ. 42:107-116, 2001.)
HCV can be classified into a number of distinct genotypes (1 to 6), and subtypes (a to c). The distribution of the genotypes and subtypes varies both geographically and between risk groups. (Robertson et al., Arch Virol. 743:2493-2503, 1998.)
The HCV genome consists of a single strand RNA about 9.5 kb encoding a precursor polyprotein of about 3000 amino acids. (Choo ei al.. Science 244:362-364, 1989, Choo et al.. Science 244:359-362, 1989.) The HCV polyprotein contains the viral proteins in the order: C-El-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B. Cleavage of the preciusor polyprotein results in mature structural and non-structural viral proteins. (Neddermann et al.. Rial Chem 375:469-476, 1997.)
As part of its infection cycle, HCV enters into a cell. Host cell LDL receptors and CD81 molecules have been identified as putative HCV receptors. The LDL receptor has been suggested to mediate* virus internalization via binding to LDL particles that are virus-associated. (Agnello et al., r'roc. Nail. Acml Sc, U.S.A. %: 12766-12771, 1999.) TheCHHl molecule has been suggested to bind HCV E2 based on recombinant envelope protein E2 from HCV genotype la. (Pileri et ai. Science 26*2:938-941, 1998.)
HCV envelope glycoprotein L2 was found to bind human hepaioma cells independently l CDK1 The receptor responsible for E2 binding to human hepatic cells was identified as the human M .ivoij'cr icccplor class H type I (SR-BI). (Scarselh ,-t ,,l . The EM HO Journal 27:5017 5025. 2002.)
SUMMARY OF THE INVENTION
The present invention features antigen binding protein that bind to a SR-BI target region identified herein as a region involved in HCV E2 binding. Identified target regions are regions bound by a single-chain antibody of SEQ ID NOs: 1, 2, 3 or 4.
Thus, a first aspect of the present invention features an isolated antigen binding protein . comprising a first variable region and a second variable region. The first and second variable regions bind one or more target regions selected from the group consisting of: the SEQ ID NO: 1 target region, the SEQ ID NO: 2 target region, the SEQ ID NO: 3 target region, and the SEQ ID NO: 4 target region.
Reference to "isolated" indicates a different form than found in nature. The different form can be, for example, a different purity than found in nature and/or a structure that is not found in nature. A structure not found in nature includes recombinant structures where different regions are combined together, for example, humanized antibodies where one or more murine CDR is inserted onto a human framework scaffold, hybrid antibodies where one or more CDR from an antibody binding protein is inserted into a different framework scaffold, and antibodies derived from natural human sequences where genes coding light and heavy variable domains were randomly combined together.
The isolated protein is preferably substantially free of serum proteins. A protein substantially free of serum proteins is present in an environment lacking most or all serum proteins.
A "variable region" has the structure of an antibody variable region from a heavy or light chain. Antibody heavy and light chain variable regions contain three complementary determining regions interspaced onto a framework. The complementary determining regions are primarily responsible for recognizing a particular epitope.
A target region defined with respect to SEQ ID NOs: 1, 2, 3 or 4, is an SR-BI region to which the corresponding single-chain antibody binds. For example, the SEQ ID NO: 1 target region is a region to which a polypeptide of SEQ ID NO: 1 binds.
A protein binding to same target region as an identified target region competes with either SEQ ID NOs: 1, 2, 3 or 4 for binding to the identified target region. For example, a protein that competes with a polypeplide of SEQ ID NO: 1 for binding to SR-BI binds to the SEQ ID NO: 1 target region.
Reference to "protein" or "polypeplide" indicates a contiguous amino acid sequence and does not provide a minimum or maximum size limitation. One or more amino acids present in the protein or polypcptide rnay contain a post-translational modification, such as glycosylation and disulfide bond formal ion.
A preferred antigen binding protein is a monoclonal antibody. Reference to a "monoclonal antibody" indicates a collection of antibodies having the same, or substantially the same.
complementary determining region, and binding specificity. The variation in the monoclonal antibodies is that which would occur if the antibodies were produced from the same construct(s).
Monoclonal antibodies can be produced, for example, from a particular hybridoma and from a recombinant cell containing one or more recombinant genes encoding the antibody. The antibody may be encoded by more than one recombinant gene where, for example, one gene encodes the heavy chain and one gene encodes the light chain.
Another aspect of the present invention describes a pharmaceutical composition. The composition contains an antigen binding protein and a pharmaceutically acceptable carrier.
Another aspect of the present invention describes a nucleic acid containing a recombinant gene encoding an antigen binding protein. A recombinant gene contains recombinant nucleic acid encoding a polypeptide along with regulatory elements for proper transcription and processing (which may include translational and post translational elements). The recombinant gene can exist independent of a host genome or can be part of a host genome.
A recombinant nucleic acid is nucleic acid that by virtue of its sequence and/or form does not occur in nature. Examples of recombinant nucleic acid include purified nucleic acid, two or more nucleic acid regions combined together providing a different nucleic acid than found in nature, and the absence of one or more nucleic acid regions (e.g., upstream or downstream regions) that are naturally associated with each other.
Another aspect of the present invention describes a method of inhibiting HCV
replication in cell. The method involves providing to the cell an effective amount of an antigen binding protein.
Another aspect of the present invention describes a method of inhibiting HCV replication in a patient. The method involves administering to the patient an effective amount of an antigen binding protein.
Another aspect of the present invention describes a recombinant cell comprising a recombinant nucleic acid encoding an antigen binding protein.
Another aspect of the present invention describes a method of producing an antigen binding piotein. The method involves growing a cell comprising a recombinant gene encoding an antigen binding under conditions where the nucleotide sequence is expressed in the cell and isolating the antigen binding protein.
Reference to "isolating" indicates separation of the protein from one or more cellular components. Preferably, the protein is substantially purified.
Unless particular terms are mutually exclusive, reference to "or" indicates cither or both possibilities. Occasionally phrases such as "and/or" are used to highlight either or both possibilities.
Reference to open-ended terms such as "comprises" allows for additional elements or steps. Occasionally phrases such as "one or more" are used with or without open-ended terms to highlight the possibility of additional elements or steps.
Unless explicitly stated reference to terms such as "a" or "an" is not limited to one. For example, "a cell" does not exclude "cells". Occasionally phrases such as one or more are used to highlight the possible presence of a plurality.
Other features and advantages of the present invention are apparent from the additional descriptions provided herein including the different examples. The provided examples illustrate different components and methodology useful in practicing the present invention. The examples do not limit the claimed invention. Based on the present disclosure the skilled artisan can identify and employ other components and methodology useful for practicing the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates the structure of an IgG molecule. "VL" refers to a light chain variable region. "Vji" refers to a heavy chain variable region. "CL" refers to a light chain constant region. "CHi", "CH2" and "CH3" are heavy chain constant regions.
Figure 2 illustrates the structure of a single-chain antibody. "VL" refers to a light chain variable region. "V|-j" refers to a heavy chain variable region.
Figures 3A and 3B provide an amino acid sequence alignment of single-chain antibodies of SEQ ID NOs: 1, 2, 3, and 4, and indicates the different complementary determining regions ("CDR"). framework regions ("FW") and linker. "SEQ 1" refers to SEQ ID NO: 1. "SEQ 2" refers to SEQ ID NO: 2. "SEQ 3" refers to SEQ ID NO: 3. "SEQ 4" refers to SEQ ID NO: 4. The CDR's can be longer than illustrated in the figure. The provided alignment eliminates common gaps in the CDR's.
Figures 4A-4D illustrate nucleic acid sequences encoding SEQ DD NOs: 1, 2, 3 and 4. Figure 4A illustrates a nucleic acid sequence (SEQ ID NO: 5) encoding SEQ ID NO: 1. Figure 4B illustrates a nucleic acid sequence (SEQ ID NO: 6) encoding SEQ ID NO: 2. Figure 4C illustrates a nucleic acid sequence (SEQ ID NO: 7) encoding SEQ ID NO: 3. Figure 4D illustrates a nucleic acid sequence (SEQ ID NO: 8) encoding SEQ ID NO: 4.
Figure 5 provides the amino acid sequence of SEQ ID NO: 9.
Figure 6 provides results illustrating the ability of single-chain antibodies of SEQ ID NOs: 1.23, and 4 to inhibit E2 protein binding to the HepG2 cell line. The vertical axis represents the percentage ol E2 protein binding to HcpG2 cells, referring to cells not pre-incubated with single-chain antibodies. As a control, an unrelated single-chain antibody was used (D5). The experiment was performed in duplicate, the averages of the duplicate samples arc shown.
Figure 7 provides results illustrating the inhibition of HCV infection of cultured human hepatocytes by the single-chain antibody of SEQ ID NO: 2 ("scFVCll"). Viral replication was measured on total RNA by quantitative PCR and expressed as number of HCV copies/350000 cells. The experiment was performed in triplicate wells and values are. shown with standard deviations. Single-chain antibody of SEQ ID NO: 2 was tested at two different concentrations: 25 and 5 jig/ml. As a control, an unrelated single-chain antibody was used (FV) at the concentration of 25 U-g/ml. As a positive control of inhibition of infection, a HCV replicase inhibitor was used ("Pol inhibitor").
Figure 8 provides results illustrating the ability of IgG4 molecules containing SEQ ID NO: 2 or SEQ ID NO: 4 variable regions to inhibit E2 protein binding to the CHO7s cell line. An unrelated IgG was used as a negative control.
DETAILED DESCRIPTION OF THE INVENTION
The present application identifies particular SR-BI regions that can be targeted to inhibit HCV E2 binding to a cell. The examples provided below illustrate that ability of single-chain antibodies of SEQ ID NOs: 1-4 to inhibit HCV E2 binding, the ability of IgG molecules containing SEQ ID NOs: 2 or 4 variable region to inhibit HCV E2 binding, and the ability of SEQ ID NO: 2 to inhibit HCV replication.
Inhibiting HCV E2 binding can have research tool and therapeutic applications. Research tool applications include using the binding protein as a tool to study HCV binding and replication, and to identify additional binding protein that bind to the same region. Therapeutic applications include using those compounds having appropriate pharmacological properties such as efficacy and lack of unacceptable toxicity to treat, or inhibit onset of, HCV in a patient.
The target SR-B1 is a glycoprotein containing a large extracellular loop anchored to the plasma membrane at both the amino and carboxyl termini by transmcmbrane domains. (Krieger Journal of Clinical Investigation 108:193-791, 2001.) SR-BI is highly expressed in the liver hepatocytes and sleroidogenic tissues, and mediates the selective cellular uptake of cholesterol and phospholipids. (Acton ct al.. Science 271:518-520, 1996, Urban et al., J. Biol. Chem. 275:33409-33415, 2000.)
SEQ ID NO: 9 provides the amino acid sequence of SR-BI used to obtain single-chain antibodies of SEQ ID NOs 1,2, 3, and 4. SEQ ID NO: 9 can be used a frame of reference for antigen binding protein.
i. Antigen Bitjding_Proiein
Antigen binding proteins contain antibody variable regions providing for specific
binding to an cpitopc. The antibody variable region can be present in, for example, a complete antibody. an antibody fragment, and a recombinant derivative of an antibody or antibody fragment.
Figures 1 and 2 provide some examples of different types of antigen binding proteins. Figure 1 illustrates a complete IgG molecule and different antibody regions. An IgG molecule contains four polypeptide chains: two longer length heavy chains and two shorter light chains. Heavy and light chains each contain a constant region and a variable region. Within the variable regions are hypervariable regions responsible for antigen specificity. (See. for example. Breitling el al., Recombinant Antibodies, John Wiley & Sons, Inc. and Spektrum Akademischer Veriag, 1999; and Lewin, Genes W, Oxford University Press and Cell Press, 1990.)
The two heavy chain carboxyl regions are constant regions joined by disulfide binding to produce an Fc region. The Fc region is important for providing biological activity such as complement and macrophage activation. Each of the two heavy chain polypeptides making up the Fc region extend into different Fab regions through a hinge region.
In higher vertebrates there are two classes of light chains and five classes of heavy chains. The light chains are either K or X. The heavy chains define the antibody class and are either a, 6, c, y, or u. For example, IgG has a y heavy chain. Subclasses also exist for different types of heavy chains such as y\, 72. Yi> and -,4. Heavy chains impart a distinctive conformation to hinge and tail regions. {Lewin, Genes FV, Oxford University Press and Cell Press, 1990.)
Subclasses can be further characterized. For example, IgG2 subtypes can be further
' •
divided into lgG2a and IgG2b- (Hahn G.S. (1982) Antibody Structure, Function and Active Sites. In Physiology of Immunoglobulins: Diagnostic and Clinical Aspects. S.E. Ritzmann (ed) Alan Liss Inc., New York; and Turner M.W. (1983) Immunoglobulins. In Immunology in Medicine. A Comprehensive Guide to Clinical Immunology. 2"* Edition. E.J. Holborow & W.G. Reeves (eds.) Grune & Stratton, London.)
Antibody fragments containing an antibody variable region include Fv, Fab, and Fat>2 regions. Each Fab region contains a light chain made up ot a variable region and a constant region, and a heavy chain region containing a variable region and a constant region. A light chain is joined to a heavy chain by disulfide bonding through constant regions. The light and heavy chain variable regions of a Fab region provide for an Fv region that participates in antigen binding.
The antibody variable region can also be part of protein containing variable regions such as single chain antibody and a minibody. A single chain antibody contains a light and a heavy variable region joined together by a linker. (Sec Figure 2.) The linker can be, for example, about 5 to 16 amino acids. A minibody is a single chain-CH3 fusion protein that self assembles into a bivalent dimcr of about 80 kDa.
Specificity of the variable region is determined by three hypervariable regions (also referred to as complementarity determining regions), that are interposed between more conserved flanking regions (also referred to as framework regions) Amino acids associated with framework
regions and complementarity determining regions can be numbered and aligned as described by Rabat e/ al. Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991.
H SEQ ID NOs: 1-4 Targeted Regions ,
The SR-B1 regions bound by SEQ ID NOs: 1,2.3 and/or 4 provide target regions for inhibiting HCV E2 binding. Single-chain antibodies of SEQ ID NOs: 1-4 do not necessarily bind to different regions. SEQ ID NOs: 1 and 4 should recognize the same epitope. SEQ ID NOs: 1 and 4 have a very high homology with one different amino acid in CDR3 and a few minor changes in the framework regions.
Antigen binding protein described herein binds to a SR-B1 target region involved in HCV E2 binding. While inhibition of HCV E2 binding is expected to occur by interacting with a site directly involved with HCV E2 binding, a region involved in HCV E2 binding can include regions not directly interacting with HCV E2 binding. For example, the targeted region may be involved in providing a proper conformation for a different region which directly binds to HCV E2.
Single-chain antibodies of SEQ ID NOs: 1-4 are examples of antigen binding proteins that can be used to inhibit HCV E2 binding. SEQ ID NOs: 1-4 can also be used to design additional antigen-binding proteins that bind to a targeted region. The design of additional binding protein can be performed, for example, using techniques involving deriviiizing SEQ ID NOs: 1-4, using of the sequence information provided in SEQ ID NOs: 1-4, or employing SEQ ED NOs: i-4 as tool to experimentally identify protein that bind to the same region.
SEQ ID NOs: 1-4 provide variable region sequences and complementary determining region sequences that can be incorporated into an antigen binding protein. Figures 3A and 3B provide the ammo acid sequence of SEQ ID NOs: 1-4 and indicate the location of different complementary determining regions, frame work regions, and a linker region.
The ability of antigen binding protein to inhibit HCV E2 binding and HCV replication can k' evaluated using methods such as those described in the Examples Infra An antigen binding protein inhibiting HCV E2 binding can be used as a starting construct to obtain additional antigen binding protein.
li-A. Single-Chain Antibody Modification
Single-chain antibodies of a known sequence, such as SEQ ID NOs: 1,2, 3 or 4. can be dcnvaii/cd to enhance stability and to enhance antigen binding. Factors effecting stability include cxposuie of hydiophobic residues that arc hidden at the interlace of a whole Ig molecule at the constant n mierl;icc; hydrophohic icj-ion exposure on the Fv surface leading to intennolccular interaction;

