Epitope of hepatitis b virus surface antigen and binding molecule specifically binding to same for neutralizing hepatitis b virus
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- CELLTRION INC
- Filing Date
- 2023-12-18
- Publication Date
- 2026-04-14
Smart Images
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Abstract
Description
[Technical field]
[0001] The present invention relates to an epitope of a hepatitis B virus surface antigen and a hepatitis B virus-neutralizing binding molecule that specifically binds to the epitope. [Background technology]
[0002] Hepatitis B virus (HBV) is a DNA virus belonging to the Hepadnaviridae family that induces acute and chronic hepatitis, and is the main cause of liver cirrhosis and liver cancer. HBV is classified into 10 serotypes based on the reaction of Hepatitis B virus surface antigen (HBsAg) to standard serum and the difference in the amino acid sequence of HBsAg, or into 8 genotypes based on the difference in gene base sequence. As of 2012, it is known that there are about 240 million chronic HBV-infected patients worldwide, and more than 500,000 people die from hepatitis B-related diseases every year. The chronic HBV infection rate among adults in Korea and China is very high at 5-8%, and 80% of adult chronic hepatitis patients, 65% of liver cirrhosis patients, and 70% of hepatocellular carcinoma patients are 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 of 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] Currently, drugs used to treat chronic hepatitis B include interferon (pegninterferon), lamivudine, adefovir dipivoxil, entecavir, and tenofovir, and all oral therapeutic agents except interferon are nucleoside / nucleotide analogues. These drugs inhibit the activity of HBV reverse transcriptase, thereby inhibiting viral DNA replication, and as a result, they have been shown to reduce the amount of HBV DNA in serum, normalize ALT values, and improve liver fibrosis.
[0004] However, when used for a long period of time, nucleoside analogues induce resistance to the drug, resulting in a decrease in efficacy and ultimately leading to the worsening of hepatitis. In the case of the most recently developed tenofovir, no resistance has been reported to date, but in the case of lamivudine, which has been the most widely used drug worldwide, it is known that the resistance incidence rate after five years reaches 70-80%. In addition, since these oral drugs cannot directly inhibit HBV infection, human plasma-derived Hepatitis B Immune globulin (HBIg) preparations are used together with oral therapeutic agents to prevent vertical infection from mothers to fetuses and reinfection in liver transplant patients.
[0005] Existing HBIg is produced by isolating antibodies from the blood of people who have antibodies against hepatitis B using advanced purification technology and removing potential sources of contamination using virus inactivation technology. However, it is difficult to secure the raw material plasma, which results in excessive import costs and the inability to flexibly meet demand. In addition, it takes a lot of time and money to remove viruses from plasma, but it is still not possible to eliminate the possibility of potential sources of infection, and there are disadvantages such as the inconvenience of administration and economic burden due to low efficacy.
[0006] In addition, it is known that most of the antibodies formed by vaccination with existing hepatitis B vaccines recognize the a determinant at amino acid positions 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 certain mutations in the a determinant that have occurred in some patients can evade the antibodies formed 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 such escape mutations of existing vaccines and HBIg. Summary of the Invention [Problem to be solved by the invention]
[0007] 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 comprising 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] A further 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 comprising 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 a 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 problem to be solved by the present invention is to provide a composition for detecting HBV, which contains the epitope or a polynucleotide encoding the epitope. [Means for solving the problem]
[0020] In order 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") includes amino acid positions 110, 118, 120 and / or 147 of HBsAg. It has also been confirmed that a sequence including these four amino acids or a part of it forms a three-dimensional structure to form an epitope to which the antibody of the present invention can bind, thereby completing the present invention.
[0021] Therefore, the present invention provides a 3- to 38-mer epitope 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). The epitope including amino acids at these positions may be used in a form bound to a carrier in order to maintain its three-dimensional structure or to improve the efficiency when used as a composition such as a vaccine. The carrier according to the present invention may be any one that is suitable for the living body and can achieve the desired effect in the present invention, and is preferably selected from peptides, serum albumin, immunoglobulin, hemocyanin, polysaccharides, etc., but is not limited thereto.
[0023] In one embodiment of the 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). Moreover, 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 has a binding affinity of less than 9×10 -10 In yet another embodiment, the binding molecule has a binding affinity of less than 8×10 -10 In yet another embodiment, the binding molecule has a binding affinity of less than 7×10 -10 In yet another embodiment, the binding molecule has a binding affinity of less than 6×10 -10 In yet another embodiment, the binding molecule has a binding affinity of less than 5×10 -10 In yet another embodiment, the binding molecule has a binding affinity of less than 4×10 -10 In yet another embodiment, the binding molecule has a binding affinity of less than 3×10 -10 In yet another embodiment, the binding molecule has a binding affinity of less than 2×10 -10 In yet another embodiment, the binding molecule has a binding affinity of less than 1×10 -10 In yet another embodiment, the binding molecule has a binding affinity of less than 1×10 -11 In yet another embodiment, the binding molecule has 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 have binding and neutralizing activity against Hepatitis B viruses of A, B, C, D, E, F, G and H genotypes.
