KR20110019044A - Hepatitis C virus-mediated liver disease prevention and treatment containing anti-NS5A antibody - Google Patents

Abstract

PURPOSE: An agent containing an anti-NS5A antibody for preventing and treating hepatitis C virus-mediated liver diseases is provided to suppress replication of hepatitis C virus. CONSTITUTION: An anti-NS5A antibody specifically binds to middle epitope of hepatitis C virus NS5A protein. The antibody is a monoclonal antibody. The N-terminal epitope of NS5A protein is amino acids 66-70. A pharmaceutical composition for preventing and treating hepatitis C virus-mediated liver diseases contains N-terminal epitope of amino acids 60-80; or anti-NS5A antibody which specifically binds to the middle epitope of amino acids 221-236. A method for diagnosing hepatitis type C comprises: a step of preparing a biological sample; a step of contacting an antibody which specifically binds to the middle epitope; a step of quantitating an antibody-antigen complex; and a step of comparing detection result with a normal control group.

Description

Pharmaceutical composition for prevention and treatment of Hepatitis C Virus-mediated liver disease containing anti-NS5A antibody

The present invention relates to an agent for preventing and treating liver disease, and more particularly, to an agent for preventing and treating hepatitis C virus mediated liver disease, including an antibody against NS5A protein.

Hepatitis C virus (HCV) was first discovered in 1989 and is one of the leading causes of liver diseases such as acute or chronic hepatitis, cirrhosis and liver cancer (Choo Q, et al, Science 244: 359-362). , 1989; Kuo, G. et al., Science, 244: 362-364, 1989). Hepatitis C virus spreads mainly by parenteral routes, and as in the case of hepatitis B virus, it is spread by needles, needles, blood transfusions, and blood products contaminated with the virus.

At least 3% of the world's population is infected with HCV, and HCV infection often progresses to cirrhosis and liver cancer [Saito I. et al., Proc. Natl. Acad. Sci. USA 1990; 87: 6547-6549; Di Bisceglie AM et al., J. Clin. Gastroenterol. 1994; 19: 222-226; Shimotohno K. Intervirology 1995; 38: 162-169. About 80% of patients with acute HCV infection develop chronic hepatitis, 20% of which develop cirrhosis and liver cancer.

The hepatitis C virus is an important pathogen for liver diseases such as hepatitis, cirrhosis and liver cancer. The prevention or treatment of the hepatitis C virus is significant, but the virus has not been developed yet. For the treatment of hepatitis C virus, interferon-α alone or interferon-α in combination with ribavirin has been used. However, these two drugs are not specific for hepatitis C virus, cause serious side effects (such as retinopathy, thyroiditis, acute pancreatitis, and depression), and are not effective for all patients with hepatitis C. Different effects occur depending on the viral strain. Therefore, there is an urgent need for the development of new antiviral agents effective in the treatment of hepatitis C virus infection that overcomes these limitations.

HCV belongs to the genus Hepacivirus in the Flaviviridae family. HCV has an envelope, within which the genome of 9.6 kb is disconnected, and in positive-sense RNA form, which is homologous to flavivirus and pestivirus [Miller RH et al., Proc . Natl Acad Sci USA 1990; 87: 2057-2061; Takamizawa A. et al., J. Virol. 1991; 65: 1105-1113. The genome is located at the 5 'end and Brown EA et al., Nucleic Acids Res 1992; 20: 5041-5045; Chen PJ et al., Virology 1992; 188: 102-113] at the 3 'end [Kolykhalov AA et al., J Virol 1996; 70: 3363-3371; Tanaka T. et al., J Virol 1996; 70: 3307-3312] encodes polyprotein precursors having untranslated regions (UTRs) and consisting of 3010 amino acids. This polyprotein is processed into at least 10 functional proteins. Core and envelope proteins (E1, E2 and p7) present at the N-terminus are cut by structural cleavage signals by host cell signal cleavage enzymes [Hijikata M. et al., Proc Natl Acad Sci USA 1991; 88: 5547-5551; Matsuura Y. et al., Virology 1994; 205: 141-150; Lin C. et al., J Virol 1994; 68: 5063-5073]. The rest (NS2, NS3, NS4A, NS4B, NS5A and NS5B) are nonstructural proteins with varying enzymatic activity, including serine proteases, RNA helicases, and RNA-dependent RNA polymerases. Processed [Grakoui A. et al., Proc Natl Acad Sci USA 1993; 90: 10583-10587; Bartenschlager R. et al., J Virol 1993; 67: 3835-3844; Tomei L. et al., J Virol 1993; 67: 4017-4026.

Nonstructural protein 5A (hereinafter referred to as "NS5A") is a phosphoprotein consisting of 447 amino acids. The NS5A protein is phosphorylated primarily at serine residues by cellular kinase and exists in two polypeptide forms, p56 and p58. These two polypeptides appear to regulate viral RNA replication. NS5A protein is a multifunctional protein involved in cell signaling regulation and antiviral resistance to interferon. Some genotype-derived NS5A interact with protein kinase (PKR), which is activated by IFN-induced double-stranded RNA, through the IFN-sensitivity-determining region (ISDR) and as an inhibitor of protein kinase. Perform the function. NS5A is located in the cytoplasm of mammalian cells and HCV infected patient cells. However, NS5A can direct heterologous proteins to the nucleus via a nuclear localization signal (NLS) at the C-terminus. NS5A protein protects against TNF-a and p53-mediated self-killing and promotes tumor growth and expression of several cell cycle regulatory genes. In addition, the NS5A protein disrupts cellular signaling pathways by selectively targeting growth-factor-receptor-bound protein 2 (Grb2) adapter proteins.

An object of the present invention is to provide an effective hepatitis C virus-mediated pharmaceutical composition for the prevention and treatment of liver disease, effective hepatitis C virus diagnostic method and diagnostic kit.

The nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) is a multifunctional protein that leads to pleiotropic responses in part by regulating cell growth and cellular signaling. We have produced monoclonal antibodies against HCV NS5A protein. The N-terminal epitope of the NS5A protein was measured at amino acids 60-80 and the epitope at the middle site was measured at amino acids 221-236. These epitopes overlap with human vesicle-associated membrane-protein-associated protein-B (hVAP-B) and TRN2 (TNF-receptor-associated factor 2) binding sites, respectively. We studied the ability of these monoclonal antibodies to inhibit viral and cellular interactions. As a result, the NS5A and hVAP-B interactions are eliminated by the monoclonal antibody E5D3 specific for amino acids 60-80, the N-terminal epitope of the NS5A protein, and the interaction of NS5A and TRAF2 is an intermediate region epitope of the NS5A protein. It was inhibited by the monoclonal antibody C6D4 specific for phosphorus amino acids 221-236. Since hVAP-B is essential for HCV replication and TRAF2 is a major messenger in the TNF signaling system, these data will help to understand the mechanisms underlying HCV-induced onset.

The present invention provides anti-NS5A antibodies that specifically bind to an N-terminal epitope of amino acids 60-80 or an intermediate epitope of amino acids 221-236 of the hepatitis C virus NS5A protein.

In addition, the present invention is characterized in that the N-terminal epitope of the NS5A protein is amino acids 66-70.

Hepatitis C virus mediated liver disease prevention and treatment comprising an anti-NS5A antibody that specifically binds to an N-terminal epitope of amino acids 60-80 or an intermediate epitope of amino acids 221-236 of the Hepatitis C virus NS5A protein. It provides a pharmaceutical composition.

In addition, the hepatitis C virus-mediated liver disease is characterized in that it is selected from the group consisting of acute hepatitis C, chronic hepatitis C, cirrhosis and liver cancer.

The present invention provides a hepatitis C diagnostic kit comprising an anti-NS5A antibody that specifically binds an N-terminal epitope of amino acids 60-80 or an intermediate epitope of amino acids 221-236 of the hepatitis C virus NS5A protein.

The kit includes a kit for an Enzyme linked immunosorbent assay (ELISA).

In addition, the present invention is characterized in that the antibody is a monoclonal antibody.

The present invention provides a biological sample selected from tissues, urine, blood, serum and plasma for antibodies that specifically bind to an N-terminal epitope of amino acids 60-80 or an intermediate epitope of amino acids 221-236 of the hepatitis C virus NS5A protein. It provides a method for diagnosing hepatitis C comprising contacting with.

In addition, the diagnostic method of the present invention

a) providing a biological sample for analysis;

b) contacting said sample with an antibody that specifically binds to an N-terminal epitope of amino acids 60-80 or an intermediate epitope of amino acids 221-236 of the hepatitis C virus NS5A protein;

c) quantitatively detecting the antigen-antibody complex; And

and d) comparing the detection result of step c) with a normal control group.

The present invention relates to a hepatitis C diagnostic kit comprising an agent for measuring the mRNA of an N-terminal epitope of amino acids 60-80 or an intermediate epitope of amino acids 221-236 or a protein thereof of the hepatitis C virus NS5A protein.

The invention also includes contacting a biological sample with an agent that measures the mRNA of an N-terminal epitope of amino acids 60-80 or an intermediate epitope of amino acids 221-236, or a protein thereof, of the hepatitis C virus NS5A protein. A method for diagnosing hepatitis C.

"Diagnosis" in the present invention means to identify the presence or characteristics of hepatitis C virus.

In one embodiment of the present invention, the N-terminal epitope of amino acids 60-80 and the intermediate epitope of amino acids 221-236 of the human hepatitis C virus NS5A protein are GLy-Ala-Gln-Ile-Ala-Gly-His-Val, respectively. 21 amino acids of -Lys-Asn-Gly-Ser-Met-Lys-Ile-Val-Gly-Pro-Lys-Thr-Cys (SEQ ID NO: 2) and Gly-Ser-Pro-Pro-Ser-Leu-Ala- It consists of 16 amino acids of Ser-Ser-Ser-Ala-Ser-Gln-Leu-Ser-Ala (SEQ ID NO: 3). However, the N-terminal epitope and the intermediate epitope amino acid sequence of the present invention are not the same in every individual, and variations may occur in part depending on the individual. The present invention is not limited to the amino acid sequences described above, and the N-terminal epitope, mutated amino acids 60-80 of human hepatitis C virus NS5A protein, and the intermediate epitope sequence, amino acids 221-236, are also within the scope of the present invention. It is apparent to those skilled in the art to which the present invention pertains.

N-terminal epitopes of amino acids 60-80 and intermediate epitopes of amino acids 221-236 of the human hepatitis C virus NS5A protein of the present invention are believed to be involved in the development of hepatitis C, and for the diagnosis, prevention and treatment of hepatitis C It can be effectively used in the composition and the like.

Antibodies of the present invention can be prepared by conventional methods known in the art using N-terminal epitopes, amino acids 60-80, and intermediate epitopes, or amino acids 221-236, of the human hepatitis C virus NS5A protein of the present invention. It can be prepared as a monoclonal antibody (polyclonal antibody) or a polyclonal antibody, using these antibodies known in the art (Enzyme Linked Immunosorbent assay (ELISA), radioimmunoassay (Radioimmunoassay; Hepatitis C can be diagnosed by confirming whether the protein is expressed in a bodily fluid sample or a tissue sample by a method such as RIA), sandwich assay, western blot or immunoblot on polyacryl gel.

The amount of protein in a biological sample can be measured by various methods.

The term "biological sample" in the present invention is preferably used for tissues, cells, and the like that can detect a difference in protein levels between N-terminal epitopes of amino acids 60-80 and intermediate epitopes of amino acids 221-236 of the human hepatitis C virus NS5A protein. Preferably liver tissue, urine, blood, plasma, serum, and the like.

In the present invention, "protein level measurement" refers to a process for checking the presence and extent of the N-terminal epitope of amino acids 60-80 and intermediate epitopes of amino acids 221-236 of the human hepatitis C virus NS5A protein in a biological sample. The amount of the protein is confirmed using an antibody that specifically binds to the protein.

