Human antibody that specifically binds to the hepatocyte receptor binding site of hepatitis B virus preS1 antigen and its use

Abstract

The present invention relates to an antibody that specifically binds to the receptor-binding motif of the Hepatitis B virus preS1 antigen, and uses thereof. Since the antibody of the present invention targets various genotypes of HBV, it can be usefully used in fields requiring neutralization of HBV or inhibition or treatment of infection with hepatitis B virus.

Description

[Technical field]

[0001] The present invention relates to a human antibody that specifically binds to the hepatocyte receptor-binding site of the Hepatitis B virus preS1 antigen, and uses thereof. [Background technology]

[0002] Hepatitis B virus (HBV) infection is one of the most serious and widespread public health problems. In 2016, there were approximately 290 million people chronically infected with HBV worldwide.

[0003] To date, a total of 10 HBV genotypes (AJ) have been identified. Genotypes A and D are distributed in Africa and Europe, genotypes B and C are mainly distributed in Asia, and genotype EJ often appears in Europe, America, and Asia. The most common genotype is AD, which accounts for approximately 90% of hepatitis B cases.

[0004] The HBV envelope contains three structurally related proteins, S (small), M (middle) and L (large). The S protein is the most abundant of these envelope proteins, the M protein is composed of preS2 and S, and the L protein is composed of preS1, preS2 and S.

[0005] Hepatocytes infected with HBV produce not only infectious HBV particles (virions) but also non-infectious spherical and filamentous subviral particles. These subviral particles are mostly composed of S protein and are generally produced in 1000 to 10000 times more than virions. These subviral particles are presumed to reduce virus-specific immune responses.

[0006] Unlike the S protein, the preS1 of the L protein is mainly present in infectious virions. In addition, amino acids 20-26 (AC, F genotype; D genotype, 9-15; E genotype, 19-25) of preS1 bind to the hepatocyte receptor NTCP (sodium taurocholate cotransporting polypeptide) to mediate HBV infection. This receptor binding site is highly conserved among HBV genotypes and therefore may be a target for the treatment and prevention of HBV infection.

[0007] Hepatitis B immune globulin (HBIG) is used for preventive medical treatment after accidental or perinatal HBV exposure. HBIG is produced by collecting serum with high anti-S protein antibody titers. However, considering the importance of the preS1 region in HBV infection, preS1-specific monoclonal antibodies (mAbs) that can inhibit HBV infection may be a promising method for the prevention and treatment of HBV infection. Clinically, the presence of anti-preS1 antibodies has been found to be associated with the recovery of hepatitis B patients. Recently, Li et al. demonstrated that a human monoclonal antibody (2H5-A14) that specifically binds to amino acids 31-38 (AC, F genotype), downstream of the hepatocyte receptor binding site (amino acids 20-26, AC, F genotype), in preS1 not only prevents HBV infection in a mouse model transplanted with human hepatocytes, but also has a therapeutic effect through its Fc-mediated effector function (Dan Li, Wenhui He, Ximing Liu, Sanduo Zheng, Yonghe Qi, Huiyu Li, Fengfeng Mao, Juan Liu, Yinyan Sun, Lijing Pan, Kaixin Du, Keqiong Ye, Wenhui Li, Jianhua Sui.2017.A potent human neutralizing antibody Fc-dependently reduces established HBV infections.eLife 6:e26738.DOI: https: / / doi.org / 10.7554 / eLife.26738).

[0008] A number of anti-preS1 mAbs with HBV neutralizing activity have been generated from mice immunized with HBV particles or recombinant preS1 antigen (Heermann K, Goldmann U, Schwartz W, Seyffarth T, Baumgarten H, Gerlich W.1984.Large surface proteins of hepatitis B virus containing the pre-s sequence.Journal of virology.52: 396-402; Pizarro JC, Vulliez-le Normand B, Riottot MM, Budkowska A, Bentley GA.2001.Structural and functional characterization of a monoclonal antibody specific for the preS1 region of hepatitis B virus.FEBS letters.509: 463-468; Kim JH, Gripon P, Bouezzedine F, Jeong MS, Chi SW, Ryu SE, Hong HJ.2015.Enhanced humanization and affinity maturation of neutralizing Anti-hepatitis B virus preS1 antibody based on antigen-antibody complex structure.FEBS letters.589: 193-200; Wi J, Jeong MS, Hong HJ.2017.Construction and characterization of an anti-hepatitis B virus preS1 humanized antibody that binds to the essential receptor binding site.Journal of microbiology and biotechnology 27, 1336-1344. etc.). However, most of the antibodies produced in this way have the problem that they do not neutralize all HBV genotypes.

Prior technical literature

Non-licensed literature

[0009]

Non-licensed literature 1

Non-licensed Document 4

[0010] Against this background, the inventors have made strenuous efforts to provide a monoclonal antibody that can bind to various HBV genotypes and is specific to the hepatocyte receptor binding site of preS1 for more efficient prevention and treatment, and as a result, have developed a human monoclonal antibody that binds to all genotypes A to F, which account for the majority of hepatitis B viruses, and binds to the hepatocyte receptor binding site to exhibit efficacy in inhibiting HBV infection, thereby completing the present invention. [Means for solving the problem]

[0011] An object of the present invention is to provide a novel antibody that specifically binds to the receptor binding site of the Hepatitis B virus preS1 antigen.

[0012] Another object of the present invention is to provide a method for producing the antibody.

[0013] Another object of the present invention is to provide a polynucleotide encoding the antibody, an expression vector containing the polynucleotide, and a host cell into which the vector has been introduced.

[0014] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating HBV infection, comprising said antibody.

[0015] Another object of the present invention is to provide a method for preventing or treating HBV infection using said antibody.

[0016] Another object of the present invention is to provide a method for providing information for diagnosing HBV infection, which comprises the step of detecting preS1 present in a biological sample isolated from an individual suspected of being infected with HBV through an antigen-antibody reaction using the antibody.

[0017] Another object of the present invention is to provide a composition for detecting HBV, which comprises the antibody.

[0018] Another object of the present invention is to provide a kit for detecting HBV, which contains the antibody. Effect of the Invention

[0019] The antibody of the present invention targets a sequence shared by various genotypes of HBV, is capable of binding to various genotypes of HBV, and has excellent binding ability to HBV, and therefore can be useful not only for detection and diagnosis of HBV infection, but also in fields where treatment is required. [Brief description of the drawings]