[0029] In addition, the binding molecule of the present invention has a 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, a 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. In this specification, 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 are capable of recognizing and binding to specific antigens. In terms of their structure, antibodies are usually Y-shaped proteins consisting of four amino acid chains (two heavy chains and two light chains). Each antibody has two main regions: a variable region and a constant region. The variable region, located at the end of the arms of the Y, binds to and interacts with the target antigen. The variable region includes complementarity determining regions (CDRs) that recognize and bind to specific binding sites on a specific antigen. The constant region located at the tail of the Y is recognized and interacted with by the immune system. A target antigen generally 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 one or more corresponding antibodies.
[0033] At the same time, the present invention includes functional variants of said 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 agents 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. Additionally, 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). As an example, the amino acid sequence of the variable regions can be varied, including but not limited to the framework, hypervariable regions, and in particular the CDR3 region. Generally, the light or heavy chain region comprises three hypervariable regions, including three CDR regions, and further conserved regions, i.e. framework regions (FRs). The hypervariable regions comprise amino acid residues from the CDRs and amino acid residues from the hypervariable loops. The functional variants within the scope of the present invention can have about 50%-99%, about 60%-99%, about 80%-99%, about 90%-99%, about 95%-99%, or about 97%-99% amino acid sequence homology with the parent antibody of the present invention. In order to optimally align the amino acid sequences to be compared and define similar or identical amino acid residues, Gap or Bestfit, known to those skilled in the art, can be used in computer algorithms.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 methods, mutagenesis using oligonucleotides, and partial mutagenesis, or by organic synthesis methods.
[0034] The invention also provides polynucleotides encoding said binding molecules. In one embodiment, the invention includes an isolated nucleic acid molecule encoding said anti-HBsAg monoclonal antibody.
[0035] The present invention further provides an expression vector comprising the polynucleotide. The expression vector may be any one selected from the group consisting of MarEx vector, which is an expression vector unique to Celltrion, and pCDNA vector, F, R1, RP1, Col, pBR322, ToL, Ti vector, cosmid, phage such as lambda, lambdoid, M13, Mu, p1P22, Qμ, T-even, T2, T3, T7, and plant virus, but is not limited thereto. All expression vectors known to those skilled in the art as expression vectors can be used in the present invention, and the selection of an expression vector should be in accordance with the properties of the target host cell. The introduction of a vector into a host cell may be performed by calcium phosphate transfection, virus infection, DEAE-dextran regulated transfection, lipofectamine transfection, or electroporation, but is not limited thereto. Those skilled in the art can select an introduction method suitable for the expression vector and host cell to be used. The expression vector may contain one or more selection markers, but is not limited thereto, and a vector without a selection marker may be used to select the presence or absence of product production. The selection marker is selected depending on the target host cell, and since this is done using a method already known to those skilled in the art, the present invention is not limited thereto. In addition, the nucleic acid molecule of the present invention may be fused by inserting a tag sequence into the expression vector to facilitate purification. The tag may include, but is not limited to, a hexa-histidine tag, a hemagglutinin tag, a myc tag, or a flag tag, and all tags known to those skilled in the art that facilitate purification can be used in the present invention.
[0036] In another embodiment of the present invention, the present invention relates to a host cell transfected with the expression vector to produce a binding molecule having neutralizing activity against Hepatitis B virus. In the present invention, the host cell may include, but is not limited to, cells of mammalian, plant, insect, fungal or cellular origin. The mammalian cell may be, but is not limited to, CHO cell, F2N cell, CSO cell, BHK cell, Bowes melanoma cell, HeLa cell, 911 cell, AT1080 cell, A549 cell, HEK293 cell, HEK293T cell, etc., and any cell that can be used as a mammalian host cell known to those skilled in the art may be used.
[0037] Furthermore, the present invention provides a composition for preventing, treating or diagnosing hepatitis B, comprising the binding molecule. The composition of the present invention may further comprise, together with the binding molecule, an interferon, an anti-HBV monoclonal antibody, an anti-HBV polyclonal antibody, a nucleoside analogue, a DNA polymerase inhibitor, an siRNA preparation or a therapeutic vaccine as an antiviral drug.
[0038] The compositions containing the binding molecules of the present invention may be formulated by conventional methods into the following forms: sterile injection solution, lyophilized dosage form, pre-filled syringe solution, oral dosage form, topical agent, suppository, etc., but are not limited thereto.
