Epitopes of hepatitis B virus surface antigen and hepatitis B virus-neutralizing binding molecules that specifically bind to these epitopes

Abstract

The present invention provides an epitope of the hepatitis B virus surface antigen and a hepatitis B virus-neutralizing binding molecule that specifically binds to the epitope. [Solution] The present invention relates to a specific epitope of the hepatitis B virus surface antigen and a hepatitis B virus-neutralizing binding molecule that binds to the epitope. The epitope provided by the present invention is generated by forming a three-dimensional structure and does not contain the a-determinant, which generates escape mutations upon administration of existing vaccines or HBIg. Therefore, compositions containing antibodies that bind to the epitope or vaccine compositions containing the epitope are highly unlikely to suffer from reduced efficacy due to escape mutations. Therefore, such antibodies or vaccine compositions are highly useful for the prevention and / or treatment of HBV.

Description

[Technical Field]

[0001] The present invention relates to epitopes of hepatitis B virus surface antigens and hepatitis B virus-neutralizing binding molecules that specifically bind to these epitopes. [Background technology]

[0002] Hepatitis B virus (HBV) is a DNA virus belonging to the Hepadnaviridae family that can cause acute and chronic hepatitis and is a major cause of liver cirrhosis and liver cancer. HBV is classified into 10 serotypes based on the response of the Hepatitis B virus surface antigen (HBsAg) to normal serum samples and differences in the amino acid sequence of HBsAg, or into 8 genotypes based on differences in the gene base sequence. As of 2012, approximately 240 million people were known to be chronically infected with HBV worldwide, and more than 500,000 people are known to die from hepatitis B-related illnesses each year. The chronic HBV infection rate among adults in Korea and China is extremely high, ranging from 5 to 8%, with 80% of adult chronic hepatitis cases, 65% of cirrhosis cases, and 70% of hepatocellular carcinoma cases being related to HBV infection. Although chronic hepatitis B has become preventable through the development and widespread use of vaccines, it remains the most important cause of chronic liver disease, and the social costs associated with liver disease are gradually increasing. Therefore, there is an urgent need to develop new forms of antiviral drugs that can prevent and treat chronic hepatitis B.

[0003] Current medications used to treat chronic hepatitis B include interferon (pegninterferon), lamivudine, adefovir dipivoxil, entecavir, and tenofovir. All oral treatments, except for interferon, are nucleoside / nucleotide analogs. These drugs inhibit viral DNA replication by suppressing the activity of HBV reverse transcriptase, resulting in a reduction in serum HBV DNA levels, normalization of ALT levels, and improvement of liver fibrosis.

[0004] However, long-term use of nucleoside analogs can induce drug resistance, resulting in reduced efficacy and ultimately worsening hepatitis. While no resistance has been reported to date to the most recently developed drug, tenofovir, the most widely used drug worldwide, the resistance rate for lamivudine is known to reach 70-80% after five years. Furthermore, because these oral medications cannot directly inhibit HBV infection, human plasma-derived hepatitis B immune globulin (HBiG) preparations are used in conjunction with oral treatments to prevent vertical transmission from mother to fetus and reinfection in liver transplant recipients.

[0005] Existing HBIg vaccines are produced by isolating antibodies from the blood of individuals with hepatitis B antibodies using advanced purification technology and then removing potential sources of contamination using viral inactivation technology. However, the difficulty in securing the raw material, plasma, leads to excessive import costs and an inability to flexibly meet demand. Furthermore, removing the virus from plasma takes time and money, and the possibility of potential sources of infection cannot be eliminated. Furthermore, low efficacy results in the inconvenience of administration and a financial burden.

[0006] Furthermore, it is known that most antibodies generated by vaccination with existing hepatitis B vaccines recognize the a determinant at amino acids 124-147 of HBsAg. Although it is true that the a determinant acts as the main neutralizing epitope for HBV, it has been reported that specific mutations within the a determinant in some patients can evade antibodies generated by vaccination with hepatitis B vaccines. Therefore, there is an increasing need to develop new antibodies and vaccines for the prevention and treatment of hepatitis B that can respond to these escape mutations in existing vaccines and HBsAg. Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, in order to solve the above problems, the present inventors developed an epitope comprising amino acid positions 110, 118, 120 and / or 147 of the hepatitis B virus surface antigen (HBsAg) and confirmed that this epitope has three-dimensional structural characteristics.

[0008] The problem to be solved by the present invention is to provide an epitope of hepatitis B virus surface antigen (HBsAg).

[0009] Another problem to be solved by the present invention is to provide a hepatitis B virus (HBV) neutralizing binding molecule that specifically binds to the epitope.

[0010] Another problem to be solved by the present invention is to provide a polynucleotide encoding the binding molecule.

[0011] Another object of the present invention is to provide an expression vector containing the polynucleotide.

[0012] Another object of the present invention is to provide a host cell that can be transfected with the expression vector to produce an HBV-neutralizing binding molecule.

[0013] Another object of the present invention is to provide a composition for preventing, treating or diagnosing hepatitis B, which comprises the binding molecule.

[0014] Another object of the present invention is to provide a polynucleotide encoding the epitope.

[0015] Another object of the present invention is to provide an expression vector containing a polynucleotide encoding the epitope.

[0016] Another object of the present invention is to provide a recombinant microorganism or virus transformed with an expression vector containing a polynucleotide encoding the epitope.

[0017] Furthermore, another problem to be solved by the present invention is to provide a method for producing an epitope, which comprises the step of culturing the recombinant microorganism or virus.

[0018] Another object of the present invention is to provide an HBV vaccine composition comprising the epitope or a polynucleotide encoding the epitope.

[0019] Another object of the present invention is to provide a composition for detecting HBV, which comprises the epitope or a polynucleotide encoding the epitope. [Means for solving the problem]

[0020] To solve the above problems, the present invention has confirmed that the epitope of a human antibody that specifically binds to HBsAg (see PCT / KR2014 / 004612, hereinafter referred to as "the antibody of the present invention") comprises amino acid positions 110, 118, 120, and / or 147 of HBsAg. Furthermore, it has been confirmed that a sequence comprising these four amino acids, or a portion thereof, forms a three-dimensional structure that forms an epitope to which the antibody of the present invention can bind, thereby completing the present invention.

[0021] Therefore, the present invention provides 3- to 38-mer epitopes selected from amino acid positions 106 to 151 of the hepatitis B virus surface antigen (HBsAg).

[0022] In one embodiment of the present invention, the epitope may include amino acids at positions 110, 118, 120, and / or 147 of the hepatitis B virus surface antigen (HBsAg). Epitopes containing amino acids at these positions may be conjugated to a carrier to maintain their three-dimensional structure or to improve their efficiency when used in compositions such as vaccines. Any carrier may be used in the present invention as long as it is biocompatible and can achieve the desired effects of the present invention. Preferably, the carrier is selected from, but not limited to, peptides, serum albumin, immunoglobulin, hemocyanin, and polysaccharides.