and hydrophilic residues in the interior of the Fv beta sheet or at the normally interface between VHand VL. (Chowdhury el at.. Engineering scFvs for Improved Stability, p. 237-254 in Recombinant Antibodies for Cancer Therapy Methods and Protocols, (Eds. Welschof and Krauss) Humana Press, Totowa, New Jersey, 2003.)
Stability can be enhanced by substituting problematic residues impacting on stability. Buried hydrophobic residue and exposed hydrophobic residues are potentially problematic. Techniques for enhancing single chain antibody stability taking into account problematic residue are well known in art. (Chowdhury et at.. Engineering scFvs for Improved Stability, p; 237-254 in Recombinant Antibodies for Cancer Therapy Methods and Protocols, (Eds. Welschof and Krauss) Humana Press, Totowa, New Jersey, 2003.)
Single chain antigen affinity can be enhanced using techniques such as site directed mutagenesis and chain shuffling. Site directed mulagenesis can be performed to substitute one or more complementary determining region amino acids and then identifying antibodies with higher affinity. (Azzazy et ai. Clinical Biochemistry 35:425-445, 2002.)
Chain shuffling can be preformed to provide new combinations of variable regions binding to an antigen. Chain shuffling can be performed by combining a single chain antibody variable region (e.g., V,,) with a repertoire of different variable region (e.g., Vu's) to produce a single chain antibody library. The resulting library contains a variable region known to be specific for the antigen and a random variable region. The library can be panned against the antigen to identify single-chain antibodies binding to the antigen with enhanced affinity.
Il.B. Antigen Binding Protein Construction Based on Variable Region Information
Variable regions and complementary determining regions from single-chain antibodies of SEQ ID NOs: 1-4 can be incorporated into antigen binding proteins. Techniques for incorporating a variable region into an antibody or an antibody fragment are well known in the art. (E.g., Azzazy ft al.. Clinical Biochemistry J5:425-445, 2002, Persic et al.. Gene 757:9-18, 1997.) An example of such a technique is as follows.
1) Separately amplify the Fv domains using PCR primers specific for the VH and VL
regions, the primers can include additional nuclcotidcs tor introducing unique restriction sites, for providing splice sites, and encoding additional ammo acids;
2) Incorporate the amplified variable encoding regions into mammalian expression
cassettes. Vj-j encoding nucleic acid can be inserted into a plasmid containing a cassette for expressing a fuiiiuin heavy (
between the leader sequence and the constant region sequence of the antibody. The intron should " contain unique restriction sites suitable for cloning the amplified FV domains; and
3) IgG production can be achieved by co-transfecting the VH and VL expression vector
in 293-EBNA.
Numerous variations of the outlined procedure can be performed to incorporate a variable region into an antibody or an antibody fragment. Such variations include, for example, using a vector encoding different types of antibody light and heavy chains or fragments thereof, using a single vector, and using different types of host cells.
Technique for grafting complementary determining regions into an antibody or antibody fragment are also well known in art. Such techniques are generally described with reference to humanizing murine antibodies by grafting murine variable regions onto a human antibody framework and, if needed making further modifications. (E.g., O'Brien et al., Humanization of Monoclonal Antibodies by CDR Grafting, p 81-100, From Meiliods in Molecular Biology Vol 207: Recombinant antibodies for Cancer Therapy: Methods and Protocols (Eds Welschof and Krauss) Humana Press, Totowa. New Jersey, 2003.)
In different embodiments the antigen binding protein is a complete antibody, an antibody fragment, or a recombinant derivative of an antibody or antibody fragment, wherein;
a) the first variable region is a Vfo region comprising a first CDR comprising amino
acids 31 -35 of SEQ ID NO: 1, a second CDR comprising amino acids 50-66 of SEQ ID NO: I, and a third CDR comprising amino acids 99-108 of SEQ ID NO: 1; and the second variable region is a Vj
region comprising a first CDR comprising amino acids 158-170 of SEQ ID NO: 1, a second CDR comprising amino acids 186-192 of SEQ ID NO: l,and a third CDR comprising amino acids 225-235 of
SEQ ID NO: 1,
b) the first variable region is a V^ region comprising a first CDR comprising amino
acids 31-37 of SEQ ID NO: 2. a second CDR comprising amino acids 52-67 of SEQ ID NO: 2, and a third CDR comprising arnino acids 100-114 of SEQ ID NO: 2; and the second variable region is a V|
region comprising a first CDR comprising ainino acids 164-176 of SEQ ID NO: 2, a second CDR comprising amino acids 192-198 of SEQ ID NO: 2, and a third CDR comprising amino acids 231-241 of
SEQ ID NO: 2;
c) the first variable region is a region comprising a first CDR comprising amino
acids 31-35 of SEQ ID NO. 3, a second CDR comprising amino acids 50-66 of SEQ ID NO: 3. and a third (.T)R comprising ainino acids 99-108 of SEQ ID NO: 3; and the second variable region is a Vj
region comprising a first CDR comprising amino acids 158-170 of SEQ ID NO: 3, a second CDR comprising aminu acids -186-192 of SEQ ID NO: 3, and a third CDR comprising amino acids 225-235 of SEQ ID NO: 3,