[0039] In another embodiment of the present invention, the present invention relates to a method for treating hepatitis B, comprising administering the composition in a therapeutically effective amount 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, but is not limited to, intravenous.
[0040] In one embodiment of the present invention, the treatment method may further include administering an anti-viral drug. The anti-viral drug may be, but is not limited to, an 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 the composition to a subject in a therapeutically effective amount. In the method of the present invention, a prophylactic agent known to those skilled in the art may be administered together. In the method of the present invention, the administration method may be oral or parenteral, and the administration route may be, but is not limited to, intravenous.
[0042] The compositions of the present invention can be administered to mammals, including humans, to prevent or treat HBV infection and diseases induced 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 in an amount of 0.001-10 mg / kg (body weight), or 0.005-1 mg / kg (body weight) per day, via parenteral route, once or in divided doses per day. In some cases, dosages lower than the ranges mentioned above may be more suitable, and larger amounts may be used without causing adverse side effects, or larger dosages may be distributed in several small doses over the course of a day.
[0043] In another embodiment of the present invention, the present invention relates to a method for diagnosing whether or not 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 of the present invention (e.g., monoclonal antibody) may be conjugated with a labeling substance as necessary for diagnostic detection, which is already 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 or not 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 of the present invention, the present invention relates to a kit for diagnosing hepatitis B virus, comprising i) the composition and ii) a container. In the diagnostic kit of the present invention, the container 2) 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 yet 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 further provides a polynucleotide encoding the epitope. The polynucleotide encoding the epitope including the amino acid position provided in the present invention can be used in the form of a gene vaccine by itself. In this case, the polynucleotide can be used by itself without a carrier, or can be carried by a viral or non-viral carrier and delivered to the body. Any viral or non-viral carrier that is known to be generally available in the technical field to which the present invention belongs can be used. Specifically, the viral carrier can be an adenovirus, an adeno-associated virus, a lentivirus, a retrovirus, etc., and the non-viral vector can be a cationic polymer, a non-ionic polymer, a liposome, a lipid, a phospholipid, a hydrophilic polymer, a hydrophobic polymer, or a complex of at least one selected from these, but is not limited thereto.
[0049] The present invention also provides an expression vector comprising a polynucleotide encoding the epitope.
[0050] The present invention further 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 of the present invention, the present invention provides a method for expressing an epitope including amino acid positions 110, 118, 120 and / or 147 of HBsAg on the surface of a microorganism or virus. In this case, a recombinant vector including a sequence encoding an inducing promoter or signal protein and various microorganisms or viruses including the recombinant vector may be used, and particularly preferred microorganisms or viruses include, but are not limited to, recombinant Escherichia coli, recombinant yeast and recombinant bacteriophage. In order to express an epitope including the amino acid positions on the surface of the microorganism or virus, a display technology well known in the art to which the present invention pertains may be used, and in particular, a polynucleotide sequence encoding an epitope including the amino acid positions may be bound to a sequence encoding a promoter or signal protein that induces expression on the surface of a microbial cell or virus, and then expressed, or a method of deleting a part of a gene site encoding a protein originally expressed on the surface and inserting a polynucleotide sequence encoding an epitope including the amino acid positions into the deleted part may be used, but is not limited to this. In this manner, an epitope containing the amino acid position expressed on the surface of a microorganism or virus can be isolated and purified as such and used for specific applications according to the present invention, and can also be used to select 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 comprising the step of culturing the recombinant microorganism or virus.
[0053] The present invention further provides an HBV vaccine composition comprising the epitope or a polynucleotide encoding the epitope. The HBV vaccine composition may further comprise a pharma- ceutically acceptable adjuvant. Any adjuvant that can promote antibody formation when administered into the body and achieve the object of the present invention may be used, including, but not limited to, aluminum salts (Al(OH)3, ALPO4), squalene, sorbitane, polysorbate 80, CpG, liposomes, cholesterol, MPL (monophosphoryl lipid A), and GLA (glucopyranosyl lipid A).