"Antibody" means a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to an N-terminal epitope of amino acids 60-80 or an intermediate epitope of amino acids 221-236 of a human hepatitis C virus NS5A protein. It includes both clone antibodies and recombinant antibodies.

Assays for measuring protein levels using antibodies include Western blot, Enzyme linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), Radioimmunodiffusion, Ouchterlony immunodiffusion, Rocket immunoelectrophoresis, Tissue immunostaining, immunoprecipitation, complement fixation assay, FACS, protein chips, and the like, are not limited to the described methods.

Through the above analysis method, the amount of antigen-antibody complex formation in the normal control group and the amount of antigen-antibody complex formation in suspected hepatitis C patients can be compared, and the N-terminal epi of amino acids 60-80 of the hepatitis C virus NS5A protein. It is possible to diagnose whether the hepatitis C suspected person is actually onset or infected by determining whether the expression level of the epitope of the top or amino acids 221-236 increases significantly.

"Antigen-antibody complex" means a combination of an N-terminal epitope, amino acids 60-80, or an intermediate epitope, amino acids 221-236, of the hepatitis C virus NS5A protein with an antibody specific thereto, and formation of an antigen-antibody complex. The amount can be measured quantitatively through the signal magnitude of the detection label. The detection label can be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules and radioisotopes, but is not limited to the materials described above. When enzymes are used as detection labels, the available enzymes include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase or alkaline phosphatase, acetyl Cholinesterase, glucose oxidase, hexokinase and the like, and are not limited to the ranges described above. Fluorescent materials include fluorescein, phycocyanin, fluorescarmine and the like, and are not limited to the above-described materials. Ligands include, but are not limited to, biotin derivatives. Luminescent materials include luciferin and the like, but are not limited thereto. The microparticles include colloidal gold and the like, but are not limited thereto. Redox molecules include, but are not limited to, quinone, 1,4-benzoquinone, hydroquinone, and the like. Radioisotopes include, but are not limited to, ³ H, ¹⁴ C, and the like.

Protein expression level measurement is preferably by using an ELISA. In ELISA, a direct sandwich ELISA using another labeled antibody that recognizes the antigen in the complex of the antibody and the antibody attached to the solid support, and reacted with another antibody that recognizes the antigen in the complex of the antibody and antigen attached to the solid support Various ELISA methods include an indirect sandwich ELISA using a labeled secondary antibody that recognizes this antibody. More preferably, the antibody is attached to a solid support and the sample is reacted, followed by enzymatic development by attaching a labeled antibody that recognizes the antigen of the antigen-antibody complex, or to an antibody that recognizes the antigen of the antigen-antibody complex. It is detected by a sandwich ELISA method in which the labeled secondary antibody is attached and enzymatically developed. Hepatitis C virus NS5A protein of the amino acid 60-80 N-terminal epitope or the intermediate epitope of amino acids 221-236 and the degree of complex formation of the antibody can be confirmed whether the onset or infection of hepatitis C.

Alternatively, one or more antibodies that specifically bind to an N-terminal epitope, amino acids 60-80 or an intermediate epitope amino acids 221-236, of the hepatitis C virus NS5A protein are arranged at a predetermined position on a substrate and immobilized at high density. Protein chips are available. In the method of analyzing a sample using a protein chip, the protein is separated from the sample, and the separated protein is hybridized with the protein chip to form an antigen-antibody complex. You can check whether or not.

Another method is to perform Western blotting using antibodies specific for the N-terminal epitope, amino acids 60-80 or intermediate epitopes amino acids 221-236, of the hepatitis C virus NS5A protein. The whole protein is isolated from the sample, which is electrophoresed to separate the protein according to size and then transferred to the nitrocellulose membrane to react with the antibody. The onset can be confirmed by confirming the amount of the generated antigen-antibody complex using a labeled antibody.

The level of the protein can be expressed as an absolute or relative difference between the N-terminal epitope, amino acids 60-80 of the hepatitis C virus NS5A protein, or the middle epitope, amino acids 221-236.

The present invention relates to a hepatitis C diagnostic kit comprising an antibody that specifically binds an N-terminal epitope of amino acids 60-80 or an intermediate epitope of amino acids 221-236 of the hepatitis C virus NS5A protein.

In addition, the present invention also includes contacting a biological sample with an antibody that specifically binds to an N-terminal epitope of amino acids 60-80 or an intermediate epitope of amino acids 221-236 of the hepatitis C virus NS5A protein. It is about a diagnostic method.

In the present invention, the production of an antibody using an N-terminal epitope of amino acids 60-80 or an intermediate epitope of amino acids 221-236 of the hepatitis C virus NS5A protein may be easily prepared using techniques well known in the art. Can be. As is well known in the art, polyclonal antibodies are collected from an animal by injecting the animal with an N-terminal epitope, amino acids 60-80, or an intermediate epitope, amino acids 221-236, of the hepatitis C virus NS5A protein. To obtain serum containing the antibody. Animals can be prepared using any animal host such as goat, rabbit, pig. Monoclonal antibodies are known in the art to which the present invention pertains, such as the hybridoma method (see Kohler & Milstein (1976) European J. Immunology 6 : 511-519) or phage antibody libraries (Clackson et al., Nature , 352 : 624-628, 1991; Marks et al, J. Mol. Biol. , 222 : 58, 1-597, 1991).

The hybridoma method utilizes cells of an immunologically suitable host animal, such as a mouse injected with an N-terminal epitope, amino acids 60-80 of the hepatitis C virus NS5A protein, or an intermediate epitope, amino acids 221-236, and the other. Uses cancer or myeloma cell lines. These two kinds of cells are fused by methods well known in the art, such as polyethylene glycol, and then antibody-producing cells are propagated by standard tissue culture methods. Antibodies specific for the N-terminal epitope, amino acids 60-80 or hepatitis C 22-236 of the hepatitis C virus NS5A protein, after obtaining a uniform cell population by subcloning by the limited dilution technique Hybridomas capable of producing C are cultured in vitro or in vivo according to standard techniques.