d) the first variable region is a Vn region comprising a first CDR comprising amino
acids 31-35 of SEQ ID NO: 4, a second CDR comprising amino acids 50-66 of SEQ ID NO: 4, and a third CDR comprising amino acids 99-108 of SEQ ID NO: 4; and the second variable region is a Vj
region comprising a first CDR comprising amino acids 158-170 of SEQ ID NO: 4, a second CDR comprising amino acids 186-192 of SEQ ID NO: 4, and a third CDR comprising amino acids 225-235 of SEQ ID NO: 4;
e) the first variable region consists of amino acids 1-119 of SEQ ED NO: 1 and the
second variable region consists of amino acids 136-245 of SEQ ID NO: 1;
f) the first variable region consists of amino acids 1-125 of SEQ ID NO: 2 and the
second variable region consists of amino acids 142-251 of SEQ ID NO: 2;
g) the first variable region consists of amino acids 1 -119 of SEQ ID NO: 3 and the
second variable region consists of amino acids 136-245 of SEQ ID NO: 3; or
h) the first variable region consists of amino acids 1-119 of SEQ ID NO: 4 and the second variable region consists of amino acids 136-245 of SEQ ID NO: 4.
II.C. Further Identification of Antigen Binding Proteins
Single-chain antibodies SEQ ID NOs: 1, 2, 3 or 4 can be used to identify additional antigen binding proteins binding to a targeted region. Identification can be performed using different techniques such as screening for antigen binding proteins that compete with SEQ ED NOs: 1, 2, 3 or 4 binding to SR-B1. mapping theepitope recognized by single-chain antibodies SEQ ED NOs: 1, 2, 3 or 4, and utilizing the epitope itself to select for additional antigen binding protein.
Antigen binding proteins for use in a competition assay can be generated using SR-B1 as an antigen Techniques for generating antigen binding protein such as a single-chain antibody, an antibody, or an antibody fragment are well known in the art. Examples of such techniques include the use of phage display technology, identification and humanization of rodent antibodies, and generation of human antibodies using a XenoMouse or Trans-Chromo mouse. (E.g.. Azzazy ct al.. Clinical biochemistry 35:425-445, 2CK)2, Bcrger et al.. Am. J. Mcd. Sci 324(1): [4-40, 2002.)
J1J. Protein Production
Antigen binding protein are preferably produced using recombinant nucleic acid
techniques or through the use of a hybridoma. Recombinant nucleic acid techniques involve constructing a nucleic acid template for protein synthesis. A hybridoma is an immortalized cell line producing the antigen binding protein.
Kecninbinunt nucleic acid encoding an antigen binding protein can IK- expressed in a host eel! that in effect serves as a factory for the encoded protein. The recombinant nucleic acid can provide a