[0054] The present invention also provides a composition for detecting HBV, which comprises the epitope or a polynucleotide encoding the epitope. Effect of the Invention
[0055] The epitope of hepatitis B virus surface antigen (HBsAg) provided by the present invention does not include key residues in the a-determinant that generate escape mutations upon administration of existing vaccines or HBIg, and therefore compositions containing antibodies that bind to the epitope or vaccine compositions containing the epitope are unlikely to suffer from reduced efficacy due to escape mutations. Therefore, such antibodies or vaccine compositions can be very useful for the prevention and / or treatment of HBV. [Brief description 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 a mutant structure was shown in which each region was deleted. [Diagram 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. [Diagram 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 in which the 15 amino acids are sequentially deleted was confirmed by phage-based ELISA. [Diagram 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 characterize the epitope of the antibody of the present invention. [Figure 6B] 1 shows the results of Western blot analysis carried out to characterize the epitope of the antibody of the present invention. [Figure 7] According to one embodiment of the present invention, the reactivity of the a determinant of antibodies 1 and 2 of the present invention against various mutant antigens was confirmed by ELISA. [Figure 8A] According to one embodiment of the present invention, this shows the results of an in vitro neutralization experiment of the 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 in cells was measured as the amount of DNA of the multiplying HBV using a real-time PCR method. [Figure 8B] According to one embodiment of the present invention, this shows the results of an in vitro neutralization experiment of the 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 in cells was measured as the amount of DNA of the multiplying HBV using a real-time PCR method. [Figure 8C]According to one embodiment of the present invention, this shows the results of an in vitro neutralization experiment of the 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 in cells was measured as the amount of DNA of the multiplying HBV using a real-time PCR method. [Figure 8D] According to one embodiment of the present invention, this shows the results of an in vitro neutralization experiment of the 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 in cells was measured as the amount of DNA of the multiplying HBV using a real-time PCR method. [Figure 9A] According to one embodiment of the present invention, this shows the results of an in vitro neutralization experiment of the 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 proliferated and released outside the cells was measured in terms of 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 the 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 proliferated and released outside the cells was measured in terms of 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 the 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 proliferated and released outside the cells was measured in terms of 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 the 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 proliferated and released outside the cells was measured in terms of 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 confirming the binding activity of antibodies 1 and 2 of the present invention against drug (lamivudine, adefovir, clevudine, entecavir)-resistant viruses by sandwich ELISA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES
[0057] The present invention will be described in detail with reference to the following 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 In order to confirm the binding site of the antibody of the present invention on HBsAg, wild-type and various deletion mutants of HBsAg were prepared, and the binding ability to these was examined by enzyme-linked immunosorbent assay (ELISA) using phages.
[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 FIG. 1). Each protein was expressed on the surface of the phage, and then the binding strength to the antibody of the present invention was 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 surface of phages, 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 (Department of Pharmacology, Konkuk University Graduate School of Medicine, Korea) containing the HBsAg gene sequence of HBV genotype C as a template. After treating with the restriction enzyme SfiI, each was inserted into a phage expression vector treated with the same restriction enzyme. The constructed plasmid was extracted using a QIAprep Spin Miniprep Kit (QIAGEN, Germany, Cat#27106), and the antibody base sequence was finally confirmed by analysis of the base sequence using the extracted DNA. The name of each completed clone and the HBsAg site are shown 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 with the antibody of the present invention To conduct binding experiments for the whole or part of the cloned HBsAg, first, the vector was inserted into expression E. coli (ER2738, Lucigen, USA, Cat#60522-2) by electroporation, and the antibiotic (ampicillin)-resistant E. coli was selectively cultured the next day. After culturing for about 10 hours, the bacteria were infected with bacteriophage, and another type of antibiotic (kanamycin) was used to selectively culture the infected E. coli. To extract the bacteriophage the next day, the E. coli was first separated by centrifugation, and the supernatant was treated with polyethylene glycol (PEG) and left on ice for 30 minutes, and the bacteriophage was separated again by centrifugation. The separated bacteriophage was dissolved, and the supernatant was filtered to extract pure bacteriophage carrying the whole or part of HBsAg on its surface.
[0064] To quantitatively evaluate the binding strength of the wild-type and mutant HBsAg exposed on the surface of bacteriophage with the antibody of the present invention, an ELISA method was used. First, an anti-human Fc antibody (Jackson Immunoresearch, USA, Cat#109-006-098) was mixed with a coating buffer (Sigma, USA, Cat#c3041) and coated on a 96-well plate at 4°C for one day, and then the antibody of the present invention was bound to it. The extracted bacteriophage was then bound to the antibody of the present invention, and the amount of bacteriophage bearing wild-type and mutant HBsAg that actually bound to the antibody of the present invention was measured using a bacteriophage M13 protein antibody (GE Healthcare, USA, 27-9421-01) with HRP enzyme (GE Healthcare, USA, 27-9421-01) and then 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 the surface in each experimental group, anti-HA antibody (Genescript, USA, Cat#A00168-100) was coated on 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 specific low binding strength with the antibody of the present invention was observed only in the bacteriophage samples from Region 1+3 expressing HBsAg on the surface lacking amino acids 101 to 160 (see FIG. 2 ), which means that the main binding site of the antibody of the present invention is contained in the Region 2 site.
[0066] Example 2: Identification of epitopes of antibodies of the present invention using serial deletion mutant antigens In order 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 the initiating amino acid 101 of Region 2, and then the serial deletion mutations were exposed on the surface of bacteriophage and the binding ability 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 reference, deletion mutations were made for each 15 amino acids. The names of the completed clones and the HBsAg sites are shown in Table 2 (see FIG. 3). The detailed cloning process is omitted since it overlaps with Example 1-1.