The phage antibody library method obtains an antibody gene for an N-terminal epitope of amino acids 60-80 or an intermediate epitope of amino acids 221-236 of the hepatitis C virus NS5A protein and expresses it in the form of a fusion protein on the surface of the phage. To prepare an antibody library in vitro and to isolate and prepare a monoclonal antibody binding to an N-terminal epitope of amino acids 60-80 or an intermediate epitope of amino acids 221-236 of the hepatitis C virus NS5A protein. to be.

Antibodies prepared by the above method can be separated by electrophoresis, dialysis, ion exchange chromatography, affinity chromatography and the like.

Antibodies included in the kits of the present invention include functional fragments of antibody molecules, as well as complete forms having two full length light chains and two full length heavy chains. The functional fragment of an antibody molecule means a fragment which retains at least antigen binding function, and includes Fab, F (ab '), F (ab') ₂ , F (ab) ₂ and Fv.

The diagnostic kit of the present invention comprising an antibody specifically binding to an N-terminal epitope of amino acids 60-80 or an intermediate epitope of amino acids 221-236 of the hepatitis C virus NS5A protein is preferably a diagnostic kit for ELISA, Antibodies specific for the control protein, and other reagents capable of detecting antigen-antibody complexes, such as labeled secondary antibodies, chromophores, enzymes conjugated with antibodies and their substrates or antibodies Other materials and the like.

In addition, the present invention provides a liver disease prevention comprising an antibody and a pharmaceutically acceptable carrier that specifically binds to an N-terminal epitope of amino acids 60-80 or an intermediate epitope of amino acids 221-236 of the hepatitis C virus NS5A protein. And pharmaceutical compositions for treatment. The pharmaceutical composition of the present invention, in addition to the antibody specifically binding to the N-terminal epitope of amino acids 60-80 or intermediate epitope of amino acids 221-236 of the hepatitis C virus NS5A protein, a pharmaceutically acceptable carrier, that is, saline, Sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome and one or more of these components can be mixed and used, and other conventional additives such as antioxidants and buffers can be added as necessary. Can be. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions, and the like, and may act specifically on target organs. Target organ specific antibodies or other ligands may be used in combination with the carriers so as to be used. Furthermore, it may be preferably formulated according to each component by a suitable method in the art or by a method disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA).

The method of administering the pharmaceutical composition of the present invention is not particularly limited, but may be parenterally or orally administered, such as intravenous, subcutaneous, intraperitoneal, or topical application, depending on the desired method. Dosage ranges depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and the severity of the disease. The daily dosage is about 0.01 to 100 mg / kg for the compound, preferably 0.1 to 10 mg / kg, preferably administered once or several times a day.

The composition for treating and preventing hepatitis C of the present invention is an antibody that specifically binds to an N-terminal epitope of amino acids 60-80 or an intermediate epitope of amino acids 221-236 of the hepatitis C virus NS5A protein of the present invention as an active ingredient. Together with a pharmaceutically acceptable carrier, excipient or optionally other additives. The pharmaceutical composition of the present invention is not particularly limited in formulation, but is preferably formulated as an injection.

According to the present invention, it is possible to provide an agent for preventing and treating HCV mediated liver disease, including a monoclonal antibody that binds to an N-terminal epitope or an intermediate epitope of HCV NS5A protein.

Hereinafter, the configuration of the present invention will be described in more detail with reference to specific embodiments of the present invention. However, it will be apparent to those skilled in the art that the scope of the present invention is not limited only to the description of the following examples.

Materials and methods

Construction of NS5A Expression Vector and Recombinant Baculovirus

HCV NS5A isolated from Koreans was used as a template, and cDNA encoding HCV NS5A (genotype 1b) was amplified by polymerase chain reaction and subcloned into XhoI and BglII sites of baculovirus transfer vector pAc-HLT. After confirming the sequence by didioxynucleotide chain termination sequencing, Spodoptera frugiperda (Sf9) cells were transferred to wild-type Autographa Californica nuclear polyhedrosis virus (AcNPV). Cotransfection was done with recombinant transition vector DNA encoding DNA and NS5A. Recombinant baculovirus was selected and amplified by plaque assays [Hwang SB, Lee CZ, Lai MM (1992) Hepatitis delta antigen expressed by recombinant baculoviruses: comparison of biochemical properties and post-translational modifications between the large and small forms. Virology 190: 413-422, Hwang SB, Park KJ, Kim YS, Sung YC, Lai MM (1997) Hepatitis C virus NS5B protein is a membrane-associated phosphoprotein with a predominantly perinuclear localization. Virology 227: 439-446. To express NS5A protein in insect cells, Sf9 insect cells were harvested 3 days after infection with recombinant baculovirus. Cell lysates were analyzed by SDS-PAGE and proteins were immunoblotted using Coomassie Brilliant Blue staining and rabbit anti-NS5A polyclonal antibodies. Deletion mutants of NS5A were prepared by PCR into the pGEX4T-1 vector to map epitopes. Wild-type and variant proteins of NS5A expressed glutathione S-transferase (GST) in these Coli BL21 (DE3) cells (Novagen).

Cells and viruses

Sf9 insect cells were cultured using Grace's insect tissue culture medium (Invitrogen) containing 10% fetal calf serum at 27 ° C. Recombinant baculoviruses are maintained as Hwang SB et al., (1992) Virology 190: 413-422, Hwang SB et al., (1997) Virology 227: 439-446 and the insect cells have a multiplicity of infection; moi) 9 was used to infect. Hybridoma cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal calf serum, 100 units / ml streptomycin and 100 units / ml penicillin.

NS5A Protein Purification

Sf9 insect cells were infected with recombinant virus expressing NS5A protein and harvested 3 days after infection. Cells were washed twice with PBS and buffer A (20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerol phosphate, 1 mM) Soluble in Na ₃ VO ₄ , 1 μg / ml leupeptin and 1 mM PMSF (phenylmethylsulfonylfluoride) Cell lysates were centrifuged at 15,000 rpm for 15 min, supernatant was SDS-PAGE and stained with Coomassie Blue. NS5A bands were cut and eluted using a Bio-Rad electroeluter (Bio-Rad, Hercules, Calif., USA) according to the manufacturer's instructions After removal of SDS, protein concentration was examined with a Bio-Rad protein assay kit. .