recombinant gene encoding the antigen binding protein that exists autonomously from a host cell genome or as part of the host cell genome.
A recombinant gene contains nucleic acid encoding a protein along with regulatory elements for protein expression. Generally, the regulatory elements that are present in a recombinant gene include a transcriptional promoter, a ribosome binding site, a terminator, and an optionally present operator. A preferred element for processing in eukaryotic cells is a polyadenylation signal. Antibody associated introns may also be present. Examples of expression cassettes for antibody or antibody fragment production are well known in art. (E.g.. Persic et ai. Gene 757:9-18, 1997, Boel et ai. J. Immunol. Methods 239:153-166, 2000, Liang et ai, J. Immunol. Methods 247:119-130, 2001.)
Expression of a recombinant gene in a cell is facilitated using an expression vector. Preferably, an expression vector, in addition to a recombinant gene, also contains an origin of replication for autonomous replication in a host cell, a selectable marker, a limited number of useful restriction enzyme sites, and a potential for high copy number. Examples of expression vectors for antibody and antibody fragment production are well known in art. (E.g.. Persic et ai. Gene 757:9-18, 1997, Boel et ai. J. Immunol. Methods 239:153-166, 2000, Liangetai. J. Immunol. Methods 247:119-130, 2001.)
If desired, nucleic acid encoding an antibody may be integrated into the host
chromosome using techniques well known in the art. (See, Ausubel, Current Protocols in Molecular Biology. John Wiley, 1987-1998, Marks et ai, International Application Number WO 95/17516, International Publication Date June 29, 1995.)
A variety of different cell lines can be used for recombinant antigen binding protein expression, including those from prokaryotic organisms (e.g.. E. coli. Bacillus, and Streptomyces) and from Eukaryotic (e.g.. yeast, Baculovirus. and mammalian). (Breitling et al.. Recombinant Antibodies, John Wiley & Sons, Inc. and Spektrum Akademischer Verlag, 1999.)
Preferred hosts for recombinant antigen binding protein expression are mammalian cells able to produce antigen binding protein with proper post translational modifications. Post translational modifications include disulfidc bond formation and glycosylation. Another type of post translational modification is signal pcptide cleavage.
Proper glycosylation can be important for antibody function. (Yoo et ai. Journal of Immunologival Methods 267:1-20, 2002.) Naturally occurring antibodies contain at least one N-linked carbohydrate attached to a heavy chain (Id.) Additional N-linked carbohydrates and O-linked carbohydrates may be present and may be important for antibody function. (Id.)
Different types of mammalian host cells can be used to provide for efficient posl-translational mcxlifications. Examples of such host cells include Chinese hamster ovary (Cho), HeLa, C6, PC 12. and myeloma cells. (Yoo «.•/ ai. Journal of Immunological Methods 267:1-20, 2002. Persic el ai. Gene /«7:9-l8, 1997.)