[0023] In one embodiment of the present invention, the epitope may be at amino acid positions 106-110, 107-111, 108-112, 109-113, 110-114, 114-118, 115-119, 119-123, 120-124, 143-147, 144-148, 145-149, 146-150, 147-151, 110-118, 118-120, 116-120, 117-121, 118-122, 120-147, 110-120, 118-147 or 110-147 of the hepatitis B virus surface antigen (HBsAg).

[0024] In the present invention, the entire amino acid sequence of the wild-type HBsAg of HBV genotype C (subtype adr) may be represented by SEQ ID NO: 3, and the sequence information can also be confirmed from GenBank No. GQ872210.1.

[0025] The present invention also provides a hepatitis B virus (HBV) neutralizing binding molecule that specifically binds to an epitope comprising one or more amino acid residues selected from the group consisting of amino acid positions 110, 118, and 120 of hepatitis B virus surface antigen (HBsAg). Furthermore, the epitope may additionally comprise amino acid position 147 of HBsAg.

[0026] In one embodiment of the present invention, the hepatitis B virus (HBV) neutralizing binding molecule that specifically binds to the epitope is 1×10 -9 In another embodiment, the binding molecule may have a binding affinity of less than 9×10 -10 In yet another embodiment, the binding molecule may have a binding affinity of less than 8×10 -10 In yet another embodiment, the binding molecule may have a binding affinity of less than 7×10 -10 In yet another embodiment, the binding molecule may have a binding affinity of less than 6×10 -10 In yet another embodiment, the binding molecule may have a binding affinity of less than 5×10 -10 In yet another embodiment, the binding molecule may have a binding affinity of less than 4×10 -10 In yet another embodiment, the binding molecule may have a binding affinity of less than 3×10 -10 In yet another embodiment, the binding molecule may have a binding affinity of less than 2×10 -10 In yet another embodiment, the binding molecule may have a binding affinity of less than 1×10 -10 In yet another embodiment, the binding molecule may have a binding affinity of less than 1×10 -11 In yet another embodiment, the binding molecule may have a binding affinity of less than 1×10 -12 It may have a binding affinity less than M.

[0027] The HBV neutralizing binding molecule of the present invention can bind to any one or more selected from the group consisting of hepatitis B virus surface antigen (HBsAg) subtypes adw, adr, ayw, and ayr, and have neutralizing activity against hepatitis B virus.

[0028] The binding molecules of the present invention bind to and have neutralizing activity against hepatitis B viruses of A, B, C, D, E, F, G, and H genotypes.

[0029] Furthermore, the binding molecule of the present invention has neutralizing activity by binding to lamivudine-, adefovir-, clevudine-, or entecavir-resistant hepatitis B virus.

[0030] In one embodiment of the present invention, the binding molecule may bind to a mutant antigen at amino acid position 101, 112, 126, 129, 133, 143, 173, 175, 184, 185, or 196 of HBsAg and have neutralizing activity against hepatitis B virus, but is not limited thereto.

[0031] In one embodiment of the present invention, the mutant antigen may be, but is not limited to, Q101R, K112R, T126N, I126S, Q129H, M133H, P143K, L173F, L175S, A184V, I185M or W196L mutant antigen.

[0032] In one embodiment of the present invention, the binding molecule is an antibody or a fragment thereof. The antibody may be, but is not limited to, a Fab fragment, an Fv fragment, a diabody, a chimeric antibody, a humanized antibody, or a human antibody. In one embodiment of the present invention, a fully human antibody that binds to HBsAg is provided. As used herein, the term "antibody" is used in the broadest sense and specifically includes intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from two or more intact antibodies, and antibody fragments that exhibit a desired biological activity. Antibodies are proteins produced by the immune system that can recognize and bind to specific antigens. Structurally, antibodies are typically Y-shaped proteins consisting of four amino acid chains (two heavy chains and two light chains). Each antibody primarily contains two regions: a variable region and a constant region. The variable region, located at the end of the Y arm, binds to and interacts with the target antigen. The variable region contains complementarity-determining regions (CDRs), which recognize and bind to specific binding sites on a specific antigen. The constant region located at the tail of the Y is recognized and interacts with the immune system. A target antigen typically has multiple binding sites, called epitopes, that are recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Therefore, one antigen can have more than one corresponding antibody.

[0033] At the same time, the present invention encompasses functional variants of the antibodies. An antibody is considered a functional variant of an antibody of the present invention if the variant can compete with the antibody of the present invention for specific binding to a subtype of hepatitis B virus or its surface antigen (HBsAg). Functional variants include derivatives with substantially similar primary structural sequences, including, but not limited to, in vitro or in vivo modifications, chemical and / or biochemical modifications not found in the parent monoclonal antibody of the present invention. Such modifications include, for example, acetylation, acylation, covalent attachment of nucleotides or nucleotide derivatives, covalent attachment of lipids or lipid derivatives, cross-linking, disulfide bond formation, glycosylation, hydroxylation, methylation, oxidation, pegylation, proteolysis, and phosphorylation. A functional variant may optionally be an antibody comprising an amino acid sequence containing one or more amino acid substitutions, insertions, deletions, or combinations thereof compared to the amino acid sequence of the parent antibody. Furthermore, functional variants may include truncated forms of the amino acid sequence at one or all of the amino or carboxy termini. The functional variants of the present invention can have the same or different, higher or lower binding affinity compared to the parent antibody of the present invention, but still bind to a subtype of hepatitis B virus or its surface antigen (HBsAg). For example, the amino acid sequences of the variable regions, including but not limited to the framework, hypervariable regions, and particularly the CDR3 region, can be varied. Generally, a light or heavy chain region contains three hypervariable regions, including three CDR regions, and a further conserved region, i.e., framework region (FR). The hypervariable region contains amino acid residues from the CDRs and the hypervariable loops. Functional variants within the scope of the present invention can have about 50% to 99%, about 60% to 99%, about 80% to 99%, about 90% to 99%, about 95% to 99%, or about 97% to 99% amino acid sequence identity with the parent antibody of the present invention. To optimally align the amino acid sequences to be compared and define similar or identical amino acid residues, computer algorithms such as Gap or Bestfit known to those skilled in the art can be used.Functional variants can be obtained by modifying a parent antibody or a portion thereof by known general molecular biology methods, including, but not limited to, PCR, mutagenesis using oligonucleotides, and partial mutagenesis, or by organic synthesis methods.

[0034] The invention also provides polynucleotides encoding the binding molecules. In one embodiment, the invention includes an isolated nucleic acid molecule encoding the anti-HBsAg monoclonal antibody.