[0020] [Figure 1] Alignment of HBV preS1 N-terminal amino acid sequences (numbered according to genotype AF and HBV genotype A). Amino acids 20-26 are the essential receptor-binding motif of preS1 and are shaded gray. Sequences contained in the Bio-preS1-L peptide are underlined. [Diagram 2]The experimental results of isolating anti-preS1 monoclonal Fab from a human Fab synthetic library are shown in Fig. 2A. The input and output titers of phages for each panning round are shown. pfu stands for plaque-forming unit. Fig. 2B shows the amplification of positive polyclonal phages determined by indirect ELISA. The fourth round of panning, which amplified the most against preS1, was selected for isolating monoclonal Fab. Fig. 2C shows the result of indirect ELISA of AbS0 Fab phage with the best antigen-binding activity against Bio-preS1-L peptide among the positive Fab clones selected from the output of the fourth panning. [Diagram 3] The antigen-binding activity of AbS0 mAb, which was converted from AbS0 Fab to IgG1 and purified, was analyzed. Figure 3A shows SDS-PAGE of AbS0 purified under non-reducing (NR, 6%) and reducing (R, 10%) conditions. M indicates protein marker, HC indicates heavy chain, and LC indicates light chain. Figure 3B shows the antigen-binding activity of purified AbS0 mAb against HBV genotype (AD) preS1 assessed by ELISA. Figure 3C shows the affinity analysis of AbS0 by BLI using Octet RED384. Recombinant preS1 of HBV genotype A was prepared in two-fold serial dilutions (25, 12.5, 6.25, 3.125, 1.5625 nM). All values ​​are expressed as mean ± SEM from multiple wells. Figure 3D shows the results of FACS analysis to exclude the possibility of nonspecific binding of the AbS0 antibody to preS1, which showed that the AbS0 antibody did not bind to the human ovarian cancer cell line SKOV3, which expresses L1CAM but not preS1. [Figure 4]The virion-binding activity and in-vitro HBV neutralization activity of AbS0 against HBV genotype D were evaluated. Figure 4A shows the immunoprecipitation of HBV genotype D particles by AbS0. HzKR127-3.2 (ref: Kim JH et al. Enhanced humanization and affinity maturation of neutralizing anti-hepatitis B virus preS1 antibody based on antigen-antibody complex structure. FEBS Lett. 589: 193-200) was used as a positive control, and mouse IgG was used as a negative control. RC DNA indicates relaxed circular DNA, and DSL DNA indicates double-stranded linear DNA. Figure 4B shows the results of pre-incubation of HBV genotype D particles with AbS0 (10, 1, 0.1 μg) or mouse IgG (10 μg) and then added to cultured HepG2-NTCP cells, and the medium was replaced every 2 days to obtain cells 7 days after infection. HBV DNA was extracted from the resulting cells and subjected to Southern blot hybridization. [Diagram 5] The figures show the results of expression, purification, and SDS-PAGE of alanine-substituted mutants of amino acids 19-34 of GST-preS1 (aa 1-56)-strep for epitope mapping of AbS0 antibody. [Figure 6] This shows the results of indirect ELISA analysis of the binding ability of AbS0 antibody to alanine substitution mutants of amino acids 19-34 of GST-preS1 (aa 1-56)-strep. [Figure 7] To analyze the preS1 antigen-binding site (paratope) of the AbS0 antibody, the antigen-binding ability of antibody mutants in which each residue in the LCDR3 (Figures 7A and 7C) and HCDR3 (Figures 7B and 7D) of the AbS0 antibody was substituted with alanine was analyzed by indirect ELISA (7A, 7B) and quantitative ELISA (7C, 7D). [Figure 8]To analyze the preS1 antigen-binding site (paratope) of the AbS0 antibody, Ala33 in HCDR1 (Fig. 8A-C) and Ser50 in HCDR2 (Fig. 8D) of the AbS0 antibody were substituted with other residues, and the antigen-binding ability of these mutant antibodies was analyzed by indirect ELISA. [Figure 9] The antigen-binding ability of AbS1 and AbS0 antibodies to GST-preS1 (aa 1-56)-strep protein of genotype A, which has the receptor-binding motif of the conserved (NPLGFFP; WT) and another sequence (NPLGFLP; preS1-L25), was analyzed by indirect ELISA. [Figure 10] The antigen-binding ability of AbS1 antibody to GST-preS1 (aa 1-56)-strep proteins of genotypes E and F was analyzed by quantitative ELISA (FIG. 10A) and indirect ELISA (FIG. 10B). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The present application will be described in more detail below. Meanwhile, each description and embodiment disclosed in the present application may be applied to each different description and embodiment. That is, all combinations of the various elements disclosed in the present application belong to the scope of the present application. Moreover, the specific description described below is not considered to limit the category of the present application. Furthermore, numerous papers and patent documents are referenced and citations are displayed throughout the present specification. The disclosure contents of the cited papers and patent documents are incorporated by reference in their entirety into the present specification to more clearly explain the state of the art to which the present application pertains and the contents of the present invention.

[0022] Additionally, those of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein, and such equivalents are intended to be encompassed by the present invention.

[0023] Throughout this specification the conventional one-letter and three-letter codes for naturally occurring amino acids are used, and the amino acids referred to herein by abbreviations are written according to the IUPAC-IUB nomenclature system.

[0024] Alanine Ala, A Arginine Arg, R Asparagine Asn, N Aspartic acid Asp, D Cysteine ​​Cys, C Glutamic acid Glu, E Glutamine Gln, Q Glycine Gly, G Histidine, H Isoleucine, Ile Leucine (Leu), L Lysine (Lys), K Methionine Met, M Phenylalanine Phe, F Proline, Pro; P Serine, Ser; S Threonine Thr, T Tryptophan Trp, W Tyrosine (Tyr), Y Valine (Val), V

[0025] To achieve the object of the present invention, one embodiment provides a novel antibody that specifically binds to the Hepatitis B virus preS1 antigen.

[0026] In this application, the term "antibody" means a protein molecule that acts as a receptor that specifically recognizes an antigen, including an immunoglobulin molecule that is immunologically reactive with a specific antigen, and includes all of polyclonal antibodies, monoclonal antibodies, whole antibodies, and antibody fragments.

[0027] The term also includes chimeric antibodies, humanized antibodies, human antibodies, and bivalent or bispecific molecules (e.g., bispecific antibodies), diabodies, triabodies, and tetrabodies. The term further includes single chain antibodies, scaps, derivatives of antibody constant regions, and artificial antibodies based on protein scaffolds that retain the binding function to FcRn. A whole antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to a heavy chain by a disulfide bond. The whole antibody includes IgA, IgD, IgE, IgM, and IgG, and IgG subtypes include IgG1, IgG2, IgG3, and IgG4. The antibody fragment means a fragment that retains the antigen-binding function, and includes Fd, Fab, Fab', F(ab')2, and Fv. The Fd means a portion of the heavy chain contained in the Fab fragment. The Fab has a structure including light and heavy chain variable regions, a light chain constant region, and the first constant region of the heavy chain (CH1 domain), and has one antigen-binding site. Fab' is different from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibodies are produced by disulfide bonds formed between cysteine ​​residues in the hinge region of Fab'. Fv (variable fragment) refers to the minimum antibody fragment that has only the heavy chain variable region and the light chain variable region. In double disulfide Fv (dsFv), the heavy chain variable region and the light chain variable region are linked by disulfide bonds, and in single chain Fv (scFv), the heavy chain variable region and the light chain variable region are generally linked by a covalent bond via a peptide linker. Such antibody fragments can be obtained using proteolytic enzymes (for example, a whole antibody can be subjected to limited cleavage with papain to obtain Fab fragments, and then cleaved with pepsin to obtain F(ab')2 fragments), or can be produced through recombinant gene technology.

[0028] The antibody of the present invention may be a monoclonal antibody.

[0029] As used herein, the term "monoclonal antibody" refers to an antibody molecule of single molecular composition obtained from a population of substantially identical antibodies; such a monoclonal antibody displays a single binding specificity and affinity for a particular epitope.

[0030] Typically, immunoglobulins have heavy and light chains, each of which contains a constant region and a variable region (also known as a domain). The variable regions of the light and heavy chains contain three hypervariable regions called complementarity-determining regions (hereinafter referred to as "CDRs") and four framework regions. The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, starting from the N-terminus, and are further identified by the chain in which the particular CDR is located.

[0031] The antibody of the present invention may be a human antibody.

[0032] In the present application, the term "human antibody" means a molecule derived from human immunoglobulin, and all amino acid sequences constituting the antibody, including the complementarity determining regions and framework regions, are entirely composed of amino acid sequences of human immunoglobulin. Human antibodies, which are usually used to treat human diseases, have at least three potential advantages. First, they interact better with the human immune system, and can destroy target cells more efficiently, for example, by complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). Second, they have the advantage that the human immune system does not recognize them as foreign. Third, they have the advantage that their half-life in the human circulation is similar to that of naturally occurring antibodies, even when smaller and less frequent doses of the drug are administered.

[0033] Furthermore, when the antibody of the present invention as described above contains a constant region, it may contain a constant region derived from IgG, IgA, IgD, IgE, IgM, or a combination or hybrid of these.