A hybridoma is an immortalized antibody producing cell line. A hybridoma can be produced using techniques such as those described in Ausubel Current Protocols in Molecular Biology, John Wiley, 1987-1998, Harlow el a\.. Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, and Kohler et al. Nature 256, 495-497, 1975.
IV. Combination Treatment
Antigen binding protein that bind to suitable SR-BI sites can be used to inhibit HCV and treat HCV patients by itself, or in combination with one or more olher anti-HGV agents. Currently approved anti-HCV agents are interferon alpha, and interferon alpha in combination with ribovarin. Different forms of interferon alpha, such as recombinant interferon and peglyated interferons, can used to treat HCV infections. (De Francesco et al., Antiviral Research 58:1-16, 2003, Walker et al., Antiviral Chemistry & Chemotherapy 14:1-21, 2003.)
A variety of different anti-HCV agents are in different phases of clinical developments. The different anti-HCV agents being developed include agents directed against different HCV targets. Examples of different HCV targets include HCV polymerase and HCV NS3-NS4A protease. (De Francesco et al.. Antiviral Research 55:1-16, 2003, Walker et al.. Antiviral Chemistry & Chemotherapy 14:1-21,2003.)
V. Administration
Guidelines for pharmaceutical administration in general are provided in, for example, Remington's Pharmaceutical Sciences 20h Edition, Ed. Gennaro, Mack Publishing, 2000; and Modem Pharmaceutics 2nd Edition, Eds. Banker and Rhodes, Marcel Dekker, Inc., 1990.
Pharmaceutically acceptable carriers facilitate storage or administration of an antigen binding protein. Substances used to stabilize protein solution formulations include carbohydrates, amino acids, and buffering salts. (Middaugh et al.. Handbook of Experimental Pharmacology 757:33-58, 1999.)
Antigen binding proteins can be administered by different routes such as subcutaneous, intramuscular, or mucosal. Subcutaneous and intramuscular administration can be performed using, for example, needles or jet-injectors Mucosal delivery, such as nasal delivery, can involve using enhancers or mucoadhcsivcs to produce a longer retention lime at adsorption sites. (Middaugh et al.. Handbook of Experimental Pharmacology 737:33-58. 1999.)
Suitable dosing regimens are preferably determined taking into account factors well known in the art including age, weight, sex and medical condition of the patient; the route of administration; the desired effect, and the particular compound employed. It is expected that a dose would consist of the range of 1.0 ug to 1.0 mg total protein, in different embodiments of the present invention the range is 0.01 mg to 1.0 ing and 0.1 nig to'1.0 nig.