Mouse immunization

Eight week old female BALB / c mice were immunized subcutaneously with 50 μg of NS5A protein mixed with an equal volume of complete Freund's adjuvant. Mice were amplified three times at intervals of two weeks with 50 μg NS5A protein in incomplete Freund's adjuvant. Mice were bled from retro-orbital plexus to check antibody preparation.

Preparation of NS5A Protein Specific Monoclonal Antibodies

Monoclonal antibodies are described in Hwang SB, Lai MMC (1993) A unique conformation at the carboxyl terminus of the small hepatitis delta antigen revealed by a specific monoclonal antibody. Virology 193: 924-931. Briefly, splenocytes were isolated from mice 3 days after the last augmentation injection and fused with gentle shaking using SP2 / 0 myeloma cells and polyethylene glycol (PEG) 1500. The PEG solution was removed by centrifugation at 1,000 rpm for 5 minutes. Cells were placed on 96-well microtiter plates and hybridoma culture supernatants were screened with anti-NS5A antibodies for immunoblot analysis with a miniblotter (Immunetics, Cambridge, MA, USA). Positive hybridomas were cloned twice with marginal dilution. Monoclonal antibodies were purified using protein-A / G Sepharose chromatography in hybridoma culture supernatants.

Immunoblot Analysis

Sf9 cells infected with recombinant baculovirus were harvested three days after infection and washed twice with PBS. Cells were resuspended in Laemmli sample buffer, boiled for 5 minutes and centrifuged for 10 minutes. Cell lysate or purified NS5A protein was isolated by electrophoresis on 10% polyacrylamide gel containing 0.5% SDS and transferred to nitrocellulose membrane at 4 ° C. for one hour. Membranes were incubated for one hour at room temperature with 5% skim milk powder and then for two hours at 4 ° C. with rabbit anti-NS5A antibody. After washing with TNT buffer (10 mM Tris HCl [pH 7.5], 150 mM NaCl, 0.05% [vol / vol] Tween 20), the membrane was incubated with horseradishperoxidase-labeled antirabbit secondary antibody for 2 hours. It was. Proteins were detected using the ECL kit (Amersham Biosciences).

GST pull-down assay

GST-fusion proteins expressed in E. coli BL21 (DE3) cells (Novagen, San Diego, CA, USA) were purified using glutathione-sepharose 4B beads (Amersham Bioscience) as directed by the manufacturer. Huh7 cells were transfected with Flag-hVAP-B or Flag-TRAF2 expressing plasmids. After 36 hours of transfection, cells were buffered A {20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerol phosphate, 1 mM Lysate in Na ₃ VO ₄ , containing 1 μg / ml leupeptin and 1 mM PMSF (phenylmethylsulfonylfluoride). Cell lysates were centrifuged at 15,000 rpm for 10 minutes and then incubated with GST-NS5A fusion protein for 3 hours at 4 ° C. Samples were washed four times with Cell Lysis Buffer A, the binding protein was isolated by SDS-PAGE, transferred to nitrocellulose membrane and immunoblot analyzed with anti-Flag monoclonal antibody (Sigma).

NS5A monoclonal antibody isotyping

Subclasses of NS5A monoclonal antibodies were determined using the IsoStrip mouse monoclonal antibody isotyping kit (Roche Diagnostics). Briefly, each monoclonal antibody was diluted with PBS in a 1: 100 ratio, 150 μl of diluted sample was added to the colored latex beads in the developing tube and incubated for 1 minute with brief agitation at room temperature. The isotyping strip was placed in a development tube and incubated for 5-10 minutes until a positive and isotype-class band was visible.

<Result>

HCV NS5A Protein Expression and Purification

CDNA encoding HCV NS5A was subcloned under the control of the polyhedrin promoter in baculovirus vectors. Subsequently, recombinant baculoviruses were prepared by simultaneously transfection with a transfer vector containing the NS5A gene (pAcHLT-NS5A) and wild-type AcNPV DNA [9, 10]. The recombinant virus produced was used for Sf9 insect cell infection and tested for protein expression. As shown in FIG. 1A, a major protein of about 60 kDa was observed, and NS5A protein was most observed in virus infected cells. Immunoblotting was performed with patient serum to confirm that the NS5A protein was correct (data not shown). Electrolyte NS5A protein from gel to prepare antigen generating NS5A protein specific monoclonal antibody, purified NS5A immunized with Coomassie blue staining (FIG. 1B, lane 2) and rabbit anti-NS5A polyclonal antibody Lotting (Figure 1c, lane 2). Rabbit anti-NS5A polyclonal antibody [16] detected only NS5A protein, but not in mock-infected cells (compare lane 2 and lane 1).

Preparation of NS5A-specific Monoclonal Antibodies from Mice Immunized with Recombinant Baculovirus-Expressing NS5A Protein

In order to understand the structural and functional properties of HCV NS5A protein, NS5A protein specific monoclonal antibodies were prepared. Mice immunized with recombinant NS5A protein were expanded three times with the same antibody and hybridoma cells were prepared by fusing splenic cells with SP2 / 0 myeloma cells. Sixteen hybridoma cell lines were selected using a multi-channel miniblotter and all hybridomas except three clones produced monoclonal antibodies (data not shown).

Identification of monoclonal antibodies that recognize the N, M and C termini of NS5A protein

In order to identify monoclonal antibodies that recognize various parts of the NS5A protein, truncated variants of the same length were prepared as shown in FIG. 2A. These variant proteins were expressed in E. coli (FIG. 2B) and the site of monoclonal antibody binding in the NS5A protein was determined. As shown in FIG. 2c, monoclonal antibodies that recognize the N-terminal region, the central M region, and the C-terminal region of the NS5A protein were identified. The major isotypes of NS5A monoclonal antibodies were determined to be IgG 1j, IgG 2aj, IgG 3aj and IgM j (Table 1).