[0068] [Table 2]
[0069] Example 2-2. Experiment to confirm the binding ability between four kinds of mutant HBsAg and the antibody of the present invention In the same manner as in Example 1-2, bacteriophages having four types of consecutive deletion mutations exposed on the surface were extracted, and ELISA was performed.
[0070] As a result, it was confirmed that the binding affinity of the antibody of the present invention was significantly reduced in Region2+3del1, which means that the main 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. Identification of the epitope of the antibody of the present invention using a single amino acid mutant antigen 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) of 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 library prepared 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 ability of the antibody of the present invention to the mutant antigen was measured three times by immunofluorescence FACS analysis and normalized based on the reactivity to wild-type HBsAg. The binding ability 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 a standard for epitope selection. That is, the mutant residues present in the clones whose binding reactivity to orb43805 of the control antibody was 55% or more (>55%WT) compared to the binding reactivity to wild-type HBsAg and whose binding reactivity to the antibody of the present invention was less than 15% (<15%WT) compared to the wild-type were selected as critical residues essential for the binding of the antibody of the present invention.
[0074] As a result of the experiment, the 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 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 experimental results of Example 2, in the case of deletion experiments, there may be structural changes due to the removal of many amino acids, and it is highly likely that some epitopes will not be identified.
[0077] In more detail, HBsAg maintains its three-dimensional structure through disulfide bonds such as C107-C138, C139-C147, etc., and it can be confirmed that when these bonds are damaged by the deletion mutation of Example 2, the original structure is destroyed, affecting the binding of the antibody. That is, in this Example, epitope residues that were not found in the deletion experiment of Example 2 were identified through single mutation experiments (see FIG. 5). However, in the case of position 147, since it is a residue that 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 normally form the structural epitopes at positions 110, 118, and 120.
[0078] Example 4: Characterization of the antibody epitopes of the invention Western blotting was performed to confirm whether the binding sites identified through Examples 1, 2, and 3 form conformational epitopes. Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis (SDS-PAGE) gels under protein denaturing conditions and Native-PAGE gels under non-denaturing conditions were used to investigate the difference in binding strength with the antibody of the present invention due to the structure of HBsAg. In particular, even under protein denaturing conditions, the presence or absence of conformational epitope formation was thoroughly examined by dividing the case into two cases: when disulfide bonds, which are important for the formation of the tertiary structure of HBsAg, were completely linearized by removing them, and when they were not, depending on whether or not a reducing agent was used.
[0079] 4-1. Western blot analysis using native-PAGE To evaluate whether the antibody of the present invention recognizes the naturally formed HBsAg structural epitope, Western blotting was performed using NativePAGE gels to which SDS was not added. 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 proteins were loaded onto Novex(R) 3-12% Bis-Tris Protein Gel (Invitrogen, USA, Cat#BN1003BOX). After 2 hours of gel running, the proteins on the gel were transferred to a PVDF membrane (Invitrogen, USA, Cat#LC2002) using NuPAGE(R) 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, and then the primary antibody was mixed in PBS-Tween 20 buffer containing 3% skim milk and refrigerated overnight on the membrane. At this time, 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 antibody of the present invention, and anti-HER2 antibody, a humanized antibody against human epidermal growth receptor 2 (HER2), was used as a negative control. After thoroughly washing the membrane with PBS-Tween 20 buffer, anti-human Fc (Thermo Scientific, USA, Cat#31413) with Horseradish Peroxidase (HRP) as a secondary antibody was mixed with PBS-Tween 20 buffer containing 3% skim milk and treated for 1 hour. After thoroughly washing with PBS-Tween 20 buffer, the enhanced chemiluminescent (ECL) substrate was treated, and the presence or absence of binding between HBsAg and each antibody tested was observed using a ChemiDoc (Bio-Rad, USA) instrument.
[0080] As a result of the experiment, HBsAg that maintained its natural tertiary structure showed a very high binding strength with the antibody of the present invention (see FIG. 6A). The specificity of this binding was confirmed by the results of an experiment using the WHO standard product as a positive control and the anti-HER2 antibody as a negative control. The WHO standard product is a polyclonal antibody purified from human blood, which contains all the antibodies that recognize the linear and structural epitopes of HBsAg, and therefore the binding was confirmed in this experiment, whereas the anti-HER2 antibody, which is a nonspecific antibody, did not bind.