VI. Examples
Examples are provided below further illustrating different features of the present invention. The examples also illustrate useful methodology for practicing the invention. These examples do not limit the claimed invention.
Example 1: Experimental Procedure to Obtain SR-B1-Specific Phage Antibodies
The CAT (Cambridge antibody technology) phage library CS was screened for single-chain antibodies binding to SR-B 1. The library provides single-chain antibodies containing a variable portion of heavy chain and light chain antibodies exposed on the surface of a filamentous phage as a fusion to the N terminus of the pUl protein. The VH region is joined to the VL region by a linker of Ser
and Gly.
To select phage displaying antibodies specifically binding to the SR-B 1 receptor, whole cells were utilized for phage enrichment. The library (10" phage) was pre-incubated with 101 CHO cells for 1 hour at room temperature, followed by centrifugation. The unbound phage present in the supernatant were recovered and incubated for 1 hour with CHO cells stably expressing the human SR-B1 receptor (SEQ ID NO: 9). Cells were then washed several limes with PBS and resuspended in eluiion buffer (triethylamina, 100 mM) for 25 minutes, followed by adjustment of the pH with Tris.HCl. Recovered phage were amplified by infecting TGI cells and subjected to other two rounds of selection as described above.
Following the third round of selection, 3 x 10s phage were recovered. A sample of these (144 phage) was tested in a cell based EL1SA, using SR-B 1 expressing CHO cells, in parallel with CHO ceils as negative control. Among these 11 phage clones were scored as SR-B1- specific. Clones were subsequently tested for the ability to HCV E2 binding.
Example 2: Inhibition of HCV E2
Clones encoding SEQ ID NOs: 1, 2, 3 and 4 produced outside the phage context were able to inhihit the binding of the HCV E2 protein to HepG2 cells HepG2 is a human hepatoma cell line.
Cells were detached and washed in phosphate buffered saline (PBS), 0.2% BSA, 10 mM Hopes (washing buffer). 4xl()3 cells were allowed to bind to different concentrations (0.5-5-20-40 jag/ml) of single chain antibodies or an unrelated single chain control D5 at room temperature for 30 minutes. Cells were then incubalcd for I hour at room temperature with recombinant soluble E2. Binding was revealed by anti-E2 rat rnAb 6/la (Pafel et al. J. Gen. Viral. 57:2873-2883, 2000) and a secondary unti rat PE-conjugalcd mAb. Fluorescence associated lo the cells was measured by FACS analysis.

As shown in Figure 6, an(i-SRBl single-chain antibodies of SEQ ID NOs: 1, 2, 3, and 4 inhibited E2 protein binding to the HepG2 cell line. The vertical axis represents the percentage of E2 protein binding to HepG2 cells, referring to cells not pre-incubated with antibodies. The experiment was performed in duplicate, the averages of the duplicate samples are shown.
Example 3 Inhibition of HCV Infection Using The Single-Chain Antibody of SEO ID NO: 2
The ability of single-chain antibodies able to inhibit HCV E2 binding to also inhibit HCV replication was illustrated using the single-chain antibody of SEQ ID NO: 2.
Isolated human hepatocytes from surgical liver resection were seeded in 24 well microplates at the density of 3xl05 cells/well. Cells were allowed to attach and recover 24 hours and then medium was replaced with fresh one containing different concentrations of ami-SRBl single-chain antibody SEQ ID NO: 2 (25 and 5 u,g/ml) or a control unrelated single chain antibody FV at the highest concentration (25 jig/ml).
Hepatocytes were preincubated 1 hour at 37°C with the indicated amounts of single-chain antibodies, then medium was replaced with fresh one containing the same amounts of single-chain antibodies and a fixed amount (100 uT) of an infectious human serum from a HCV chronic patient. Cells were incubated 18 hours with the virus to allow infection, then washed and incubated for four days. Total RNA was extracted and viral replication was measured by quantitative RT-PCR.
Typically, 104 to JO5 copies of genomes/well are detected after four days from infection. To be sure thai the measured viral RNA derived from active replication, a small molecule inhibitor of the viral replicasc was included as a positive control. As shown in Figure 7, anti-SRBl single chain of SEQ ID NO: 2 (scFVCll) can block HCV infection of cultured human hepatocytes.
Example 4: 1^0production from Single-Chain Antibodies
lpCi4 molecules were produced containing SEQ ID NO: 2 or SEQ ID NO: 4 variable regions SEQ ID NOs: 2 or 4 variable regions were separately amplified by PCR using primers specific for the Vjj ami the VL regions. The primers contained additional nuclcotidcs for the introduction of
unique restriction sites, and bases representing splice sites or coding for additional amino acids.
The amplified products were introduced into two separate mammalian expression vectors: Vj \ w;is inserted in the pEU8.2 containing a cassette for the expression of human gamma 4 heavy chain, while Vj was introduced into the vector pEU 4.2 expressing the constant region of the human lambda lij'hi chain Both veclois carry an intron between the leader sequence and the constant icgion sequentf of the antibody. The intron contains unique restriction sites suitable for cloning the amplified vaiiable domains