N-terminal Epitope Mapping of NS5A Protein

N-terminal fragment variants were constructed to determine epitope locations of the NS5A protein N-terminal region (FIG. 3A), and E. Wild type and variant proteins in Collie were expressed in GST-fusion protein form (data not shown). Each variant protein was identified by immunoblotting with rabbit anti-NS5A polyclonal antibody (data not shown). As shown in Figure 3b, monoclonal antibody E5D3 bound to amino acids 1-80 and 1-110 NS5A variant proteins. However, this monoclonal antibody did not bind to variants comprising amino acids 1-59 (FIG. 3B, lane 4) and variants comprising amino acids 81-49 (FIG. 3B, lane 7). These results indicate that the N-terminal epitope of NS5A is located at amino acid 60-80 sites. We also identified this epitope using synthetic peptides comprising amino acid 60-80 sites of NS5A (data not shown).

Intermediate Epitope Mapping in NS5A Proteins

Various variants were constructed and expressed in E. coli to determine epitope location of the intermediate region of NS5A protein as shown in FIG. 3C (data not shown). Wild-type and variant proteins were analyzed by SDS-PAGE on 10% polyacrylamide gels and each variant protein was identified by immunoblotting with rabbit anti-NS5A polyclonal antibody. 3D shows that monoclonal antibody C6D4 binds to NS5A variant protein consisting of amino acids 150-298 and 221-276. However, this monoclonal antibody did not bind to variants consisting of amino acids 150-220 (FIG. 3D, lane 3) and 254-298 (FIG. 3D, lane 5). Additional deletion data shows that the middle epitope of NS5A is located at amino acids 221-236 of NS5A. We isolated a monoclonal antibody (H7E8) that recognizes the C-terminal portion of NS5A (FIG. 2E). However, the isotype of monoclonal antibody H7E8 was IgM and the epitope of this monoclonal antibody could not be determined.

Monoclonal antibody E5D3 inhibits protein interactions between NS5A and hVAP-B.

It has been reported that HCV NS5A interacts with hVAP-B and two separate sites of NS5A (amino acids 66-70 and 340-344) are essential for interaction with hVAP-B [Hamamoto I, Nishimura Y, Okamoto T, Aizaki H, Liu M, Mori Y, Abe T, Suzuki T, Lai MMC, Miyamura T, Moriishi K, Matsuura Y (2005) Human VAP-B is involved in hepatitis C virus replication through interaction with NS5A and NS5B. J Virol 79: 13473-13482. VAP-B is an essential host factor for HCV replication by binding to NS5A. To test whether monoclonal antibody E5D3 can block the interaction between NS5A and hVAP-B, GST-NS5A was preincubated with monoclonal antibody E5D3 and then incubated with Huh7 cell lysate containing Flag hVAP protein for three hours. It was. Antigen antibody complexes and hVAP-B proteins were pulled down using GST beads and bound proteins were immunoblotted with Flag antibodies. Indeed, NS5A protein has been reported by Hamamoto et al. J Virol 79: 13473-13482, as well as hVAP-B (Fig. 4a, lane 2). However, NS5A was unable to bind hVAP-B in the presence of monoclonal antibody E5D3 and binding was inhibited dose dependently depending on the amount of monoclonal antibody E5D3 added (FIG. 4B). Since the E5D3 epitope is in the amino acid 60-80 region of NS5A, this result is sufficient to inhibit the interaction of NS5A with hVAP-B by blocking the N-terminal binding site comprising amino acids 60-80 with monoclonal antibody E5D3. Say yes.

Monoclonal antibody C6D4 inhibits the interaction of NS5A with TRAF2.

It has been reported that NS5A interacts with TRAF2 protein and alters TNF signaling in host cells [16, 17]. Intermediate sites of NS5A (amino acids 148-301) and the TRAF domain of TRAF2 were measured to interact. We therefore investigated whether mAb monoclonal antibody C6D4 could inhibit the interaction between NS5A and TRAF2. GST-NS5A was first incubated with monoclonal antibody C6D4, and then Huh7 cell lysate containing Flag-TRAF2 protein was incubated for three hours. Samples were pulled down using GST beads and bound proteins were immunoblotted with Flag antibodies. As shown in FIG. 5A, NS5A protein interacted with TRAF2 (lane 2). In the presence of monoclonal antibody C6D4, NS5A was no longer bound to TRAF2, and binding was inhibited in a dose dependent manner depending on the degree of addition of monoclonal antibody C6D4 (FIG. 5B). A2C8 and E5D4 monoclonal antibodies blocked binding of TRAF2 and NS5A (data not shown).

HCV NS5A is a pleiotropic protein involved in RNA replication and host cell signaling modifications. In this respect NS5A is of interest, and we have produced monoclonal antibodies that recognize various parts of NS5A and expressed the NS5A protein in insect cells using a recombinant baculovirus expression system.

We selected and epitope mapped monoclonal antibodies of different isotypes. One is monoclonal antibody E5D3 which specifically binds to the N-terminal site (amino acids 60-80), and the other is monoclonal antibody C6D4 which specifically binds to the intermediate site (amino acids 221-236). NS5A performs several functions in HCV-induced development. NS5A allows for viral replication using host factors. As an example, HCV NS5A interacts with hVAP-B to enhance viral RNA replication. In addition, NS5A interacts with cellular signaling transducers to avoid cellular immune responses. Our previous study revealed that NS5A interacts with the TRAF2 adapter protein to alter TNF signaling in host cells. Amino acids 60-80, the N-terminal epitope of NS5A protein, and amino acids 221-236, the epitope in the middle region, are respectively hVAP-B (human vesicle-associated membrane-protein-associated protein-B) and TRAF2 (TNF-receptor- associated factor 2) The monoclonal antibody E5D3 specific for the N-terminal epitope of the NS5A protein and dose-dependently inhibited the interaction of NS5A with hVAP. Previous studies have reported that two separate portions of NS5A (amino acids 51-75 and 325-349) are essential for interaction with hVAP-B [Hamamoto I, Nishimura Y, Okamoto T, Aizaki H, Liu M, Mori Y, Abe T, Suzuki T, Lai MMC, Miyamura T, Moriishi K, Matsuura Y (2005) Human VAP-B is involved in hepatitis C virus replication through interaction with NS5A and NS5B. J Virol 79: 13473-13482. However, the data of the present invention indicate that blocking only the portion containing amino acids 66-70 of NS5A can inhibit the interaction of NS5A with hVAP-B. In addition, the inventors have found that monoclonal antibody C6D4 specific for amino acids 221-236, the epitope of the NS5A intermediate region, dose-dependently inhibits binding between NS5A and TRAF2. Since the C6D4 epitope was mapped to NS5A amino acids 221-236, this result means that TRAF2 binds to the 221-236 region of NS5A.