[0035] The present invention also provides an expression vector containing the polynucleotide. The expression vector may be selected from the group consisting of Celltrion's proprietary MarEx vector, the commercially available pCDNA vector, F, R1, RP1, Col, pBR322, ToL, and Ti vectors; cosmids; phages such as lambda, lambdoid, M13, Mu, p1P22, Qμ, T-even, T2, T3, and T7; and plant viruses, but is not limited thereto. All expression vectors known to those skilled in the art can be used in the present invention, and the selection of an expression vector should be based on the characteristics of the target host cell. Introduction of the vector into the host cell may be performed by calcium phosphate transfection, viral infection, DEAE-dextran-mediated transfection, lipofectamine transfection, or electroporation, but is not limited thereto. Those skilled in the art can select an appropriate introduction method for the expression vector and host cell used. The expression vector may contain one or more selectable markers, but is not limited thereto. Alternatively, a vector without a selectable marker can be used to select for product production. The selection of a selectable marker is determined by the target host cell, and methods known to those skilled in the art are used, so the present invention is not limited thereto. Furthermore, to facilitate purification, the nucleic acid molecules of the present invention can be fused by inserting a tag sequence into the expression vector. Examples of such tags include, but are not limited to, a hexa-histidine tag, a hemagglutinin tag, a myc tag, or a flag tag. Any tag known to those skilled in the art that facilitates purification can be used in the present invention.

[0036] In another embodiment, the present invention relates to a host cell that is transfected with the expression vector to produce a binding molecule having hepatitis B virus-neutralizing activity. In the present invention, the host cell may be of mammalian, plant, insect, fungal, or cellular origin, but is not limited thereto. Examples of the mammalian cell include, but are not limited to, CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK293 cells, and HEK293T cells. Any cell known to those skilled in the art that can be used as a mammalian host cell can be used.

[0037] The present invention also provides a composition for preventing, treating, or diagnosing hepatitis B, comprising the binding molecule. The composition of the present invention may additionally comprise, in addition to the binding molecule, an interferon, an anti-HBV monoclonal antibody, an anti-HBV polyclonal antibody, a nucleoside analog, a DNA polymerase inhibitor, an siRNA preparation, or a therapeutic vaccine as an antiviral drug.

[0038] Compositions containing the binding molecules of the present invention may be formulated by conventional methods into forms such as, but not limited to, sterile injection solutions, lyophilized formulations, pre-filled syringe solutions, oral formulations, topical preparations, or suppositories.

[0039] In another embodiment of the present invention, the present invention relates to a method for treating hepatitis B, comprising administering a therapeutically effective amount of the composition to a subject infected with hepatitis B virus. In the treatment method of the present invention, a therapeutic agent known to those skilled in the art can be administered together. In the treatment method of the present invention, the administration method can be divided into oral and parenteral, and the administration route can be, for example, intravenous, but is not limited thereto.

[0040] In one embodiment of the present invention, the treatment method may further include administering an antiviral drug. The antiviral drug may be, but is not limited to, interferon, a nucleoside / nucleotide analog, an anti-HBV monoclonal antibody, an anti-HBV polyclonal antibody, a DNA polymerase inhibitor, a siRNA formulation, or a therapeutic vaccine. The nucleoside / nucleotide analog may be, but is not limited to, lamivudine, entecavir, clevudine, or adefovir dipivoxil.

[0041] In another embodiment of the present invention, the present invention relates to a method for preventing hepatitis B, comprising administering a therapeutically effective amount of the composition to a subject. In the method of prevention of the present invention, a prophylactic agent known to those skilled in the art can be administered together. In the method of prevention of the present invention, the administration method can be divided into oral and parenteral, and the administration route can be, for example, intravenous, but is not limited thereto.

[0042] The compositions of the present invention can be administered to mammals, including humans, to prevent or treat HBV infection and diseases caused by HBV infection. The dosage of the binding molecule (e.g., antibody) depends on the subject being treated, the severity of the disease or condition, the rate of administration, and the discretion of the prescribing physician. As an active ingredient, the binding molecule can be administered to mammals parenterally in a daily dose of 0.001 to 10 mg / kg (body weight), or 0.005 to 1 mg / kg (body weight) in a single dose or in divided doses. In some cases, lower doses than the above-mentioned ranges may be more appropriate, and larger doses may be used without causing adverse side effects, or larger doses may be distributed over several small doses throughout the day.

[0043] In another embodiment, the present invention relates to a method for diagnosing whether a patient is infected with hepatitis B virus, comprising the steps of: i) contacting a sample with the composition; and ii) detecting a reaction between the composition and the sample. In the diagnostic method of the present invention, the binding molecule (e.g., monoclonal antibody) of the present invention may be conjugated with a labeling substance for diagnostic detection, as necessary, which is well known to those skilled in the art.

[0044] In the diagnostic method of the present invention, the sample may be any one selected from the group consisting of sputum, saliva, blood, sweat, lung cells, mucus from lung tissue, respiratory tissue, and saliva of a subject, but is not limited thereto, and the sample can be prepared by a conventional method known to those skilled in the art.

[0045] In another embodiment of the present invention, the present invention relates to a method for providing information for diagnosing whether a patient is infected with hepatitis B virus, comprising the steps of: i) contacting a sample with the composition; and ii) detecting a reaction between the composition and the sample.

[0046] In another embodiment, the present invention relates to a diagnostic kit for hepatitis B virus, comprising i) the composition and ii) a container. In the diagnostic kit of the present invention, the container (ii) contains a solid support. The binding molecule of the present invention may be attached to the solid support, which may be porous or non-porous, planar or non-planar.

[0047] In another embodiment, the present invention relates to a method for detecting the presence or absence of hepatitis B virus, comprising the step of contacting a sample derived from a patient with the composition described above.

[0048] The present invention also provides a polynucleotide encoding the epitope. The polynucleotide encoding the epitope containing the amino acid position provided by the present invention can be used in the form of a genetic vaccine. The polynucleotide can be used without a carrier, or it can be delivered to the body via a viral or non-viral carrier. Any viral or non-viral carrier known to be commonly used in the art of the present invention can be used. Specifically, viral carriers include adenoviruses, adeno-associated viruses, lentiviruses, and retroviruses. Non-viral vectors include, but are not limited to, cationic polymers, non-ionic polymers, liposomes, lipids, phospholipids, hydrophilic polymers, hydrophobic polymers, and complexes of at least one of these.

[0049] The present invention also provides an expression vector comprising a polynucleotide encoding the epitope.

[0050] The present invention also provides a recombinant microorganism or virus transformed with the expression vector. In one embodiment, the recombinant microorganism or virus may be a recombinant Escherichia coli, a recombinant yeast, or a recombinant bacteriophage.

[0051] In one embodiment, the present invention provides a method for expressing an epitope containing amino acid positions 110, 118, 120, and / or 147 of HBsAg on the surface of a microorganism or virus. A recombinant vector containing an inducible promoter or a sequence encoding a signal protein, and various microorganisms or viruses containing the recombinant vector, may be used. Particularly suitable microorganisms or viruses include, but are not limited to, recombinant Escherichia coli, recombinant yeast, and recombinant bacteriophage. To express an epitope containing the amino acid positions on the surface of the microorganism or virus, well-known display technologies can be used. In particular, a polynucleotide sequence encoding the epitope containing the amino acid positions can be linked to a promoter or a sequence encoding a signal protein that induces expression on the surface of a microbial cell or virus, or a polynucleotide sequence encoding the epitope containing the amino acid positions can be inserted into the deleted gene encoding the protein originally to be expressed on the surface. In this manner, an epitope containing the amino acid position expressed on the surface of a microorganism or virus can be isolated and purified and used for specific applications according to the present invention, and can also be used to select and obtain antibodies that specifically bind to an epitope containing the amino acid position in a surface-expressed state.