IgG production was achieved by co-transfecting the Vj-j and VL expression vectors into 293-EBNA cells (Invitrogen), using Lipofectamine 2000 reagent (Invitrogcn) and collecting the supernatant for up to eight days. IgG's were purified from culture medium using Hi-Trap protein A columns, Amersham, following manufacturers instruction.
Example 5: Inhibition of HCV E2 Protein Binding Using Anti-SRB I IeG
Cho7s cells stably expressing the human SR-B1 receptor (Scarselli el ai, Tlie EMBO Journal 2/f/9j:5017-5025, 2002) were used to evaluate the ability of anti-SRBl IgG containing the SEQ ID NO: 2 or 4 variable regions to inhibit HCV E2 binding. Cells were detached and washed in phosphate buffered saline (PBS), 0.2% BSA. 10 mM Hepes (washing buffer). 4 x 105 cells were allowed to bind to different concentrations (60-12-2.4 ng/ml) of antibodies at room temperature for 30 minutes. Cells were then incubated for 1 hour at room temperature with recombinam soluble E2 (carrying a His-Tag).
Binding was revealed by an anti-penta His biotin conjugate and streptavidin-R-PE. Fluorescence associated to the cells was measured by FACS analysts. As shown in Figure 8, anti-SRBl IgG4 SEQ ID NOs: 2 and 4 inhibit E2 protein binding to the Cho7s cell line. Unrelated IgGs were used as a negative control
Other embodiments are within the following claims. While several embodiments have been shown and described, various modifications may be made without departing from the spirit and scope of ihe present invention

WHAT IS CLAIMED IS:
1. An isolated antigen binding protein comprising a first variable region and a
second variable region, wherein said first and said second variable regions bind one or more target
regions selected from the group consisting of the SEQ ID NO: 1 target region, the SEQ ID NO: 2 target
region, the SEQ ID NO: 3 target region, and the SEQ ID NO: 4 target region.
2. The antigen binding protein of claim 1, wherein either:
a) said first variable region is a Vj, region comprising a first CDR comprising amino
acids 31-35 of SEQ ID NO: I, a second CDR comprising amino acids 50-66 of SEQ ID NO: 1. and a third CDR comprising amino acids 99-108 of SEQ ID NO: 1; and said second variable region is a Vj
region comprising a first CDR comprising amino acids 158-170 of SEQ ED NO: I, a second CDR comprising amino acids 186-192 of SEQ ID NO: 1, and a third CDR comprising amino acids 225-235 of
SEQ ID NO: 1;
b) said first variable region is a Vfo region comprising a first CDR comprising amino
acids 31 -37 of SEQ ID NO: 2, a second CDR comprising amino acids 52-67 of SEQ ED NO: 2, and a third CDR comprising the amino acids 100-114 of SEQ ID NO: 2; and said second variable region is a V|
region comprising a first CDR comprising amino acids 164-176 of SEQ ID NO: 2, a second CDR comprising amino acids 192-198 of SEQ ID NO: 2, and a third CDR comprising amino acids 231-241 of SEQ ID NO: 2;
c) said first variable region is a Vf, region comprising a first CDR comprising amino
acids 31 -35 of SEQ ID NO: 3, a second CDR comprising amino acids 50-66 of SEQ ID NO: 3, and a third CDR comprising amino acids 99-108 of SEQ ID NO: 3; and said second variable region is a V|
region comprising a first CDK comprising amino acids 158-170 of SEQ ID NO: 3, a second CDR comprising amino acids 186-192 of SEQ ID NO. 3, and a third CDR comprising amino acids 225-235 of SEQ ID NO. 3; or
d) said first variable icgion is a Vf, region comprising a first CDR comprising amino
acids 31 -35 of SI:Q ID NO: 4, a second CDR comprising amino acids 50-66 of SEQ ID NO: 4, and a third CDR comprising amirio acids 99-108 ot SEQ ID NO: 4; and said second variable region is a V|
region comprising a first CDR comprising amino acids 158-170 of SEQ ID NO: 4, a second CDR comprising amino acids 186-192 of SEQ ID NO: 4, and a third CDR comprising amino acids 225-235 of SI-Q IJ) NO: 4.
3. The binding protein of claim 2, wherein said binding protein is an antibody 01 a
fragment thereof