We have prepared monoclonal antibodies specific for NS5A from mice immunized with NS5A protein expressed in recombinant baculovirus. Proteins expressed in insect cells infected with recombinant baculovirus are subject to posttranslational modifications (Hwang SB, Lee CZ, Lai MM (1992) Hepatitis delta antigen expressed by recombinant baculoviruses: comparison of biochemical properties and post- translational modifications between the large and small forms. Virology 190: 413-422], NS5A expressed in insect cells is also phosphorylated. Thus, monoclonal antibodies of the invention can recognize and detect phosphorylated NS5A protein.

1 expresses wild-type NS5A protein in insect cells. a: Sf9 insect cells were infected with mock-infected (lane 1) or recombinant baculovirus expressing HCV NS5A protein (lane 2) and harvested 3 days after infection. Total cell lysates were electrophoresed on SDS containing 15% polyacrylamide gels and proteins stained with Coomassie Blue. b: NS5A protein was electroeluted in cell lysate and electrophoresed on SDS-containing 10% polyacrylamide gel and stained with Coomassie Blue. c: Purified NS5A protein was isolated, electrotransferred to nitrocellulose membrane and immunoblotted with rabbit anti-HCV NS5A antibody. M: molecular weight marker, lane 1: mock-infected cells.

Figure 2 identifies monoclonal antibodies that recognize the N-terminus, middle region and C-terminus of NS5A protein. a: Schematic of N, M, C variant structure of HCV NS5A protein cut to the same length. b: GST and GST-NS5A fusion proteins expressed in E. coli. Partially purified GST-NS5A fusion protein was isolated by SDS-PAGE and stained with Coomassie Blue. c: Monoclonal antibody E5D3, which recognizes the N-terminus of NS5A protein, confirmed by immunoblotting. d: monoclonal antibody C6D4 recognizing the intermediate region of NS5A protein. e: monoclonal antibody H7E8 recognizing the C-terminus of NS5A protein.

3 is the location of N and M epitopes in NS5A protein. a: Structural diagram of HCV NS5A variant used for N-terminal domain epitope mapping of NS5A protein. The binding results are shown in the right panel. b: Immunoblot GST-NS5A fusion protein with monoclonal antibody E5D3. c: Structure diagram of HCV NS5A variant used for intermediate domain epitope mapping of NS5A protein. The binding results are shown in the right panel. d: immunoblot analysis of GST-NS5A fusion protein with monoclonal antibody C6D4.

4 inhibits the interaction between NS5A and hVAP-B with monoclonal antibody E5D3. a: E5D3 specifically inhibits the interaction between NS5A and hVAP-B. GST-NS5A fusion protein expressed in E. coli was incubated with monoclonal antibody E5D3 or C6D4 at 4 ° C. for 30 min and then incubated with cell lysate containing Flag-labeled hVAP-B protein for 3 hours. Antigen-antibody and protein complexes were pulled down using GST beads and immunoblotted with Flag antibodies (top panel). NS5A and hVAP-B expression was confirmed by antibody immunoblotting using anti-NS5A antibodies (middle panel) or anti-Flag antibodies against hVAP-B protein (bottom panel). b: Monoclonal antibody E5D3 dose-dependently inhibited the interaction of NS5A with hVAP-B. GST-NS5A was incubated at 4 ° C. for 30 minutes with increasing amount of monoclonal antibody E5D3, and then incubated with Flag-labeled hVAP-B protein in Huh7 cells for 3 hours. Samples were pulled down using GST beads and immunoblotted with Flag antibodies (top panel). Both NS5A (center panel) and hVAP-B (bottom panel) proteins were detected in this manner. IB: immunoblotting.

5 shows that monoclonal antibody C6D4 inhibits the interaction between NS5A and TRAF2. a: Monoclonal antibody C6D4 specifically inhibits the interaction between NS5A and TRAF2. The GST-NS5A fusion protein expressed in E. coli was incubated with monoclonal antibody C6D4 or E5D3 at 4 ° C. for 30 minutes as a control and then incubated with cell lysate containing Flag labeled TRAF2 protein for 3 hours. . Samples were pulled down using GST beads and immunoblotted with Flag antibodies (top panel). NS5A and TRAF2 expression was confirmed by immunoblotting using cell lysates with anti-NS5A antibodies (middle panel) or anti-flag antibodies against TRAF2 protein (bottom panel). b: Monoclonal antibody C6D4 inhibited the interaction between NS5A and TRAF2 in a dose dependent manner. The GST-NS5A fusion protein was incubated at 4 ° C. for 30 minutes with increasing amount of monoclonal antibody C6D4 and then incubated with Flag-labeled TRAF2 protein expressed in Huh7 cells for 3 hours. Antigen-antibody and protein complexes were pulled down to GST beads and immunoblotted with Flag antibodies (top panel). Both NS5A (middle panel) and TRAF2 (bottom panel) were detected using the same cell lysate in the same manner as above. IB: immunoblotting.