[0034] In the present application, the term "combination" refers to a combination of a polypeptide encoding a single-chain immunoglobulin constant region of the same origin with a single-chain polypeptide of a different origin when forming a dimer or multimer. For example, a dimer or multimer can be formed from two or more constant regions selected from the group consisting of IgG, IgA, IgD, IgE, and IgM constant regions.

[0035] In the present application, the term "hybrid" means that a single-chain immunoglobulin heavy chain constant region contains sequences corresponding to immunoglobulin heavy chain constant regions of two or more different origins. An example of such a domain hybrid may be a domain hybrid consisting of 1 to 4 domains selected from the group consisting of CH1, CH2, CH3, and CH4 of IgG, IgA, IgD, IgE, and IgM.

[0036] In this application, the term "hepatitis B virus" is a term used interchangeably with HBV, which is the virus that causes hepatitis B when infected by an immune response against it.

[0037] In the present application, the term "preS1 antigen" refers to a protein encoded by the S gene, which is one of the four genes S, C, X, and P of HBV DNA. Specifically, the S gene is a gene that encodes a surface antigen, preceded by a Pre-S region, which is divided into a PreS1 region and a PreS2 region. These encode the surface proteins S (small), M (middle), and L (large) proteins. The S protein is encoded by the S gene, the M protein is composed of preS2 and S protein, and the L protein is composed of preS1, preS2, and S protein, and is mainly present in infectious virus particles (virions). Thus, preS1 constitutes the surface protein of the virus and is mainly present in infectious virions.

[0038] The antibodies provided by the present invention may be capable of binding to HBV of various genotypes, specifically, to all of genotypes A, B, C, D, E and F. In one embodiment, the antibodies provided by the present invention may bind to a receptor binding motif sequence shared by preS1 genotypes A, B, C, D, E and F, but are not limited thereto.

[0039] In the present application, the term "antibody that binds to the hepatitis B virus preS1 antigen" refers to an antibody that binds to preS1, a surface antigen of HBV, and exhibits neutralizing activity against HBV and / or inhibitory activity against HBV infection.

[0040] In one embodiment, the antibody provided by the present invention may bind to amino acids at specific positions in the preS1 antigen, which may correspond to positions 22, 23, and 25 of SEQ ID NO:19.

[0041] SEQ ID NO: 19 is merely a reference sequence for identifying the position to which the antibody of the present invention binds in any preS1 antigen, and the preS1 antigen of the present invention is not limited to SEQ ID NO: 19, and the corresponding positions of 22, 23, and 25 in any preS1 antigen protein based on SEQ ID NO: 19 can be confirmed through sequence alignment. For such sequence alignment, the Needleman-Wunsch algorithm, the Needle program in the EMBOSS package, multiple sequence alignment, etc., known in the art, can be used, but are not limited thereto.

[0042] The amino acid sequence of the preS1 antigen can be obtained from a publicly known database, and may include, for example, the sequence of SEQ ID NO: 17 or 18. The amino acids at positions 22, 23, and 25 may be the amino acids at positions 3, 4, and 6, respectively, in the amino acid sequence of SEQ ID NO: 17 or 18, but are not limited thereto.

[0043] The amino acids at positions 22, 23 and 25 of SEQ ID NO: 19 are the binding site of the antibody of the present invention, and the "epitope" of the preS1 antigen recognized by the antibody of the present invention can be expressed as the positions corresponding to positions 22, 23 and 25 of SEQ ID NO: 19. Meanwhile, the positions corresponding to positions 22, 23 and 25 of SEQ ID NO: 19 may be included in the binding site of the preS1 antigen to the hepatocyte receptor. Thus, the antibody of the present invention can inhibit the binding of the preS1 antigen to the hepatocyte receptor.

[0044] As an embodiment according to any one of the above-mentioned embodiments, the antibody may bind to leucine (Leu22) corresponding to position 22, glycine (Gly23) corresponding to position 23, and phenylalanine (Phe25) or leucine (Leu25) corresponding to position 25, based on SEQ ID NO: 19.

[0045] As an embodiment according to any one of the above-mentioned embodiments, the antibody may have tyrosine at position 8 of the LCDR3 sequence of SEQ ID NO: 5 and leucine at position 6 of the HCDR3 sequence of SEQ ID NO: 9 as a paratope.

[0046] As an embodiment according to any one of the above-mentioned embodiments, the antibody may be an antibody having, as a paratope, amino acid 1 of the HCDR2 sequence shown in SEQ ID NO: 8. The amino acid 1 of SEQ ID NO: 8 may be alanine or leucine.

[0047] In the present invention, the term "paratope" refers to the region or area on an antibody or antibody fragment to which the antibody or antibody fragment specifically binds to an antigen, i.e., where the antibody or antibody fragment physically contacts the antigen.

[0048] As an example of any one of the above-mentioned embodiments, the antibody may be an antibody that specifically binds to preS1, comprising a light chain variable region comprising a light chain CDR1 (LCDR1) of SEQ ID NO: 3, a light chain CDR2 (LCDR2) of SEQ ID NO: 4, and a light chain CDR3 (LCDR3) of SEQ ID NO: 5; and a heavy chain variable region comprising a heavy chain CDR1 (HCDR1) of SEQ ID NO: 7, a heavy chain CDR2 (HCDR2) of SEQ ID NO: 8, and a heavy chain CDR3 (HCDR3) of SEQ ID NO: 9.

[0049] As an example of any one of the above-mentioned embodiments, the antibody may be an antibody that specifically binds to preS1, comprising a light chain variable region comprising a light chain CDR1 (LCDR1) of SEQ ID NO: 3, a light chain CDR2 (LCDR2) of SEQ ID NO: 4, and a light chain CDR3 (LCDR3) of SEQ ID NO: 6; and a heavy chain variable region comprising a heavy chain CDR1 (HCDR1) of SEQ ID NO: 10, a heavy chain CDR2 (HCDR2) of SEQ ID NO: 11 or 12, and a heavy chain CDR3 (HCDR3) of SEQ ID NO: 13.

[0050] In one embodiment according to the above-mentioned embodiments, the light chain variable region of the antibody may be composed of the amino acid sequence set forth in SEQ ID NO: 1. Specifically, it may be composed of the amino acid sequence set forth in SEQ ID NO: 14, but is not limited thereto.

[0051] In one embodiment according to the above-mentioned embodiments, the heavy chain variable region of the antibody may be composed of the amino acid sequence set forth in SEQ ID NO: 2. Specifically, it may be composed of the amino acid sequence set forth in SEQ ID NO: 15 or 16, but is not limited thereto.

[0052] In one embodiment according to any one of the above-mentioned embodiments, the antibody may comprise, but is not limited to, a light chain variable region set forth in SEQ ID NO: 1 and a heavy chain variable region set forth in SEQ ID NO: 2. Specifically, the antibody may comprise, but is not limited to, a light chain variable region set forth in SEQ ID NO: 14; and a heavy chain variable region set forth in SEQ ID NO: 15 or 16.

[0053] In one embodiment of the present invention, an antibody that specifically binds to the preS1 region, including the light chain variable region of sequence number 14 and the heavy chain variable region of sequence number 15, was named AbS0, and it was confirmed that AbS0 exhibits excellent binding ability for all of HBV genotypes A, B, C and D.

[0054] Furthermore, it was confirmed that tyrosine at position 8 of the LCDR3 sequence of SEQ ID NO: 6, leucine at position 6 of the HCDR3 sequence of SEQ ID NO: 13, and serine at position 1 of the HCDR2 sequence of SEQ ID NO: 11 are the paratopes of the AbS0 antibody, and when serine at position 1 of HCDR2 in the AbS0 antibody was replaced with another amino acid (alanine), it was confirmed that an antibody having the HCDR2 sequence of SEQ ID NO: 12 showed superior binding ability compared to AbS0, and the antibody having the HCDR2 sequence of SEQ ID NO: 12 was named AbS1 antibody.