1
4. The binding protein of claim 3, wherein either:
a) said first variable region consists of amino acids 1-119 of SEQ ID NO: 1 and said
second variable region consists of amino acids 136-245 of SEQ ID NO: 1;
b) said first variable region consists of amino acids 1-125 of SEQ ID NO: 2 and said
second variable region consists of amino acids 142-251 of SEQ ID NO: 2,
c) said third Vfo region consists of amino acids 1-119 of SEQ ID NO: 3 and said third Vj
region consists of amino acids 136-245 of SEQ ED NO: 3; or
d) said fourth Vfo region consists of amino acids 1-119 of SEQ ID NO: 4 and said fourth
V| region consists of amino acids 136-245 of SEQ ID NO: 4.
5. The binding protein of claim 4, wherein said binding protein is an antibody.
6. The binding protein of claim 5, wherein said antibody is a monoclonal antibody.
7. The binding protein of claim 3, wherein said binding protein is a single chain
antibody wherein said Vh region is joined to said V| region by an amino acid linker about 5 to 16 amino
acids in length.
8. The binding protein of claim 7, wherein either:
a) said first variable region consists of amino acids 1-119 of SEQ ED NO: 1 and said
second variable region consists of amino acids 136-245 of SEQ ID NO: 1;
b) said first variable region consists of amino acids 1-125 of SEQ ID NO: 2 and said
second variable region consists of amino acids 142-251 of SEQ ID NO: 2;
c) said first variable region consists of amino acids 1-119 of SEQ ID NO: 3 and said
second variable region consists of amino acids 136-245 of SEQ ID NO: 3; or
d) said first variable region consists of amino acids 1-119 of SEQ ID NO: 4 and said
second variable region consists of amino acids 136-245 of SEQ ID NO: 4.

9. A nucleic acid comprising a recombinant gene encoding the antigen binding
protein ol any one of claims 1 -X.
10. A pharmaceutical composition comprising the binding protein of any one of
claims 1-8 and a pharmaceulically acceptable carrier

11. A method of inhibiting HCV replication in cell comprising the step of providing
to said cell an effective amount of the binding protein of any one of claims 1-8.
12. A method of treating HCV in a patient comprising the step of administering to
said patient an effective amount of the binding protein of any one of claims 1-8.
13. The method of claim 12, wherein said patient is a human infected with HCV.
14. A recombinant cell comprising the nucleic acid of claim 9, wherein said
nucleotide sequence is expressed in said cell.
15. A method of producing an antigen binding protein comprising the step of
growing the recombinant cell of claim 14 under conditions wherein said nucleotide sequence is
expressed in said cell and isolating said antigen binding protein.
16. The Invention substantially such as herein before described.

Documents:

769-delnp-2007-Abstract-(04-06-2013).pdf

769-delnp-2007-abstract.pdf

769-delnp-2007-assignment.pdf

769-delnp-2007-Claims-(25-02-2014).pdf

769-delnp-2007-claims.pdf

769-delnp-2007-Correspondence Others-(14-12-2012).pdf

769-delnp-2007-Correspondence Others-(18-12-2013).pdf

769-delnp-2007-Correspondence Others-(20-12-2013).pdf

769-delnp-2007-Correspondence Others-(25-02-2014).pdf

769-delnp-2007-Correspondence Others-(27-07-2012).pdf

769-delnp-2007-Correspondence-Others-(04-06-2013).pdf

769-delnp-2007-Correspondence-Others-(13-06-2013).pdf

769-delnp-2007-correspondence-others.pdf

769-delnp-2007-description (complete).pdf

769-delnp-2007-Drawings-(04-06-2013).pdf

769-delnp-2007-drawings.pdf

769-delnp-2007-form-1.pdf

769-delnp-2007-Form-2-(04-06-2013).pdf

769-delnp-2007-form-2.pdf

769-delnp-2007-Form-3-(13-06-2013).pdf

769-delnp-2007-Form-3-(14-12-2012).pdf

769-delnp-2007-Form-3-(18-12-2013).pdf

769-delnp-2007-Form-3-(27-07-2012).pdf

769-delnp-2007-form-3.pdf

769-delnp-2007-form-5.pdf

769-delnp-2007-gpa.pdf

769-delnp-2007-pct-101.pdf

769-delnp-2007-pct-210.pdf

769-delnp-2007-pct-220.pdf

769-delnp-2007-pct-237.pdf

769-delnp-2007-pct-304.pdf

769-delnp-2007-Petition-137-(25-02-2014).pdf

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Patent Number

259637

Indian Patent Application Number

769/DELNP/2007

PG Journal Number

13/2014

Publication Date

28-Mar-2014

Grant Date

20-Mar-2014

Date of Filing

31-Jan-2007

Name of Patentee

ISTITUTO DI RICERCHE DI BIOLOGIA MOLECOLARE P ANGELETTI SPA

Applicant Address

VIA PONTINA KM.30.600 I-00040 POMEZIA ITALY

Inventors:

#	Inventor's Name	Inventor's Address
1	VITELLI, ALESSANDRA	VIA PONTINA KM.30.600 I-00040 POMEZIA ITALY
2	CORTESE, RICCARDO	VIA PONTINA KM.30.600 I-00040 POMEZIA ITALY
3	LUZZAGO, ALESSANDRA	VIA PONTINA KM.30.600 I-00040 POMEZIA ITALY
4	NICOSIA, ALFREDO	VIA PONTINA KM.30.600 I-00040 POMEZIA ITALY

PCT International Classification Number

C07K 16/28

PCT International Application Number

PCT/EP2005/007160

PCT International Filing date

2005-07-01

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	60/586,356	2004-07-08	U.S.A.