<110> Industry Academic Foundation Corporation, Hallym University <120> Pharmaceutical composition for preventinion and treatment of          Hepatitis C Virus-mediated liver disease containing anti-NS5A          antibody <130> HallymU-sbhwang-NS5A <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 451 <212> PRT <213> Hepatitis C virus <400> 1 Asp Thr Ser Trp Leu Arg Asp Val Trp Asp Trp Val Cys Thr Val Leu   1 5 10 15 Ser Asp Phe Arg Val Trp Leu Gln Ala Lys Leu Leu Pro Arg Leu Pro              20 25 30 Gly Ile Pro Phe Phe Ser Cys Gln Thr Gly Tyr Arg Gly Val Trp Ala          35 40 45 Gly Asp Gly Val Cys His Thr Thr Cys Thr Cys Gly Ala Val Ile Ala      50 55 60 Gly His Val Lys Asn Gly Thr Met Lys Ile Thr Gly Pro Lys Thr Cys  65 70 75 80 Ser Asn Thr Trp His Gly Thr Phe Pro Ile Asn Ala Thr Thr Thr Gly                  85 90 95 Pro Ser Thr Pro Arg Pro Ala Pro Ser Tyr Gln Arg Ala Leu Trp Arg             100 105 110 Val Ser Ala Glu Asp Tyr Val Glu Val Arg Arg Leu Gly Asp Arg His         115 120 125 Tyr Val Val Gly Val Thr Ala Glu Gly Leu Lys Cys Pro Cys Gln Val     130 135 140 Pro Ala Pro Glu Phe Phe Thr Glu Ile Asp Gly Val Arg Leu His Arg 145 150 155 160 Tyr Ala Pro Pro Cys Lys Pro Leu Leu Arg Asp Glu Val Thr Phe Ser                 165 170 175 Val Gly Leu Ser Thr Tyr Ala Ile Gly Ser Gln Leu Pro Cys Glu Pro             180 185 190 Glu Pro Asp Val Thr Val Val Thr Ser Met Leu Thr Asp Pro Thr His         195 200 205 Ile Thr Ala Glu Thr Ala Ala Arg Arg Leu Lys Arg Gly Ser Pro Pro     210 215 220 Ser Leu Ala Ser Ser Ser Ala Ser Gln Leu Ser Ala Pro Ser Leu Lys 225 230 235 240 Ala Thr Cys Thr Thr Ser Lys Asp His Pro Asp Met Glu Leu Ile Glu                 245 250 255 Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly Asn Ile Thr Arg Val             260 265 270 Glu Ser Glu Asn Lys Val Val Val Leu Asp Ser Phe Glu Pro Leu Thr         275 280 285 Ala Glu Tyr Asp Glu Arg Glu Ile Ser Val Ser Ala Glu Cys His Arg     290 295 300 Pro Pro Arg His Lys Phe Pro Pro Ala Leu Pro Ile Trp Ala Arg Pro 305 310 315 320 Asp Tyr Asn Pro Pro Leu Ile Gln Ala Trp Gln Met Pro Gly Tyr Glu                 325 330 335 Pro Pro Val Val Ser Gly Cys Ala Ile Ala Pro Pro Lys Pro Ala Pro             340 345 350 Ile Pro Pro Pro Arg Arg Lys Arg Leu Val Arg Leu Asp Glu Ser Thr         355 360 365 Val Ser His Ala Leu Ala Gln Leu Ala Asp Lys Val Phe Val Glu Ser     370 375 380 Ser Ser Asp Pro Gly Pro Ser Ser Asp Ser Gly Leu Ser Ile Ala Ser 385 390 395 400 Pro Val Pro Pro Ala Pro Thr Thr Ser Asp Asp Ala Cys Ser Glu Ala                 405 410 415 Glu Ser Tyr Ser Ser Met Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro             420 425 430 Asp Leu Ser Ser Gly Ser Trp Ser Thr Val Ser Asp Gln Asp Asp Val         435 440 445 Val Cys Cys     450 <210> 2 <211> 21 <212> PRT <213> Hepatitis C virus <400> 2 Gly Ala Gln Ile Ala Gly His Val Lys Asn Gly Ser Met Arg Ile Val   1 5 10 15 Gly Pro Arg Thr Cys              20 <210> 3 <211> 16 <212> PRT <213> Hepatitis C virus <400> 3 Gly Ser Pro Pro Ser Leu Ala Ser Ser Ser Ala Ser Gln Leu Ser Ala   1 5 10 15  

Claims (11)

An anti-NS5A antibody that specifically binds an N-terminal epitope of amino acids 60-80 or an intermediate epitope of amino acids 221-236 of the hepatitis C virus NS5A protein. The method according to claim 1, The antibody is an anti-NS5A antibody, characterized in that the monoclonal antibody. The method according to claim 1, N-terminal epitope of the NS5A protein is an anti-NS5A antibody, characterized in that amino acids 66-70. Hepatitis C virus-mediated prevention and treatment of hepatitis C virus-mediated liver disease comprising an anti-NS5A antibody that specifically binds to an N-terminal epitope of amino acids 60-80 or an intermediate epitope of amino acids 221-236 of the hepatitis C virus NS5A protein. Composition. The method according to claim 4, The N terminal epitope of the NS5A protein is amino acid 66-70, characterized in that the composition. The method according to claim 4, The antibody is a composition characterized in that the monoclonal antibody. The method according to claim 4, The hepatitis C virus-mediated liver disease is a pharmaceutical composition for preventing and treating hepatitis C virus-mediated liver disease, characterized in that selected from the group consisting of acute hepatitis C, chronic hepatitis C, liver cirrhosis and liver cancer. Contacting an antibody specifically binding to an N-terminal epitope, amino acids 60-80 or an intermediate epitope, amino acids 221-236, of the hepatitis C virus NS5A protein with a biological sample selected from tissues, urine, blood, serum and plasma Hepatitis C diagnostic method comprising the step. The method according to claim 8, a) providing a biological sample for analysis; b) contacting said sample with an antibody that specifically binds to an N-terminal epitope of amino acids 60-80 or an intermediate epitope of amino acids 221-236 of the hepatitis C virus NS5A protein; c) quantitatively detecting the antigen-antibody complex; And d) comparing the detection result of step c) with a normal control group. A kit for assaying for the presence of hepatitis C virus comprising an anti-NS5A antibody that specifically binds an N-terminal epitope of amino acids 60-80 or an intermediate epitope of amino acids 221-236 of the hepatitis C virus NS5A protein. The method according to claim 10, The antibody is a kit, characterized in that the monoclonal antibody.

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