[0081] Meanwhile, the molecular weight of HBsAg is known to be around 23kd, and this experiment revealed that the molecular weight of HBsAg bound to antibodies was very large. However, since it is well known that native HBsAg assembles autonomously and forms a specific shape (22nm subviral particles), this can be concluded as a natural phenomenon (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, reacted at 95°C for 5 minutes, and then loaded onto a 4-20% Mini-PROTEAN TGX Precast gel for gel running. Two types of loading samples were prepared. One was added with a reducing agent (NuPAGE Sample Reducing Agent (10x), LifeTechnologies, USA) to induce complete denaturation of the protein, and the other was incompletely denatured without the reducing agent, maintaining the disulfide bond of HBsAg. After running, HBsAg was transferred to a nitrocellulose (NC) membrane, blocked, and refrigerated overnight with primary antibodies. The primary antibodies used were the antibody of the present invention, WHO standard, HBIg (Hepabig, Green Cross, Korea), and anti-HER2 antibody. The subsequent steps are the same as those described above, so they will be omitted.
[0084] As a result of the experiment, it was confirmed that the antibody of the present invention does not bind to HBsAg that has been completely linearized by removing even the disulfide bonds, but can bind well to HBsAg that has only been partially denatured. In the case of the WHO standard and HBIg used as positive controls, since they are polyclonal antibodies as described in Example 4-1, they contain antibodies that recognize linear epitopes, and it was revealed that they bind well to HBsAg that has been completely linearized under reducing conditions. In the case of the anti-HER2 antibody, there was no binding at all, regardless of the structure of HBsAg. It is a well-known fact that disulfide bonds formed inside 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 maintenance of the tertiary structure of HBsAg by disulfide bonds is essential for the binding of the antibody of the present invention, and it can be concluded that the antibody of the present invention recognizes a conformational epitope of HBsAg.
[0086] Example 5: Binding activity study of the antibody of the present invention against various mutant antigens of the a determinant ELISA was performed to confirm the binding activity of the antibody 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, and are not only found in Hepatitis B Immune globulin (HBIg) or vaccine escape mutations reported in chronic hepatitis B patients, but also cause problems such as the inability to measure surface antigens in diagnosis (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 the presence or absence of reactivity.
[0088] [Table 5]
[0089] As a result of the experiment, it was confirmed that the antibodies 1 and 2 of the present invention have the binding ability to various mutant HBsAg a determinants. This indicates that the antibodies of the present invention may be free from the influence of the a 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] In addition, in the case of amino acid 147, it is a residue that is important for the formation of the structure, and it is known that mutation of this residue has a serious effect on infectivity, so it is expected that the occurrence rate of mutations is actually very low.
[0091] Therefore, a vaccine composition comprising an epitope at positions 110, 118, 120 and / or 147, or an antibody that binds to said epitope, is 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 neutralization experiments against HBV are a method to evaluate the neutralizing power of antibodies by measuring the amount of virus inside and outside the cells at the time when the virus is most actively multiplying, and to what extent the infection is inhibited depending on the treatment conditions of each antibody when the virus is infected into human hepatocytes. The amount of virus inside the cells was measured by the amount of HBV DNA multiplying, and the amount of virus multiplied and released outside the cells was measured by the amount of HBV DNA and HBsAg in the medium. HBV DNA was quantified by real-time PCR using a TaqMan probe, and HBsAg was quantified by chemoluminescent immunoassay (CLIA) method.
[0094] 6-1. Primary in vitro neutralization experiment Human hepatocytes required for hepatitis B virus infection were prepared from chimeric mice with humanized liver tissue (uPA / SCID mice with humanized liver) one day before virus inoculation by a two-step collagenase perfusion method. The isolated hepatocytes were plated at 4 × 10 per well on a 24-well plate coated with type I collagen. 5The hepatocytes were placed in each well, and 500 μl of DMEM (Gibco, USA, 11965) containing 10% FBS (Atlas Biologicals, USA, F0500A), 1x penicillin / streptomycin (Gibco, USA, 15140), and 20 mM HEPES (Gibco, USA, 15630) was used as the medium. The prepared hepatocytes were cultured for 24 hours in a humidified cell incubator at 37°C with 5% CO2.
[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), which were produced in chimeric mice with humanized liver tissue, and the HBVs were mixed with the antibody of the present invention and administered 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 Using dHCGM medium (DMEM + 10% FBS, NaHCO3 44mM, L-proline 15ug / ml, insulin 0.25ug / ml, dexamethasone 50nM, EGF 5ng / ml, Asc-2p 0.1mM, DMSO 2%), the virus and each antibody were mixed to a final volume of 100μl and reacted 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 ug / ml.
[0097] B. Virus inoculation 25 μl of 40% PEG (Sigma, USA, P1458) was mixed with 125 μl of dHCGM medium, and then the virus / antibody mixture prepared in A was added to prepare a final inoculation mixture of 250 μl. After removing the medium from the prepared cells, the inoculation mixture was added and then cultured for 24 hours.