[0055] Such human antibodies that bind to preS1 of the present invention provide higher affinity and lower immunogenicity than existing antibodies and exhibit excellent neutralizing and infection-suppressing activity against HBV, and therefore the antibodies of the present invention can be used in any application in which antigen recognition against preS1 or neutralization and infection suppression of HBV can be useful.

[0056] Another aspect of the invention provides a method for producing the antibody.

[0057] The antibody of the present invention can be easily produced by known antibody production techniques. For example, a method for producing a monoclonal antibody can be carried out by producing a hybridoma using B lymphocytes obtained from an immunized animal (Koeher and Milstein, 1976, Nature, 256:495) or by using a phage display technique, but the antibody can be easily produced by other known antibody production techniques without being limited thereto.

[0058] The antibody library using the phage display technology is a method of expressing an antibody on the surface of a phage by obtaining an antibody gene from a B lymphocyte without producing a hybridoma. The phage display technology overcomes many of the existing difficulties associated with generating monoclonal antibodies by B cell immortalization. In general, the phage display technology includes the steps of: 1) inserting an oligonucleotide with a random sequence into a gene site corresponding to the N-terminus of the p or p of the coat protein of a phage; 2) expressing a fusion protein of a part of a natural coat protein and a polypeptide encoded by the oligonucleotide with the random sequence; 3) treating a substance capable of binding to the polypeptide encoded by the oligonucleotide; 4) eluting the antibody phage particles bound to the substance using a low pH or a molecule that is competitive with the binding; 5) amplifying the phage eluted by panning in a host cell; 6) repeating the above method to obtain a desired amount; and 7) determining the sequence of an active antibody from the DNA sequence of the phage clone selected by panning.

[0059] The method for producing the monoclonal antibody of the present invention may be carried out using a phage display technique. Those skilled in the art can easily carry out each step of the method for producing the monoclonal antibody of the present invention by referring to known phage display techniques, for example, methods known in the art such as those described in the papers of Barbas et al. (METHODS: A Companion to Methods in Enzymology 2:119, 1991 and J. Virol. 2001 Jul;75(14):6692-9) and Winter et al. (Ann. Rev. Immunol. 12:433, 1994). Phages used to construct the antibody library include, but are not limited to, fd, M13, f1, If1, Ike, Zj / Z, Ff, Xf, Pf1 or Pf3 phages as filamentous phages. In addition, vectors used for expressing heterologous genes on the surface of the filamentous phages include, but are not limited to, phage vectors such as fUSE5, fAFF1, fd-CAT1, or fdtetDOG, or phagemid vectors such as pHEN1, pComb3, pComb8, or pSEX, etc. In addition, helper phages used to provide wild-type coat proteins required for successful reinfection of recombinant phages for amplification include, but are not limited to, M13K07 or VSCM13.

[0060] Polynucleotides encoding the monoclonal antibodies or phage display clones of the present invention can be easily isolated and sequenced using conventional procedures. For example, oligonucleotide primers designed to specifically amplify the heavy and light chain coding regions from phage template DNA can be used. Once the polynucleotides are isolated, they can be placed into an expression vector, which can then be introduced into a suitable host cell to produce the desired monoclonal antibody from the transformed host cell (i.e., transformant). Thus, the method for producing the human monoclonal antibody of the present invention can be, but is not limited to, a method for producing a human monoclonal antibody, which includes amplifying a polynucleotide encoding the human monoclonal antibody in an expression vector comprising the polynucleotide encoding the human monoclonal antibody.

[0061] Another aspect of the invention provides a polynucleotide encoding the antibody, an expression vector comprising the polynucleotide, and a host cell into which the vector has been introduced.

[0062] The antibody is as described above.

[0063] The expression vector comprising the polynucleotide encoding the antibody provided by the present invention may be a vector capable of replicating and / or expressing the polynucleotide in eukaryotic or prokaryotic cells, including, but not limited to, mammalian cells (e.g., human, monkey, rabbit, rat, hamster, mouse cells, etc.), plant cells, yeast cells, insect cells, or bacterial cells (e.g., E. coli, etc.), and more specifically, may be a vector operably linked to a suitable promoter so that the nucleotide can be expressed in a host cell, and comprising at least one selection marker. For example, the polynucleotide may be introduced into a phage, plasmid, cosmid, mini-chromosome, virus, or retrovirus vector.

[0064] The expression vector containing a polynucleotide encoding the antibody may be an expression vector containing a polynucleotide encoding either the heavy chain or the light chain of the antibody, or an expression vector containing all of the polynucleotides encoding the heavy chain or the light chain.

[0065] The host cell into which the expression vector provided by the present invention is introduced may be, but is not limited to, bacterial cells, such as Escherichia coli, Streptomyces, Salmonella typhimurium, etc., yeast cells, fungal cells, such as Pichia pastoris, etc., insect cells, such as Drosophila and Spodoptera Sf9 cells, etc., animal cells, such as CHO (Chinese hamster ovary cells), SP2 / 0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), Bowes melanoma cells, HT-1080, BHK (baby hamster kidney cells), HEK (human embryonic kidney cells), PERC.6 (human retinal cells), etc., transformed with the expression vector, or plant cells.

[0066] In the present application, the term "introduction" refers to a method of transferring a vector containing a polynucleotide encoding the antibody into a host cell. Such introduction can be performed by various methods known in the art, such as calcium phosphate DNA coprecipitation, DEAE-dextran mediated transfection, polybrene mediated transfection, electric shock, microinjection, liposome fusion, lipofectamine and protoplast fusion. Transduction also refers to the delivery of a target substance into a cell by infection using virus particles. In addition, a vector can be introduced into a host cell by gene gun bombardment or the like. In the present invention, introduction can be used interchangeably with transformation.

[0067] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating HBV infection, comprising the antibody. Specifically, the composition may be for preventing or treating hepatitis B.

[0068] In this application, the term "hepatitis B" refers to a disease in which inflammation of the liver occurs due to an immune response caused by HBV infection. The antibody of the present invention exhibits excellent neutralizing ability and infection suppressing ability against HBV, and therefore can be used for the prevention or treatment of hepatitis B.

[0069] In this application, the term "prevention" means any action that suppresses or delays the onset of HBV infection by administering the composition, and the term "treatment" means any action that ameliorates or beneficially alters the symptoms of HBV infection by administering the composition.

[0070] The pharmaceutical composition may further comprise a pharma- ceutically acceptable carrier.

[0071] In this application, the term "pharmaceutical acceptable carrier" refers to a carrier or diluent that does not stimulate the organism and does not inhibit the biological activity and properties of the administered compound. Pharmaceutical carriers acceptable for compositions formulated into liquid solutions include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components that are sterile and suitable for the body, and other common additives such as antioxidants, buffers, bacteriostatic agents, etc. can be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants can be added to formulate the compositions into injection forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets.

[0072] The pharmaceutical composition may be in various oral or parenteral dosage forms. When formulated, it is prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, or surfactant. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations are prepared by mixing one or more compounds with at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate, talc, etc. are also used. Liquid preparations for oral administration include suspensions, internal solutions, emulsions, syrups, etc., and may contain various excipients such as wetting agents, sweeteners, flavorings, preservatives, etc. in addition to water and liquid paraffin, which are commonly used simple diluents. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspension solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. Suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin, etc.

[0073] The pharmaceutical composition may have any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, suspensions, oils, lyophilized preparations, and suppositories.

[0074] The compositions of the present invention are administered in a pharma- ceutically effective amount.

[0075] In this application, the term "pharmacologically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment, and the level of an effective dose can be determined by factors including the type and severity of the individual, age, sex, type of cancer, drug activity, sensitivity to the drug, administration time, administration route and excretion rate, treatment duration, concurrently used drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. It may also be administered singly or multiple times. It is important to administer an amount that provides maximum effect at a minimum amount without side effects, taking into consideration all of the above factors, and this can be easily determined by those skilled in the art.