[0052] The present invention also provides a method for producing an epitope, which comprises the step of culturing the recombinant microorganism or virus.

[0053] The present invention also provides an HBV vaccine composition comprising the epitope or a polynucleotide encoding the epitope. The HBV vaccine composition may further comprise a pharmaceutically acceptable adjuvant. Any adjuvant that enhances antibody formation upon administration to the body and achieves the objectives of the present invention may be used, including, but not limited to, aluminum salts (Al(OH)3, ALPO4), squalene, sorbitan, polysorbate 80, CpG, liposomes, cholesterol, monophosphoryl lipid A (MPL), and glucopyranosyl lipid A (GLA).

[0054] The present invention also provides a composition for detecting HBV, which comprises the epitope or a polynucleotide encoding the epitope. [Effects of the Invention]

[0055] The epitope of hepatitis B virus surface antigen (HBsAg) provided by the present invention does not contain key residues in the a determinant that generate escape mutations upon administration of existing vaccines or HBsAg, and therefore compositions containing antibodies that bind to this epitope or vaccine compositions containing the epitope are unlikely to suffer from reduced efficacy due to escape mutations. Therefore, such antibodies or vaccine compositions are highly useful for the prevention and / or treatment of HBV. [Brief explanation of the drawings]

[0056] [Figure 1] In order to identify the binding site of the antibody of the present invention, wild-type HBsAg (226a.a) was divided into three regions, amino acid sequences 1 to 100 (Region 1), 101 to 160 (Region 2), and 161 to 226 (Region 3), and the mutant structures were each deleted. [Figure 2]1 shows the results of an experiment in which the binding ability of the antibody of the present invention to wild-type HBsAg and three mutant HBsAg lacking Regions 1, 2, and 3 was confirmed by phage-based ELISA. [Figure 3] 1 shows the structures of four mutant HBsAg mutants in which 15 amino acids have been sequentially deleted in order to identify the epitope of the antibody of the present invention. [Figure 4] The results are from an experiment in which the binding ability of the antibody of the present invention to four mutant HBsAg mutants in which the 15 amino acids are sequentially deleted was confirmed by phage-based ELISA. [Figure 5] The epitopes and disulfide bond positions are shown on the HBsAg model. [Figure 6A] 1 shows the results of Western blot analysis carried out to investigate the characteristics of the antibody epitope of the present invention. [Figure 6B] 1 shows the results of Western blot analysis carried out to investigate the characteristics of the antibody epitope of the present invention. [Figure 7] 1 shows the results of confirming the reactivity of antibodies 1 and 2 of the present invention to various mutant antigens of the a determinant using ELISA, according to one embodiment of the present invention. [Figure 8A] According to one embodiment of the present invention, this shows the results of an in vitro neutralization experiment of antibodies 1 and 2 of the present invention against four genotypes of hepatitis B virus, A, B, C, and D, in which the amount of intracellular virus was measured as the amount of DNA of the multiplying HBV using real-time PCR. [Figure 8B] According to one embodiment of the present invention, this shows the results of an in vitro neutralization experiment of antibodies 1 and 2 of the present invention against four genotypes of hepatitis B virus, A, B, C, and D, in which the amount of intracellular virus was measured as the amount of DNA of the multiplying HBV using real-time PCR. [Figure 8C]According to one embodiment of the present invention, this shows the results of an in vitro neutralization experiment of antibodies 1 and 2 of the present invention against four genotypes of hepatitis B virus, A, B, C, and D, in which the amount of intracellular virus was measured as the amount of DNA of the multiplying HBV using real-time PCR. [Figure 8D] According to one embodiment of the present invention, this shows the results of an in vitro neutralization experiment of antibodies 1 and 2 of the present invention against four genotypes of hepatitis B virus, A, B, C, and D, in which the amount of intracellular virus was measured as the amount of DNA of the multiplying HBV using real-time PCR. [Figure 9A] According to one embodiment of the present invention, this shows the results of an in vitro neutralization experiment of antibodies 1 and 2 of the present invention against four genotypes of hepatitis B virus, A, B, C, and D, in which the amount of virus multiplied and released outside the cells was measured as the amount of HBsAg using a chemiluminescent immunoassay (CLIA) method. [Figure 9B] According to one embodiment of the present invention, this shows the results of an in vitro neutralization experiment of antibodies 1 and 2 of the present invention against four genotypes of hepatitis B virus, A, B, C, and D, in which the amount of virus multiplied and released outside the cells was measured as the amount of HBsAg using a chemiluminescent immunoassay (CLIA) method. [Figure 9C] According to one embodiment of the present invention, this shows the results of an in vitro neutralization experiment of antibodies 1 and 2 of the present invention against four genotypes of hepatitis B virus, A, B, C, and D, in which the amount of virus multiplied and released outside the cells was measured as the amount of HBsAg using a chemiluminescent immunoassay (CLIA) method. [Figure 9D] According to one embodiment of the present invention, this shows the results of an in vitro neutralization experiment of antibodies 1 and 2 of the present invention against four genotypes of hepatitis B virus, A, B, C, and D, in which the amount of virus multiplied and released outside the cells was measured as the amount of HBsAg using a chemiluminescent immunoassay (CLIA) method. [Figure 10]The binding activity of antibodies 1 and 2 of the present invention to 15 HBV surface antigen serum samples (derived from patients with seven hepatitis B virus genotypes A, B, C, D, E, F, and H) was confirmed by sandwich ELISA. [Figure 11] 1 shows the results of sandwich ELISA confirming the binding activity of antibodies 1 and 2 of the present invention against drug (lamivudine, adefovir, clevudine, entecavir)-resistant viruses. DETAILED DESCRIPTION OF THE INVENTION [Example]

[0057] The present invention will be described in more detail below with reference to examples. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the invention is not limited by the examples. The literature cited in the present invention is incorporated by reference into the specification of the present invention.

[0058] Example 1: Identification of the binding site of the antibody of the present invention using deletion mutant antigens To identify the binding site on HBsAg of the antibody of the present invention, wild-type and various deletion mutants of HBsAg were prepared, and their binding ability to these was examined by enzyme-linked immunosorbent assay (ELISA) using phage.

[0059] The experiment was carried out in two steps. First, HBsAg (226a.a) was divided into three regions, namely, amino acid sequences 1-100, 101-160, and 161-226, and mutant and wild-type expression vectors were prepared by deleting each region (see Figure 1). Each protein was then expressed on the surface of phage, and its binding to the antibody of the present invention was then measured.