[0098] C. Medium replacement and culture, preparation of analytical samples After inoculation with the virus, hepatocytes were cultured for a total of 12 days, with cell washing and medium replacement 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 amount of dHCGM medium was added. In the case of medium replacement on day 7, 300 μl and 30 μl of the existing culture medium were collected for the quantification of extracellular HBsAg and HBV DNA newly produced and excreted from the cells, respectively, and stored at -20°C until analysis.
[0099] After 12 days of incubation, both the cells and the culture medium were used for intracellular / extracellular virus quantitative analysis. The culture medium was collected separately for HBsAg measurement and HBV DNA measurement in the same manner as before, and the cells were collected by washing each well once with 500 μl of DMEM + 10% FBS, and then dissolving them by adding 500 μl of SMITEST (Medical & Biological Laboratories Co., Ltd.) solution. HBV DNA was extracted according to the manufacturer's (Medical & Biological Laboratories Co., Ltd.) protocol.
[0100] D. Sample Analysis HBV DNA quantification was performed by real-time PCR using TaqMan probes, TaqMan PCR Core Reagents (Life Technologies, USA), and the 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 Figs. 8A to 8D and 9A to 9D, categorized by virus genotype according to each measurement item.
[0104] First, a comparative analysis of the amount of intracellular HBV DNA according to the treatment concentration of each antibody was performed. Since the antibodies 1 and 2 of the present invention were selected based on their binding ability to the adr subtype of HBsAg, which is classified as genotype C, it was confirmed that both of them have strong neutralizing ability against genotype C. In a situation where the HB Ig used as a positive control showed a 400-fold reduction in the amount of HBV DNA compared to the anti-HER2 antibody used as a negative control, the antibody 2 of the present invention, which is 1 / 10 of the treatment amount, showed the same level of reduction in viral DNA in the 1 ug / ml treatment sample. In the case of the antibody 1 of the present invention, it was revealed that the antibody 1 maintains a relatively high level of neutralizing ability by showing a 100-fold reduction in HBV DNA even in a treatment with a low concentration of 0.1 ug / 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 group, HBIg, and against the D genotype, the neutralizing potency was maintained high even at low concentrations of 1 ug / ml (antibody 2 of the present invention) or 0.1 ug / ml (antibody 1 of the present invention) (Figures 8A to 8D).
[0105] It was revealed that the above-mentioned characteristics of the neutralizing activity of antibodies 1 and 2 of the present invention against the four genotypes A, B, C, and D were very similarly reflected in the quantitative results of extracellular HBsAg measured in the culture medium (Figures 9A to 9D).
[0106] To summarize the above results, in vitro neutralizing efficacy verification was conducted against four HBV genotypes, 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, the antibodies were tested using a World Health Organization (WHO) reference panel (1 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 2ug / ml by dispensing 100μl each 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 by treating with phosphate buffer solution (Teknova, USA, D5120) containing 3% bovine serum albumin (BSA). After washing again, 100μl each of 15 serum samples, which are HBsAg genotype panel, was dispensed and incubated at 37℃ for 90 minutes. At this time, each serum sample was appropriately diluted with phosphate buffer solution (Teknova, USA, D5120) containing 1% BSA so that it had an absorbance of about 0.8 to 1.2 at 450 / 620 nm. To detect HBsAg that reacted with the antibody, rabbit anti-HBV surface antigen antibody (Thermo Scientific, USA, PA1-73087) labeled with peroxidase was treated at 37°C for 60 minutes. The color development, reaction termination, and absorbance measurement were performed in the same manner as in Example 5. The reactivity of the two antibodies to each genotype HBV surface antigen was analyzed graphically using Excel (Microsoft, USA) (Figure 10).
[0109] As a result of the analysis, it was confirmed that both the antibodies 1 and 2 of the present invention bind well to 15 HBsAg samples. As mentioned above, this surface antigen sample is a serum actually produced from the blood of a patient, and covers seven genotypes from A to H, excluding type G, among all eight genotypes of HBV. In addition, in the case of types A, B, C, D, and F, which have multiple subgenotypes, two to three samples of the predominantly widespread subgenotypes and subtypes (serotypes) are included for each genotype, which means that the WHO HBV genotype panel used in the experiment represents the majority of HBV genotypes that are prevalent all over the world. Genotype G is missing from the panel, but in the case of G, no subgenotype has been reported so far, and it is classified as subtype (serotype) adw2, so the binding activity of the antibodies 1 and 2 of the present invention against type 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 are capable of binding to all genotypes of HBV that are prevalent worldwide and thereby exhibit neutralizing activity.