[0076] Another aspect of the invention provides a method of preventing or treating HBV infection using the antibodies.

[0077] In particular, the method may comprise administering the antibody to an individual suspected of being infected with HBV.

[0078] The method is a method for preventing or treating HBV infection comprising the step of administering to an individual infected with or infected with HBV a pharmaceutical composition further comprising an antibody and a pharma- ceutical acceptable carrier, the pharma-ceutical acceptable carrier being as described above.

[0079] The individual includes, without limitation, mammals, birds, etc., including cows, pigs, sheep, chickens, dogs, humans, etc., and includes individuals whose HBV infection is treated by administering the composition of the present invention. In this case, the antibody may be administered singly or multiple times in a pharma- ceutically effective amount. In this case, the antibody may be administered in the form of a liquid, powder, aerosol, capsule, enteric coated tablet or capsule, or suppository. The route of administration includes, but is not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration, etc.

[0080] The method may also be used to prevent or treat hepatitis B, specifically, but is not limited to, for the treatment of HBV infection with any one or more selected from HBV genotypes A, B, C, D, E and F.

[0081] Another aspect of the present invention is the use of the antibody in the manufacture of a medicament for preventing or treating HBV infection. The medicament may also be used for the prevention or treatment of hepatitis B, and may be used to treat HBV infection of any one or more selected from HBV genotypes A, B, C, D, E, and F, but is not limited thereto. The antibody and the prevention or treatment of HBV infection are as described above.

[0082] Another aspect of the present invention provides a method for providing information for diagnosing HBV infection, comprising the step of detecting preS1 present in a biological sample isolated from an individual suspected of being infected with HBV through an antigen-antibody reaction using the antibody.

[0083] The method may be a method for diagnosing HBV infection, and may be used for diagnosing Hepatitis B.

[0084] The antibody and HBV are as described above.

[0085] The method for providing information for the diagnosis of HBV involves reacting the preS1-specific antibody of the present invention with a biological sample isolated from an individual suspected of being infected with HBV, and detecting the formation of an antigen-antibody complex to detect preS1, thereby providing information for the diagnosis of HBV infection.

[0086] Specifically, the method may include the steps of: (a) treating a biological sample isolated from an individual suspected of being infected with HBV with the antibody and detecting preS1 through an antigen-antibody reaction; and (b) comparing the level of preS1 detected in (a) with a control group, and if the level of preS1 is higher than that of the control group, determining that the patient is infected with HBV.

[0087] As used herein, the term "biological sample" includes, but is not limited to, tissues, cells, whole blood, serum, plasma, tissue autopsy samples (brain, skin, lymph nodes, spinal cord, etc.), cell culture supernatants, ruptured eukaryotic cells, and bacterial expression systems, etc. These biological samples, either manipulated or unmanipulated, can be reacted with the antibodies of the present invention to determine the presence of preS1 or HBV infection.

[0088] In the present application, the term "antigen-antibody complex" refers to a conjugate of preS1 antigen in a sample and the antibody according to the present invention that recognizes the antigen-antibody complex, and the formation of such an antigen-antibody complex can be detected by any method selected from the group consisting of a colorimetric method, an electrochemical method, a fluorimetric method, a luminometry method, a particle counting method, a visual assessment method, and a scintillation counting method. However, the method is not necessarily limited to these, and various applications and uses are possible.

[0089] In the present invention, various labels can be used to detect the antigen-antibody complex, specific examples of which can be selected from the group consisting of enzymes, fluorescent substances, ligands, luminescent substances, microparticles, and radioisotopes, but are not necessarily limited to these.

[0090] Enzymes used as detection labels include acetylcholinesterase, alkaline phosphatase, β-D-galactosidase, horseradish peroxidase, and β-lactamase, while fluorescent substances include fluorescein, Eu 3+ ,EU 3+The ligand includes a biotin derivative, etc.; the luminescent material includes an acridinium ester, an isoluminol derivative, etc.; the fine particles include gold colloid, colored latex, etc.; the radioisotope includes 57 Co, 3 H, 125 I, 125 I-Bolton Hunter Reagent, and the like, but are not limited to these.

[0091] Specifically, the detection method may be one using an enzyme-linked immunosorbent assay (ELISA) for the antigen-antibody complex. The enzyme-linked immunosorbent assay (ELISA) includes various ELISA methods, such as direct ELISA using a labeled antibody that recognizes the antigen attached to a solid support, indirect ELISA using a labeled secondary antibody that recognizes the capture antibody in the antibody complex that recognizes the antigen attached to a solid support, direct sandwich ELISA using another labeled antibody that recognizes the antigen in the antibody-antigen complex attached to a solid support, and indirect sandwich ELISA using a labeled secondary antibody that recognizes the antibody after reacting with another antibody that recognizes the antigen in the antibody-antigen complex attached to a solid support.

[0092] The antibodies may carry a detectable label, or if they do not, the monoclonal antibodies may be captured and identified with another antibody that carries a detectable label.

[0093] Another aspect of the present invention provides a composition for detecting HBV, comprising the antibody of the present invention. The antibody is as described above.

[0094] The detection of HBV may be detection of preS1 of HBV.

[0095] In one embodiment, the composition may further include a tool, device, or reagent included in a typical detection composition for detecting the presence or absence of an antigen protein in a biological sample using an antibody, such as a tool for collecting a sample to be tested or applying a detection reagent, or a reagent or device for detecting antigen-antibody binding.

[0096] In another aspect, the present invention provides a kit for detecting HBV, which comprises the above-mentioned antibody. The detection of HBV is as described above.

[0097] In one embodiment, the kit may be in the form of a hepatitis diagnosis kit. The composition and hepatitis B diagnosis are as described above.

[0098] Another aspect of the present invention is use of the antibody for producing a composition for detecting HBV. The antibody and HBV are as described above.

[0099] The present invention will be described in more detail with reference to the following examples and experimental examples. However, these examples and experimental examples are provided for illustrative purposes only and the scope of the present invention is not limited to these examples and experimental examples.

[0100] Example 1: Production of preS1 antigen GST-preS1(1-119), GST-preS1(1-56), and preS1(1-119) of HBV genotype C were prepared using the method disclosed in Biotechnology letters. 17: 871-876 (Kim HS, Hong HJ. 1995. Efficient expression, purification and characterization of hepatitis B virus preS1 protein from Escherichia coli). In addition, biotinylated synthetic preS1 peptide (biotin-SGSGSNPLGFFPDHQLDP, Bio-preS1-L peptide) containing NTCP binding motif (20-NPLGFFP-26) was prepared by AnyGen, Inc. (South Korea). The recombinant preS1 antigen and Bio-preS1-L peptide thus prepared were used as antigens for panning of the antibody library. To analyze the binding of the isolated antibodies to preS1 antigens of various HBV genotypes, GST-preS1(1-56)-strep tag of HBV genotype AF was expressed in E. coli and purified. The sequence of preS1(1-56) of genotype AF is shown in Figure 1.

[0101] Example 2: Selection of Fabs specific to the hepatocyte receptor binding site of preS1 from a phage-displayed human synthetic Fab library To isolate Fabs specific for the hepatocyte receptor binding site of HBV preS1, a human synthetic Fab phage display library was panned against the four preS1 antigens.

[0102] Specifically, a human synthetic Fab library (diversity 1.35×10) was prepared by standard panning procedures for 1, 2, 3, and 4 rounds against the four preS1 antigens, GST-preS1(1-119), GST-preS1(1-56), Bio-preS1-L peptide, and preS1(1-119) in Example 1, respectively. 9) were panned. Each preS1 antigen was coated onto an immunotube (streptavidin-coated plate in the case of Bio-preS1-L peptide) and stored overnight at 4°C. The antibody library phages were incubated with the antigen, and unbound phages were washed with 0.1% PBST (0.1% Tween 20 in PBS). Bound phages were eluted with 10 μg / ml trypsin solution for 30 min at 37°C. E. coli TG1 cells were infected with the eluted phages, grown at 37°C for 1 h, and incubated at 37°C overnight. The amplified phages were used for the next round of panning, with increasingly more extensive washing for each round. The input and output phage numbers for each round were calculated by dilution and CFU (Figure 2A).