[0060] Example 1-1. Preparation of wild-type HBsAg and three mutant HBsAg expression vectors To express wild-type HBsAg and its three-part deletion mutant proteins on the phage surface, cloning was performed using a phage expression vector. The detailed experimental method is as follows: To clone the HBsAg wild-type and site-specific deletion mutants, the corresponding genes were amplified by polymerase chain reaction (PCR) using an HBV vector containing the HBsAg gene sequence of HBV genotype C (Department of Pharmacology, Konkuk University Graduate School of Medicine, Korea) as a template. The resulting vectors were digested with the restriction enzyme SfiI and then inserted into phage expression vectors digested with the same restriction enzyme. The constructed plasmids were extracted using a QIAprep Spin Miniprep Kit (QIAGEN, Germany, Cat. #27106), and the extracted DNA was used for sequencing to confirm the antibody sequence. The names of the resulting clones and the HBsAg sites are listed in Table 1.

[0061] [Table 1]

[0062] In this example, the entire amino acid sequence of the wild-type HBsAg of HBV genotype C (subtype adr) is represented by SEQ ID NO: 3, and the sequence information can also be confirmed from GenBank No. GQ872210.1.

[0063] Example 1-2. Experiment to confirm the binding ability of wild-type HBsAg and three mutant HBsAg to the antibody of the present invention To conduct binding experiments with the cloned HBsAg fragment or fragments, we first electroporated the vector into expression E. coli (ER2738, Lucigen, USA, Cat. #60522-2) and selectively cultured ampicillin-resistant E. coli the following day. After approximately 10 hours of culture, we infected the E. coli with bacteriophage and used another antibiotic, kanamycin, to selectively culture the infected E. coli. The next day, we centrifuged the E. coli and separated the bacteriophage. The supernatant was treated with polyethylene glycol (PEG), placed on ice for 30 minutes, and then centrifuged again to separate the bacteriophage. The isolated bacteriophage was thawed, and the supernatant was filtered to extract pure bacteriophage bearing either the entire or partial HBsAg fragment on its surface.

[0064] To quantitatively evaluate the binding strength of the antibodies of the present invention to wild-type and mutant HBsAg exposed on the surface of bacteriophage, we used an ELISA technique. First, an anti-human Fc antibody (Jackson Immunoresearch, USA, Cat. #109-006-098) was mixed with coating buffer (Sigma, USA, Cat. #c3041) and coated onto a 96-well plate at 4°C for 1 day. The antibodies of the present invention were then allowed to bind to the plate. The extracted bacteriophage was then bound to the antibodies of the present invention. The amount of bacteriophage bearing wild-type and mutant HBsAg that actually bound to the antibodies of the present invention was measured using a bacteriophage M13 protein antibody conjugated with HRP enzyme (GE Healthcare, USA, 27-9421-01) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS, KPL, USA, Cat. #50-62-00). To measure the amount of bacteriophage expressing HBsAg on their surface in each experimental group, anti-HA antibody (Genescript, USA, Cat# A00168-100) was coated onto a 96-well plate at 4°C for 1 day, and the extracted bacteriophage was bound to the antibody and measured in the same manner.

[0065] As a result, a low binding affinity of the antibody of the present invention was observed specifically only in the bacteriophage sample from Region 1+3, which expresses on its surface HBsAg lacking amino acids 101 to 160 (see Figure 2), indicating that the main binding site of the antibody of the present invention is contained in Region 2.

[0066] Example 2: Identification of epitopes for antibodies of the present invention using serially deleted mutant antigens To identify the epitope site based on the main binding site of the antibody of the present invention identified in Example 1, serial deletion mutations were made by sequentially truncating 15 amino acids from amino acid 101, the initiating amino acid of Region 2, and then the mutations were exposed on the surface of bacteriophage and their binding to the antibody of the present invention was confirmed by ELISA.

[0067] Example 2-1. Preparation of four mutant HBsAg expression vectors Using the Region 2+3 clone as a base, deletion mutations were made in groups of 15 amino acids. The names of the resulting clones and the HBsAg site are shown in Table 2 (see Figure 3). The detailed cloning process is omitted as it overlaps with Example 1-1.

[0068] [Table 2]

[0069] Example 2-2. Experiment to confirm the binding strength between four types of mutant HBsAg and the antibody of the present invention Bacteriophages with four types of consecutive deletion mutations exposed on the surface were extracted in the same manner as in Example 1-2, and ELISA was performed.

[0070] As a result, we confirmed that the binding affinity of the antibody of the present invention was significantly reduced in Region2+3del1, which indicates that the major antigenic determinant (epitope) of the antibody of the present invention is located in the region from amino acids 101 to 115 (see Figure 4).

[0071] Example 3. Epitope determination of the antibody of the present invention using single amino acid mutant antigens To more precisely identify the epitope of the antibody of the present invention, random mutations were introduced into HBsAg (ADR subtype) using the shotgun mutagenesis method (see, e.g., J Am Chem Soc. 2009;131(20):6952-6954) by Integral Molecular, USA, and the binding ability of the antibody of the present invention to each mutant antigen was measured. The characteristics of the mutant antigen library used in the experiment are shown in Table 3. Each clone of the constructed library was expressed in HEK-293T cells cultured in a 384-well plate.

[0072] [Table 3]

[0073] Example 3-1: Confirmation of antibody epitope The binding activity of the antibodies of the present invention to the mutant antigens was measured three times by immunofluorescence FACS analysis and normalized to the reactivity to wild-type HBsAg. Furthermore, the binding activity results using orb43805, a mouse monoclonal antibody against HBsAg, as a control antibody were used to confirm the reliability of the experimental results and set standards for epitope selection. Specifically, mutated residues present in clones whose binding reactivity to orb43805 with the control antibody was 55% or more (>55%WT) compared to the binding reactivity to wild-type HBsAg and whose binding reactivity to the antibodies of the present invention was less than 15% (<15%WT) compared to the wild-type were selected as critical residues essential for binding of the antibodies of the present invention.

[0074] As a result of the experiment, core residues for the antibody of the present invention were derived from a total of four mutant antigens, and it was confirmed that the epitope of the antibody of the present invention includes amino acid positions 110, 118, 120, and 147 of HBsAg. Specific experimental results are shown in Table 4.

[0075] [Table 4]

[0076] Of these, amino acid position 110 is also included in the binding site identified through the experiment in Example 2, and therefore can be concluded to be the core epitope for the antibody of the present invention. Although positions 118, 120, and 147 are not within the binding site identified in the experiment in Example 2, in the case of deletion experiments, there is a possibility that structural changes may occur due to the removal of multiple amino acids, and it is highly likely that some epitopes will not be identified.

[0077] More specifically, HBsAg maintains its three-dimensional structure through disulfide bonds such as C107-C138 and C139-C147. It was confirmed that the loss of these bonds through the deletion mutations in Example 2 disrupts the original structure, affecting antibody binding. Specifically, in this example, single mutation experiments were used to identify epitope residues not identified in the deletion experiments in Example 2 (see Figure 5). However, because position 147 forms a disulfide bond that is important for maintaining the three-dimensional structure of HBsAg, it can also participate in binding with the antibody of the present invention by helping to ensure the normal formation of the structural epitopes at positions 110, 118, and 120.