[0111] [Table 8]
[0112] Example 8: Investigation of binding characteristics against various drug-resistant viruses The binding characteristics between the antibodies 1 and 2 of the present invention and resistance mutations against lamivudine (LMV), adefovir (ADV), clevudine (CLV), and entecavir (ETV), which are HBV polymerase inhibitors widely used in patients with chronic hepatitis B, were confirmed using the same sandwich ELISA method as that used in Example 7. All resistance mutation 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, and were strains whose 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 as shown in Table 9.
[0113] Each of the HBV expression vectors thus prepared was transduced into Huh7 cell line cultured in a T75 flask (BD BioScience, 353136) using Lipofectamine2000 (Life Technologies, 11698019), and the cells were cultured for 3 days to produce the virus. The produced virus was concentrated using Centricon (Millipore, USA), and the amount of virus in each sample was compared with the amount of HBsAg using a Monolisa HBsAg Ultra (BioRad, 72346) ELISA kit, and the samples were appropriately diluted to have similar values and used in the experiment.
[0114] As a result of the experiment, it was revealed 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 FIG. 11). Here, the binding activity to ADV resistant viruses appears to be relatively low, but this is believed to be due to the fact that the production amount of the sample itself was slightly lower than that of the other samples.
[0115] This means that the antibodies 1 and 2 of the present invention can have binding activity and neutralizing efficacy not only against one type of drug-resistant virus used in the experiment, but also against most of the resistant viruses that occur against the drug. This is because, as can be seen from Table 9, the mutations that cause drug resistance in HBV are related to specific amino acid mutations in the reverse transcriptase (RT) domain of HBV polymerase, and such mutations occur very specifically for each drug. Such specific polymerase mutations are accompanied by specific mutations in HBsAg due to the characteristics of HBV sharing genes. For example, the rtM204I mutation in polymerase causes the W196L mutation in HBsAg, and the rtA181V mutation causes the L173F mutation in HBsAg. It was confirmed that the antibodies 1 and 2 of the present invention bind well regardless of these surface antigen mutations associated with drug resistance mutations.
[0116] Furthermore, each of the resistant viruses used in this experiment has 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 having mutations at the Q101R, K112R, I126S, L175S, A184V, and I185M positions of HBsAg.
[0117] [Table 9]
[0118] Example 9: Antigen-antibody binding affinity measurement To measure the antigen-antibody binding affinity of the antibody of the present invention, a surface plasmon resonance (SPR) assay was used. Specifically, the binding affinity between the antibody of the present invention and recombinant HBsAg (adr subtype, ProspecBio) was determined by surface plasmon resonance analysis using an analysis 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. HBsAg protein (50 μg / ml) diluted in 10 mM sodium acetate (pH 5.0) was directly immobilized on a CM5 research biosensor chip at about 500 RU using an amine coupling kit according to the manufacturer's guidelines and procedures. Non-reactive sites on the surface of the biosensor were blocked with ethanolamine. For reaction analysis, Biacore T200 control software, Biacore T200 evaluation software were used. The antibody of the present invention was diluted in HBS-EP buffer. In the course of the assay, all measurements were performed using the surface of the biosensor without immobilized HBsAg 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 with 3-fold serial dilutions, and using 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 reaction rate constant by the following equation: KD = Kd / Ka. The binding was calculated and recorded as a function of time and the reaction rate constant.
[0119] The experimental results are shown in Table 10, and confirmed that the antibody of the present invention has high binding affinity to HBsAg.
[0120] [Table 10]
Claims
1. A 3- to 38-mer epitope selected from amino acid positions 106-151 of the hepatitis B virus surface antigen (HBsAg).
2. The epitope according to claim 1, characterized in that the epitope is 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).
3. The epitope according to claim 1 or 2, characterized in that the epitope is located at amino acid positions 110-120 or 110-147 of the Hepatitis B virus surface antigen (HBsAg).
4. 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 the hepatitis B virus surface antigen (HBsAg).
5. The binding molecule of claim 4, wherein the epitope additionally includes amino acid position 147.
6. The binding molecule is 1×10 －９ 6. The binding molecule of claim 4 or 5, having a binding affinity of less than M.
7. The binding molecule according to claim 4 or 5, characterized in that the binding molecule is an antibody or a fragment thereof.
8. The binding molecule of claim 7, wherein the antibody is a human monoclonal antibody.
9. A polynucleotide encoding the epitope according to any one of claims 1 to 3.
10. An expression vector comprising the polynucleotide of claim 9.
11. A recombinant microorganism or virus transformed with the expression vector according to claim 10.
12. A method for producing an epitope comprising culturing the recombinant microorganism or virus of claim 11.
13. 4. An HBV vaccine composition comprising the epitope according to any one of claims 1 to 3 or a polynucleotide encoding the same.
14. 14. The vaccine composition of claim 13, further comprising a pharma- ceutically acceptable adjuvant.
15. A composition for detecting HBV, comprising the epitope according to any one of claims 1 to 3 or a polynucleotide encoding the same.