[0103] To determine whether antibody clones against the preS1 antigen were amplified as the panning proceeded, indirect ELISA was performed on the output phages from each round, and it was confirmed that Fab clones binding to the recombinant preS1 antigen and Bio-preS1-L peptide were amplified from the third panning onwards (Figure 2B).

[0104] Therefore, to select the Fab clones with the highest activity against the preS1 antigen, after the fourth round of panning, 188 phage Fab clones were randomly selected and subjected to indirect ELISA using the Bio-preS1-L peptide common to the preS1 antigens of HBV genotype (AD) (Fig. 2C).

[0105] Specifically, after four rounds of panning, a total of 188 colonies were randomly selected and cultured, and then infected with helper phages to obtain monoclonal Fab phages. The phages were then subjected to indirect ELISA to confirm positive clones. In the indirect ELISA, 100ng of BSA and Bio-PreS1-L peptide were used as immobilized antigens, and horseradish peroxidase (HRP)-conjugated anti-M13 (1:5000 v / v, GE Healthcare) was used as the secondary antibody. In the quantitative ELISA, anti-M13 antibody (100ng / well, GE Healthcare) and anti-human kappa HRP (1:2000 v / v, Novex) were used as immobilized and secondary antibodies, respectively.

[0106] As a result of the analysis, it was confirmed that the AbS0 Fab phage showed the highest antigen-binding ability to the Bio-PreS1-L peptide, and also showed high binding ability to the preS1 (1-119) antigen of Example 1 (Figure 2C).

[0107] Example 3: Conversion of AbS0 Fab to human IgG1 and analysis of antigen-binding ability The AbS0 Fab was converted to human IgG1 and expressed in HEK293F cells, and the protein was purified from the culture supernatant.

[0108] 3-1 Conversion of AbS0 Fab to human IgG1 To convert the selected Fab into an IgG1 form, the sequences of the heavy chain variable region (VH) and kappa light chain variable region (VK) were amplified by PCR and then linked to IgG heavy and light chain leader sequences, respectively, using recombinant PCR. The resulting VH and VK sequences were subcloned into the EcoRI-ApaI and HindIII-BsiWI sites of the pdCMV-dhfrC expression plasmid containing the human Cγ1 and Cκ genes to produce the pdCMV-dhfrC-AbS0 expression plasmid.

[0109] 3-2 Antibody purification The expression plasmid prepared in 3-1 was introduced into 30 mL of HEK293F cells at a ratio of 1:4 (30 μg:120 μg) using polyethyleneimine (PEI). The transformed cells were cultured for 7 days, and affinity chromatography was performed using Protein A as the culture supernatant. The antibody concentration was then determined using a UV-Vis Spectrophotometer, and the purity of the purified antibody was verified by SDS-PAGE (Figure 3A).

[0110] 3-3 Analysis of binding activity To analyze the antigen-binding activity of purified AbS0 mAb to preS1 antigens of various HBV genotypes, each well was coated with GST-preS1(1-56)-strep (30 nM / well) of genotype AD (Figure 1), and then reacted with serially diluted AbS0 antibody. Bound AbS0 antibody was detected using anti-human IgG Fc-HRP (1:10,000 v / v, Jackson) as the secondary antibody.

[0111] As a result, it was confirmed that the purified AbS0 antibody bound well to all recombinant preS1 antigens of genotype AD (Figure 3B), confirming that the AbS0 antibody exhibited binding activity to the preS1 antigen of genotype AD.

[0112] 3-4 Analysis of antigen-binding affinity of AbS0 Antibody affinity was determined by Bio-Layer Interferometry using Octet RED384 (ForteBio). Specifically, anti-human Fc-coated biosensor tips (AHC, ForteBio) were activated by stirring a 96-well microtiter plate (Greiner Bio-One) at 1000 rpm for 20 min in 0.1% PBA (0.1% BSA in PBS). AbS0 antibody (1 μg / ml, 200 μl) was added and captured for 10 min, and washed with 0.1% PBA for 2 min. HBV genotype A GST-preS1(1-56)-strep antigen was subjected to 2-fold serial dilution (25, 12.5, 6.25, 3.125, 1.5625 nM) with 0.1% PBA, and the antibody bound to the chip was subjected to a 5 min association step and a 10 min dissociation step, respectively. Measurements were corrected for baseline drift by excluding the value of a control sensor (antibody-captured AHC sensor) exposed only to running buffer without antibody. The operating temperature was maintained at 30°C. Data were analyzed using ForteBio data analysis software version 7.1 with a 1:1 interaction model (fitting global, Rmax unlinked by sensor).

[0113] As a result, the affinity of the AbS0 antibody was confirmed to be 0.5 nM.

[0114] [Table 1]

[0115] 3-5 Evaluation of antigen-binding specificity of AbS0 antibody To exclude the possibility of nonspecific binding of the AbS0 antibody to the preS1 antigen, flow cytometry was performed using the human ovarian cancer cell line SKOV3 (Min, J.-K. et al., Clin. Cancer Res. 16(14);3571-80, 2010), which expresses L1CAM but not preS1.

[0116] Specifically, cultured SKOV3 cells (2x10 5 ) AbS0 or Ab417 (L1CAM-specific human antibody: Cho S.et al., MABS 8(2), 414-425, 2016) was added as a positive control in 100μg (in 0.1% PBA) and incubated at 4℃ for 1 hour, then washed twice with 0.1% PBA. Anti-human IgG Fc-fluorescein isothiocyanate (FITC) conjugate (1:2000v / v, BD Pharmingen) was added to the cells and incubated at 4℃ for 1 hour, then propidium iodide (1:200v / v, Sigma) was added and the cells were analyzed using a FACSCalibur (Becton Dickinson). The experiment showed that Ab417 bound to SKOV3, but AbS0 antibody did not bind to SKOV3 cells (Figure 3D).

[0117] Through this, it was confirmed that the AbS0 antibody specifically binds to the preS1 antigen.

[0118] Example 4: Evaluation of HBV neutralizing activity of AbS0 antibody 4-1 Analysis of immunoprecipitation To confirm whether AbS0 antibody binds to HBV virions, immunoprecipitation analysis was performed using HBV genotype D.

[0119] The existing anti-preS1 humanized antibody HzKR127-3.2, which has been shown to neutralize HBV, was used as a positive control for HBV genotype D, and mouse IgG was used as a negative control. After immunoprecipitation, viral DNA was extracted and measured by Southern blotting.

[0120] Specifically, for the production of HBV particles of HBV genotype D, HepG2 cells were cultured in 6-well plates at 6Х10 5 The cells were seeded at a density of 100 μg / ml and cultured at 37°C. The next day, HepG2 cells were transfected with pHBV1.2 (HBV genotype D) and incubated for 4 days. The culture supernatants were collected and incubated with 1 μg / ml AbS0, HzKR127-3.2 (positive control), or mouse IgG (negative control) at 4°C overnight, followed by immunoprecipitation with 20 μl of protein A beads at 4°C for 6 h. The immunoprecipitated complexes were washed three times with PBS, and viral DNA was detected as follows: to remove the transfected plasmid DNA, the immune complexes were treated with DNase I (Sigma) and mung bean nuclease (Takara, Kusatsu, Shiga, Japan) for 20 min at 37°C. To prepare core-associated HBV DNA, the precipitated immune complexes were digested with Proteinase K (20 mg / ml, Roche) in the presence of 0.5% SDS for 2–3 h at 37°C, and HBV DNA was extracted with phenol / chloroform / isoamyl alcohol (25:24:1) (Sigma) and purified with ethanol and 3 M sodium acetate. The purified DNA was separated on a 1% agarose gel, and HBV DNA was detected by Southern blotting.