[0078] Example 4: Characterization of antibody epitopes of the present invention Western blotting was performed to confirm whether the binding sites identified in Examples 1, 2, and 3 form conformational epitopes. Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis (SDS-PAGE) gels, which are protein-denaturing conditions, and native-PAGE gels, which are non-denaturing conditions, were used to investigate differences in binding strength with the antibodies of the present invention depending on the structure of HBsAg. In particular, even under protein-denaturing conditions, the presence or absence of conformational epitope formation was closely examined by using or not using a reducing agent, dividing HBsAg into two groups: one in which disulfide bonds, which are important for the formation of the tertiary structure, were removed to completely linearize it, and the other in which they were not.

[0079] 4-1. Western blot analysis using native-PAGE To evaluate whether the antibodies of the present invention recognize the structural epitopes of naturally formed HBsAg, Western blotting was performed using NativePAGE gels containing no SDS. TM Sample Buffer (Invitrogen, USA, Cat#BN2003) and NativePAGE TM After mixing with 5% G-250 Sample Additive (Invitrogen, USA, Cat# BN2004), NativePAGE was performed. TMThe samples were loaded onto Novex® 3-12% Bis-Tris Protein Gel (Invitrogen, USA, Cat# BN1003BOX). After approximately 2 hours of gel running, proteins in the gel were transferred to a PVDF membrane (Invitrogen, USA, Cat# LC2002) using NuPAGE® Transfer Buffer (Invitrogen, USA, Cat# NP0006). The membrane was blocked with phosphate-buffered saline (PBS)-Tween 20 buffer containing 5% skim milk for 1 hour, after which the primary antibody was mixed with 3% skim milk in PBS-Tween 20 buffer and the membrane was refrigerated overnight. The World Health Organization (WHO) standard (WHO International Standard for anti-HBs immunoglobulin, human (code: 07 / 164)) was used as a positive control for the antibodies of the present invention, and a humanized anti-HER2 antibody against human epidermal growth receptor 2 (HER2) was used as a negative control. After thoroughly washing the membrane with PBS-Tween 20 buffer, the secondary antibody, horseradish peroxidase (HRP)-conjugated anti-human Fc (Thermo Scientific, USA, Cat. #31413), was mixed in PBS-Tween 20 buffer containing 3% skim milk and incubated for 1 hour. After thoroughly washing with PBS-Tween 20 buffer, the membrane was treated with enhanced chemiluminescent (ECL) substrate and then examined for binding between HBsAg and each antibody tested using a ChemiDoc (Bio-Rad, USA) instrument.

[0080] As a result of the experiment, HBsAg that maintained its native tertiary structure exhibited very high binding affinity with the antibody of the present invention (see Figure 6A). The specificity of this binding was confirmed by the results of experiments using the WHO standard as a positive control and the anti-HER2 antibody as a negative control. The WHO standard is a polyclonal antibody purified from human blood, which contains all antibodies that recognize the linear and structural epitopes of HBsAg, and therefore binding was confirmed in this experiment, while the nonspecific anti-HER2 antibody did not bind.

[0081] Meanwhile, the molecular weight of HBsAg is known to be around 23 kD, but this experiment revealed that the molecular weight of HBsAg bound to antibodies was much larger. However, this can be concluded as a natural phenomenon, as it is well known that native HBsAg assembles autonomously and forms a specific form (22 nm subviral particles) (Ira Berkower et al., J Virol., Mar 2011; 85(5): 2439-2448).

[0082] 4-2. Western blot analysis using SDS-PAGE To more closely analyze the structural characteristics of the epitope for the antibody of the present invention, Western blot was performed using SDS-PAGE gel and reducing agent. The overall experimental procedure was the same as in Example 4-1 and is briefly as follows.

[0083] First, the HBsAg solution was mixed with SDS-PAGE sample buffer and incubated at 95°C for 5 minutes. The gel was then loaded onto a 4-20% Mini-PROTEAN TGX Precast Gel and run. Two loading samples were prepared: one containing a reducing agent (NuPAGE Sample Reducing Agent (10x), Life Technologies, USA) to induce complete protein denaturation, and the other containing no reducing agent, which maintained the disulfide bonds of HBsAg and resulted in incomplete denaturation. After running, the HBsAg was transferred to a nitrocellulose (NC) membrane, blocked, and refrigerated overnight with primary antibodies. Primary antibodies used were the antibody of the present invention, a WHO standard, HBIg (Hepabig, Green Cross, Korea), and an anti-HER2 antibody. The subsequent steps are identical to those described above and are therefore omitted here.

[0084] The experimental results confirmed that the antibodies of the present invention did not bind to HBsAg that had been completely linearized, even down to the disulfide bonds, but could bind well to HBsAg that had only been partially denatured. As described in Example 4-1, the WHO standard and HB1g used as positive controls are polyclonal antibodies, and contain antibodies that recognize linear epitopes, demonstrating their ability to bind well to HBsAg that had been completely linearized under reducing conditions. The anti-HER2 antibody did not bind at all, regardless of the HBsAg structure. It is well known that disulfide bonds formed within or between proteins play an important role in the tertiary structure of HBsAg (Mangold CM et al., Arch Virol., 1997;142(11):2257-67).

[0085] Therefore, it is clear that the maintenance of the tertiary structure of HBsAg by disulfide bonds is essential for the binding of the antibodies of the present invention, and it can be concluded that the antibodies of the present invention recognize a conformational epitope of HBsAg.

[0086] Example 5: Binding activity of the antibody of the present invention to various mutant antigens of the a determinant ELISA was performed to confirm the binding activity of the antibodies of the present invention against four mutant antigens on the a determinant. These antigens have mutations at amino acid positions 126, 129, 133, and 143, respectively. These antigens were not only discovered as escape mutations against Hepatitis B Immune globulin (HBIg) or vaccines reported in chronic hepatitis B patients, but also pose problems such as making it difficult to measure surface antigens in diagnostics (Horvat et al., Labmedicine, vol. 42(8):488-496, 2011). Recombinant proteins of these antigens were purchased from ProspecBio.

[0087] FIG. 7 shows the reactivity of antibodies 1 and 2 of the present invention with mutant antigens of the a determinant, and the results are summarized in Table 5, classified as positive (+) or negative (-) depending on whether or not there was reactivity.

[0088] [Table 5]

[0089] Experimental results confirmed that antibodies 1 and 2 of the present invention retain binding ability to HBsAg with various α-determinant mutations. This indicates that the antibodies of the present invention may be free from the influence of the α-determinant (amino acids 124-147), which shows a high mutation rate, because they recognize epitopes at positions 110, 118, 120, and / or 147 of HBsAg.

[0090] Furthermore, in the case of amino acid 147, it is a residue that is important for the formation of the structure, and mutation of this residue is known to have a serious impact on infectivity, so it is expected that the mutation rate will actually be very low.

[0091] Therefore, vaccine compositions containing epitopes at positions 110, 118, 120 and / or 147 or antibodies that bind to said epitopes are unlikely to suffer from reduced efficacy due to escape mutations and can be usefully used for the prevention or treatment of HBV.

[0092] Example 6: In vitro neutralization efficacy against hepatitis B virus In order to verify the neutralizing ability of the antibody of the present invention against various genotypes of hepatitis B virus, an in vitro neutralization assay was carried out.

[0093] In vitro HBV neutralization experiments evaluate the neutralizing power of antibodies by measuring the amount of intracellular and extracellular virus at the time of maximum viral replication, when human hepatocytes are infected with the virus, to determine the extent to which infection is inhibited depending on the treatment conditions of each antibody. The amount of intracellular virus was measured by the amount of HBV DNA replicating, and the amount of virus released into the extracellular space by the amount of HBV DNA and HBsAg in the culture medium. HBV DNA was quantified by real-time PCR using a TaqMan probe, and HBsAg was quantified by chemoluminescent immunoassay (CLIA).

[0094] 6-1. Primary in vitro neutralization experiment Human hepatocytes, required for hepatitis B virus infection, were prepared from chimeric mice (uPA / SCID mice with humanized liver) one day before virus inoculation using a two-step collagenase perfusion method. The isolated hepatocytes were plated at 4 × 10 per well in a 24-well plate coated with type I collagen. 5Hepatocytes were plated in wells, each well containing 500 μl of DMEM (Gibco, USA, 11965) medium containing 10% FBS (Atlas Biologicals, USA, F0500A), 1x penicillin / streptomycin (Gibco, USA, 15140), and 20 mM HEPES (Gibco, USA, 15630). The hepatocytes were cultured at 37°C in a 5% CO2 humidified cell incubator for 24 hours.

[0095] Viral infection was performed using HBV of four genotypes, A (Genebank accession number: AB246345.1), B (Genebank accession number: AB246341), C (Genebank accession number: AB246338.1), and D (Genebank accession number: AB246347), produced in chimeric mice with humanized liver tissue. The virus was mixed with the antibody of the present invention and used at 2 × 10 per well. 6 The cells were treated with the virus at a concentration of 100 μg / ml. The detailed procedure is as follows:

[0096] A. Preparation of the virus inoculum mixture The virus and each antibody were mixed in dHCGM medium (DMEM + 10% FBS, NaHCO3 44 mM, L-proline 15 μg / ml, insulin 0.25 μg / ml, dexamethasone 50 nM, EGF 5 ng / ml, Asc-2p 0.1 mM, DMSO 2%) to a final volume of 100 μl, and incubated at room temperature for 1 hour. 6 The antibody of the present invention was diluted to four concentrations: 10, 1, 0.1, and 0.01 μg / ml.

[0097] B. Virus inoculation 25 μl of 40% PEG (Sigma, USA, P1458) was mixed with 125 μl of dHCGM medium, and the virus / antibody mixture prepared in A was then added to prepare a final inoculation mixture of 250 μl. After removing the medium from the prepared cells, the inoculation mixture was added and the cells were then cultured for 24 hours.

[0098] C. Medium exchange and incubation, preparation of analytical samples After virus inoculation, hepatocytes were cultured for a total of 12 days, with cell washing and medium changes on days 1, 2, and 7. After removing the existing culture medium, cells were washed with 500 μl of DMEM + 10% FBS, and the same volume of dHCGM medium was added. After the medium change on day 7, 300 μl and 30 μl of the existing culture medium were collected for quantification of newly produced and secreted extracellular HBsAg and HBV DNA, respectively, and stored at -20°C until analysis.

[0099] After 12 days of incubation, both cells and culture medium were used for intracellular and extracellular virus quantification. Culture medium was collected for HBsAg and HBV DNA measurements in the same manner as previously described. Cells were collected by washing each well once with 500 μl of DMEM + 10% FBS and then lysing in 500 μl of SMITEST (Medical & Biological Laboratories Co., Ltd.) solution. HBV DNA was extracted according to the manufacturer's protocol.

[0100] D. Sample Analysis HBV DNA quantification was performed by real-time PCR using a TaqMan probe, TaqMan PCR Core Reagents (Life Technologies, USA), and an ABI Prism 7500 sequence detector system (Applied Biosystems, USA). HBsAg quantification was performed using an automated system, ARCHITECT (Abbott, USA), using CLIA procedures.

[0101] [Table 6]

[0102] [Table 7]

[0103] The experimental results for antibodies 1 and 2 of the present invention are shown in Figures 8A to 8D and 9A to 9D, categorized by virus genotype according to each measurement item.

[0104] First, a comparative analysis of intracellular HBV DNA levels according to the treatment concentration of each antibody confirmed that antibodies 1 and 2 of the present invention, which were selected based on their binding ability to the adr subtype of HBsAg, which is classified as genotype C, both have strong neutralizing activity against genotype C. While HB1g, used as a positive control, reduced HBV DNA levels by more than 400-fold compared to the anti-HER2 antibody used as a negative control, antibody 2 of the present invention, treated with 1 μg / ml, which is 1 / 10 of the treatment amount, also reduced viral DNA to the same level. Antibody 1 of the present invention maintained a relatively high level of neutralizing activity, reducing HBV DNA by up to 100-fold even at a low concentration of 0.1 μg / ml. Furthermore, antibodies 1 and 2 of the present invention showed excellent efficacy, particularly against the A and B genotypes, with maximum neutralizing potency more than twice that of the positive control HBIg, and against the D genotype, the neutralizing potency remained high even at low concentrations of 1 μg / ml (antibody 2 of the present invention) or 0.1 μg / ml (antibody 1 of the present invention) (Figures 8A to 8D).

[0105] The above-mentioned characteristics of the neutralizing potency of antibodies 1 and 2 of the present invention against the four genotypes A, B, C, and D were found to be very similarly reflected in the quantification results of extracellular HBsAg measured in the culture medium (Figures 9A to 9D).

[0106] To summarize the above results, in vitro neutralizing efficacy testing was conducted against four genotypes of HBV: A, B, C, and D, and the results confirmed that antibodies 1 and 2 of the present invention have high levels of neutralizing activity against all viruses used.

[0107] Example 7: Investigation of binding characteristics to surface antigens of various genotype viruses derived from chronic hepatitis B patients In order to confirm whether the antibodies 1 and 2 of the present invention can actually bind to and neutralize various genotypes of viruses prevalent worldwide, we tested them against a World Health Organization (WHO) reference panel (1) consisting of surface antigens of various genotypes of viruses derived from patient sera. stSandwich ELISA was performed using the WHO International Reference Panel for HBV Genotypes for HBsAg Assays (PEI code 6100 / 09). Detailed information on the standard is shown in Table 8, and the experimental method is as follows.