[0121] As a result, HBV DNA in the relaxed circular (RC) and double-stranded linear (DSL) forms was detected together with AbS0 or HzKR127-3.2 antibody precipitates, but HBV DNA was not detected by mouse IgG (FIG. 4A).

[0122] Through this, it was confirmed that the AbS0 antibody binds to HBV genotype D virions.

[0123] 4-2 Evaluation of HBV neutralizing activity of AbS0 To test whether AbS0 could neutralize HBV infection, genotype D HBV particles were pre-incubated with different concentrations of the antibody and then added to HepG2-NTCP cells overexpressing the cellular receptor NTCP.

[0124] Specifically, the HBV neutralizing activity of AbS0 was analyzed using a HepG2 cell line overexpressing NTCP (referred to as HepG2-NTCP). HepG2-NTCP cells were cultured in 6-well plates at 6x10 5 The cells were seeded at 100 ng / ml and cultured at 37°C. The next day, HepG2-NTCP cells were infected with genotype D HBV particles (~2000 viral genome equivalents per cell) in primary hepatocyte maintenance medium (PMM) containing 4% polyethylene glycol (PEG) and 2.5% DMSO.

[0125] For neutralization analysis, HBV particles were preincubated with diluted AbS0 antibody (10, 1, 0.1 μg) or mouse IgG (10 μg) at room temperature for 1 h, respectively, and then added to cultured HepG2-NTCP cells, and the cells were cultured in PMM medium containing 2.5% DMSO, which was replaced every 2 h every day for 7 days. Intracellular HBV DNA was extracted from infected HepG2-NTCP cells and subjected to Southern blotting.

[0126] As a result, it was confirmed that AbS0 antibody showed HBV neutralizing effect in a dose-proportional manner, whereas mouse IgG showed no neutralizing effect (FIG. 4B).

[0127] Through this, it was confirmed that AbS0 has the ability to neutralize HBV infection.

[0128] The above results suggest that the novel antibody AbS0 of the present invention can bind to all HBV genotype AD and effectively neutralize the virus of the genotype, and is effective in preventing infection with the virus of the genotype and treating patients with hepatitis caused by the virus infection. In addition, it also suggests that the presence or absence of infection with the HBV genotype AD can be specifically detected.

[0129] The light chain variable region of the novel antibody AbS0 of the present invention is shown in SEQ ID NO:14, and the heavy chain variable region is shown in SEQ ID NO:15.

[0130] Example 5: Determination of fine epitopes of AbS0 antibody To understand the mechanism of AbS0 antibody's HBV neutralization efficacy, we precisely determined the binding site (fine epitope) of AbS0 within preS1 and analyzed whether the amino acid sequence of this fine epitope is conserved among HBVs.

[0131] 5-1 Alanine scanning mutagenesis of PreS1(19-34) The AbS0 antibody was discovered by panning a human antibody library using the preS1(20-32) peptide of genotype C. We assumed that the antibody binds to the preS1(20-32) region. To analyze which residues the AbS0 antibody binds to, we created mutants in which each residue of preS1(19-34) of genotype C was replaced with alanine, and analyzed the antigen-binding ability of the AbS0 antibody to each mutant.

[0132] Specifically, DNA was synthesized by substituting each residue of preS1(19-34) with alanine using recombinant PCR with primers containing the mutant sequence, and then digested with BamHI and EcoRI (NEB) restriction enzymes, and subcloned into the BamHI-EcoRI site of the pGST-preS1(1-56)-strep expression plasmid containing the wild type preS1 sequence to prepare an expression plasmid having alanine substitution mutants instead of wild type preS1(1-56). Then, to express each mutant, recombinant E. coli DH5α was cultured at 18°C ​​for 19 hours in a medium containing 0.2 mM IPTG. Then, the cells were disrupted by sonication, and the GST-preS1(1-56)-strep protein was purified by affinity chromatography using glutathione beads (Genscript), and the purity of the purified protein was confirmed by 12% SDS-PAGE (Figure 5).

[0133] 5-2. Analysis of antigen-binding ability of antibodies against alanine-substituted mutants of preS1 The antigen-binding ability of AbS0 antibody against each alanine substitution mutant was analyzed by indirect ELISA (FIG. 6).

[0134] Specifically, each well was coated with mutated GST-preS1(1-56)-strep antigen (200 ng) and then incubated with serially diluted AbS0 antibodies. Bound AbS0 antibodies were detected with anti-human IgG Fc-HRP (1:10,000 v / v, Thermo) as the secondary antibody.

[0135] As a result, it was confirmed that the L22A, G23A, and F25A mutants completely lost antibody binding activity compared to the wild-type preS1 antigen, indicating that the AbS0 antibody directly binds to Leu22, Gly23, and Phe25 of the preS1 antigen.

[0136] Example 6: Determination of epitope binding site (paratope) of AbS0 antibody and identification of mutants with increased affinity To identify amino acid residues in the CDRs of AbS0 antibody that directly bind to antigen, the CDR residues were subjected to alanine scanning mutagenesis and site-directed mutagenesis, and the antigen-binding ability of the mutants was then analyzed.

[0137] 6-1. Alanine scanning mutagenesis of HCDR3 and LCDR3 of AbS0 antibody To prepare mutants in which each residue in HCDR3 (V95, I96, Y97, S99, L100 based on SEQ ID NO: 15) and LCDR3 (S91, Y92, S93, S94, Y96 based on SEQ ID NO: 14) of AbS0 antibody was replaced with alanine, VH or VL genes were synthesized by recombinant PCR using primers containing the mutant sequences. In the case of HCDR3 mutants, each synthesized VH gene was cleaved with EcoRI and ApaI restriction enzymes and then subcloned into the EcoRI-ApaI site of an expression vector (pCMV-dhfr-AbS0H) containing the heavy chain gene of AbS0 antibody. In the case of LCDR3 mutants, each synthesized VL gene was cleaved with HindIII and BsiWI restriction enzymes and then subcloned into the HindIII-BsiWI site of an expression vector (pCMV-dhfr-AbS0k) containing the light chain gene of AbS0 antibody. A total of 10 expression plasmids for alanine substitution mutants were constructed as follows.

[0138] [Table 2]

[0139] To produce each mutant, the expression plasmid was mixed with polyethyleneimine (PEI) at a ratio of 1:4 (30μg:120μg) and then introduced into HEK293F cells. The transformed cells were cultured for 7 days to express the mutant antibodies, and the culture supernatant was collected and analyzed for their antigen binding ability by indirect ELISA. Specifically, each well was coated with recombinant GST-preS1(1-56)-strep antigen (100ng) of HBV genotype C, and serially diluted AbS0 antibody or AbS0 mutant was bound to it, followed by addition of anti-human IgG Fc-HRP (1:10,000v / v, Thermo) as the secondary antibody for reaction.

[0140] The concentration of AbS0 antibody or AbS0 variants used in the indirect ELISA was determined by quantitative ELISA. Each well was coated with anti-human IgG kappa antibody (100 ng, Thermo), and serially diluted AbS0 antibody or variants were bound to the antibody and reacted with anti-human IgG Fc-HRP (1:10,000 v / v, Thermo) as a secondary antibody.

[0141] As a result, HCDR3 mutant L100A and LCDR3 mutant Y96A had no or very low antigen-binding activity, while the other six mutants (HCDR3 V95A, S99A; LCDR3 S91A, Y92A, S93A, S94A) showed the same antigen-binding activity as wild-type AbS0 (Figure 7). CDR3 mutants I96A and Y97A were not expressed and therefore could not be analyzed. The results confirmed that HCDR3 Leu100 and LCDR3 Tyr96 of AbS0 are highly involved in antigen binding.