[0108] The two antibodies were adsorbed at a concentration of 2 μg / ml in 100 μl aliquots into each well of a 96-well microtiter plate (Nunc, Denmark, 449824) coated with anti-human IgG Fcγ(gamma) antibody (Jackson ImmunoResearch, USA, 109-006-098). After washing, the plate was blocked with phosphate buffer solution (Teknova, USA, D5120) containing 3% bovine serum albumin (BSA). After washing again, 100 μl aliquots of 15 serum samples representing the HBsAg genotype panel were added and incubated at 37°C for 90 minutes. Each serum sample was appropriately diluted with phosphate buffer solution (Teknova, USA, D5120) containing 1% BSA to achieve an absorbance of approximately 0.8-1.2 at 450 / 620 nm. To detect HBsAg that had reacted with the antibody, the sample was treated with peroxidase-labeled rabbit anti-HBV surface antigen antibody (Thermo Scientific, USA, PA1-73087) at 37°C for 60 minutes. Color development, reaction termination, and absorbance measurement were performed using the same methods as in Example 5. The reactivity of the two antibodies to each genotype of HBV surface antigen was analyzed graphically using Excel (Microsoft, USA) (Figure 10).

[0109] Analysis confirmed that both Antibodies 1 and 2 of the present invention bound well to 15 HBsAg samples. As mentioned above, these surface antigen samples were serum samples prepared from actual patient blood and covered seven of the eight HBV genotypes, A through H, excluding G. Furthermore, for types A, B, C, D, and F, which have multiple subgenotypes, two to three samples of predominantly prevalent subgenotypes and subtypes (serotypes) were included for each genotype. This means that the WHO HBV genotype panel used in the experiment essentially represents the majority of HBV genotypes prevalent worldwide. Although genotype G was omitted from the panel, no subgenotype has been reported for G to date, and it is classified as subtype (serotype) adw2. Therefore, the binding activity of Antibodies 1 and 2 of the present invention to G can be inferred from the experimental results for the five adw2 samples included in the panel.

[0110] Therefore, the fact that antibodies 1 and 2 of the present invention showed excellent binding activity in all 15 samples means that the two antibodies can bind to all genotypes of HBV that are prevalent worldwide and thereby demonstrate neutralizing power.

[0111] [Table 8]

[0112] Example 8: Investigation of binding characteristics against various drug-resistant viruses The binding characteristics of antibodies 1 and 2 of the present invention to resistance mutations against lamivudine (LMV), adefovir (ADV), clevudine (CLV), and entecavir (ETV), HBV polymerase inhibitors commonly used in chronic hepatitis B patients, were confirmed using a sandwich ELISA assay similar to that used in Example 7. All resistance mutant viruses, including the wild-type virus, used in the experiment were cloned at the Department of Pharmacology, Konkuk University Graduate School of Medicine, using HBV DNA obtained from the blood of patients who had developed resistance to the drug treatment. Drug resistance was experimentally confirmed through transduction experiments using Huh7 or HepG2 cell lines (Ahn et al., Journal of Virology, 88(12):6805-6818, 2014). All viruses were genotype C, and the characteristics of each virus are shown in Table 9.

[0113] Each of the prepared HBV expression vectors was transduced into Huh7 cells cultured in a T75 flask (BD BioScience, 353136) using Lipofectamine 2000 (Life Technologies, 11698019) and cultured for 3 days to produce virus. The virus produced was concentrated using Centricon (Millipore, USA), and the viral load of each sample was compared with the HBsAg load using a Monolisa HBsAg Ultra (BioRad, 72346) ELISA kit. The samples were then appropriately diluted to obtain similar values ​​for use in the experiment.

[0114] The experimental results demonstrated that both antibodies 1 and 2 of the present invention have binding activity to lamivudine (LMV), adefovir (ADV), clevudine (CLV), and entecavir (ETV)-resistant viruses at the same level as wild-type viruses (see Figure 11). Here, the binding activity to ADV-resistant viruses appears to be relatively low, which is believed to be due to the production volume of this sample being slightly lower than that of the other samples.

[0115] This indicates that antibodies 1 and 2 of the present invention may have binding activity and neutralizing efficacy not only against one type of drug-resistant virus used in the experiments, but also against most of the resistant viruses that arise against those drugs. This is because, as can be seen in Table 9, mutations that confer drug resistance to HBV are associated with specific amino acid mutations in the reverse transcriptase (RT) domain of HBV polymerase, and these mutations occur very specifically for each drug. Due to the nature of HBV, which shares genes, these specific polymerase mutations are often accompanied by specific HBsAg mutations. For example, the rtM204I mutation in the polymerase results in the W196L mutation in HBsAg, and the rtA181V mutation results in the L173F mutation in HBsAg. Antibodies 1 and 2 of the present invention were confirmed to bind well despite these surface antigen mutations associated with drug resistance mutations.

[0116] Furthermore, each of the resistant viruses used in this experiment had many non-specific surface antigen mutations in addition to the resistance-specific surface antigen mutations mentioned above (see Table 9). The results of this experiment demonstrate that antibodies 1 and 2 of the present invention can bind to and neutralize viruses with mutations at the Q101R, K112R, I126S, L175S, A184V, and I185M positions of HBsAg.

[0117] [Table 9]

[0118] Example 9: Measurement of antigen-antibody binding affinity Surface plasmon resonance (SPR) assays were used to measure the antigen-antibody binding affinity of the antibodies of the present invention. Specifically, the binding affinity of the antibodies of the present invention to recombinant HBsAg (adr subtype, ProspecBio) was determined by SPR analysis using an assay buffer HBS-EP (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20) at 25°C on a Biacore T200 (GE Healthcare) instrument. Approximately 500 RU of HBsAg protein diluted in 10 mM sodium acetate (pH 5.0) was directly immobilized onto a CM5 research biosensor chip using an amine coupling kit according to the manufacturer's guidelines and procedures. Unreactive sites on the biosensor surface were blocked with ethanolamine. The Biacore T200 Control Software and Biacore T200 Evaluation Software were used for reaction analysis. The antibodies of the present invention were diluted in HBS-EP buffer. During the assay process, a biosensor surface without immobilized HBsAg was used as a control. The binding and dissociation rate constants, Ka (M-1s-1) and Kd (s-1), were determined at a flow rate of 30 μl / min. The rate constants were obtained by performing reaction binding measurements at antibody concentrations ranging from 2.46 to 200 nM using a three-fold serial dilution and buffer as a control. The equilibrium dissociation constant, KD (M), for the reaction between the antibody and the target antigen was then calculated from the rate constant using the following equation: KD = Kd / Ka. Binding was recorded as a function of time and the rate constant.

[0119] The experimental results are shown in Table 10, and confirmed that the antibodies of the present invention have high binding affinity to HBsAg.

[0120] [Table 10]

Claims

1. 1. A method for screening for a binding molecule for diagnosing a disease caused by hepatitis B virus, excluding a binding molecule that binds to amino acid position 119 of HBsAg, comprising: The equilibrium dissociation constant KD of the binding molecule for an epitope including amino acid positions 110, 118, 120, and 147 of the hepatitis B virus surface antigen (HBsAg) is 1 x 10 －９ When it is less than M, determining that the binding molecule is useful for diagnosing a disease caused by hepatitis B virus; Screening methods.

2. 2. The screening method according to claim 1, wherein the hepatitis B virus surface antigen (HBsAg) has the amino acid sequence shown in SEQ ID NO:

1.

3. The screening method according to claim 1 or 2, wherein the binding molecule is an antibody.

4. The screening method according to claim 3, wherein the antibody is a human monoclonal antibody.

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