[0142] 6-2 Site-directed mutations in HCDR1 and HCDR2 of AbS0 antibody In general, the amino acid at position 33 of HCDR1 and the amino acid at position 50 of HCDR2 in antibodies are highly diverse and different from antibody to antibody, and are known to be involved in antigen binding. Therefore, in the case of AbS0 antibody, site-directed mutagenesis was performed to analyze whether the residues at positions 33 and 50 are involved in antigen binding. (Positions 33 and 50 are the positions described based on SEQ ID NO: 15.)

[0143] In the case of AbS0 antibody, the 33rd amino acid of HCDR1 was alanine, and to examine whether this residue was involved in antigen binding, the alanine was replaced with 8 other residues (S, P, V, T, G, D, L, N), and the 50th amino acid was serine, and replaced with 6 other residues (A, Y, E, F, V, P). To prepare mutant genes, recombinant PCR was performed using degenerate primers, and the synthesized VH gene was cleaved with EcoRI and ApaI restriction enzymes, and then subcloned into the EcoRI-ApaI site of pdCMV-dhfr-AbS0 to obtain expression plasmids for the mutants. Analysis of the base sequence confirmed that a total of 14 mutants were obtained. Each expression plasmid was introduced into HEK293F cells in the same manner as in 6-1, and the culture supernatant obtained after 7 days of culture was analyzed by indirect ELISA and quantitative ELISA as in 6-1.

[0144] As a result, in the case of the heavy chain HCDR1 amino acid at position 33, substitution of alanine with asparagine (A33N, FIG. 8A), leucine (A33L, FIG. 8B), or aspartic acid (A33D, FIG. 8C) did not show antigen-binding ability, and the mutant substituted with glycine (A33G, FIG. 8C) also showed a significantly reduced antigen-binding ability compared to the wild-type AbS0 antibody. This result indicates that the heavy chain HCDR1 amino acid at position 33 is involved in antigen binding. On the other hand, the three mutants A33S (FIG. 8A), A33V, and A33P (FIG. 8B) showed antigen-binding ability similar to that of the wild-type antibody.

[0145] In the case of mutants of serine 50 of HCDR2, the S50A mutant showed slightly increased antigen binding activity compared to AbS0 antibody, and the antigen binding activity of the S50Y mutant was greatly reduced, while the S50F and S50E mutants showed no antigen binding activity (Figure 8D). The antigen binding ability of the S50V and S50P mutants could not be analyzed because their expression was not detected. The results indicate that the 50th residue of HCDR2 plays an important role in antigen binding, and that alanine is more optimal for antigen binding than serine.

[0146] To analyze whether glutamine at position 52a of HCDR2 of AbS0 antibody (the fourth amino acid based on SEQ ID NO:11) is also involved in antigen binding, mutants were prepared in which glutamine was replaced with four other amino acids (Y, A, F, M) and transiently expressed in HEK293F cells. As a result, the mutants (Q52aA, A52aF, A52aM) showed the same antigen binding ability as AbS0 antibody, but the expression of the Q52aY mutant was not detected and could not be analyzed. From this result, it was inferred that amino acid 52a of HCDR2 is not involved in the interaction with the preS1 antigen.

[0147] Based on the results of Examples 5 and 6, it was estimated that in the AbS0 antibody, Ala33 of HCDR1, Ser50 of HCDR2, Leu100 of HCDR3, and Tyr96 of LCDR3 directly bind to Leu22, Gly23, and Phe25 of preS1. Furthermore, through the experiment of Example 6, a mutant S50A with improved antigen-binding ability compared to AbS0 was obtained, and this mutant antibody was named "AbS1."

[0148] Example 7: Analysis of antigen-binding properties of AbS1 antibody 7-1 Comparison of the binding ability of AbS1 and AbS0 antibodies to preS1 antigens of genotypes A and C Since recombinant GST-preS1(1-56)-strep antigen of HBV genotype C was used to analyze the antigen-binding ability of AbS0 antibody and mutants in Example 6-2, in order to analyze whether AbS1 antibody has higher binding ability than AbS0 to preS1 antigens of other genotypes, indirect ELISA was performed against recombinant GST-preS1(1-56)-strep antigen of genotype A. As a result, it was confirmed that AbS1 exhibits higher antigen-binding ability to preS1 of genotype A than AbS0 (FIG. 9).

[0149] 7-2 Analysis of the binding ability of AbS1 antibody to preS1 antigens with other receptor-binding motifs Because the epitope of the AbS1 antibody (Leu22, Gly23, and Phe25 of preS1) is included in the HBV receptor-binding motif, we analyzed the sequences of the receptor-binding site (aa 20-26, genotype A) of the preS1 gene of 9639 genes (as of April 30, 2021) registered in the HBV database (https: / / hbvdb.lyon.inserm.fr / HBVdb / ) as of April 30, 2021, to estimate whether the AbS1 antibody can bind to a wide range of HBVs present on the planet. As a result, 96.6% of the preS1 sequences had the NPLGFFP sequence as a receptor-binding motif, and 2.6% had NPLGFLP, which has leucine instead of phenylalanine at position 25 (Table 3).

[0150] [Table 3]

[0151] Therefore, to analyze whether the AbS1 antibody also binds to NPLGFLP, we expressed and purified GST-preS1(1-56)-strep antigen of genotype A containing the NPLGFLP sequence and performed indirect ELISA. The results confirmed that the AbS1 antibody bound well to NPLGFLP, although its binding ability was slightly lower than that to NPLGFLP (Figure 9).

[0152] From the above results, it was confirmed that the AbS1 antibody shows higher antigen binding ability than AbS0 for preS1 antigens of genotypes A and C, and it was predicted that both antibodies would also bind well to HBV that has the hepatocyte receptor binding motif of the NPLGFLP sequence.

[0153] 7-3 Analysis of the binding ability of AbS1 antibody to preS1 antigens of genotypes E and F Since the above examples confirmed that AbS1 antibody binds to preS1 antigens of HBV genotypes AD and mutants, quantitative ELISA (FIG. 10A) and indirect ELISA (FIG. 10B) were performed against recombinant GST-preS1(1-56)-strep antigens of genotypes E and F to confirm whether AbS1 antibody can also bind to preS1 antigens of genotypes E and F. In this case, the F25A mutant preS1 antigen of genotype C in Example 5 was used as a negative control. As a result, it was confirmed that AbS1 binds well to preS1 of genotypes E and F (FIG. 10).

[0154] The above results indicate that the novel antibodies AbS0 and AbS1 of the present invention can bind to both HBV genotype AF and the hepatocyte receptor binding motif of preS1, and therefore have the mechanism of action of an HBV entry inhibitor that not only neutralizes HBV by blocking the process of HBV entering hepatocytes, but also effectively inhibits HBV infection, and can be useful in the prevention and treatment of HBV infection.

[0155] From the above description, it will be understood by those skilled in the art to which the present invention pertains that the present invention may be embodied in other specific forms without changing the technical idea or essential features of the present invention. In this regard, it should be understood that the above-described embodiments are merely illustrative and not limiting. The scope of the present invention should be interpreted as including any modified or altered forms derived from the meaning and scope of the claims below, and their equivalent concepts, rather than the above detailed description.

Claims

1. An antibody that binds to an amino acid at a specific position in the preS1 antigen of HBV (Hepatitis B virus), The specific positions in the preS1 antigen correspond to positions 22, 23, and 25 of SEQ ID NO:

19.

2. The antibody according to claim 1 , which has tyrosine at position 8 of the LCDR3 sequence of SEQ ID NO: 5 and leucine at position 6 of the HCDR3 sequence of SEQ ID NO: 9 as paratopes.

3. The antibody according to claim 1 , which has the first amino acid of the HCDR2 sequence shown in SEQ ID NO: 8 as a paratope.

4. The antibody of claim 3, wherein the first amino acid of the HCDR2 sequence shown in SEQ ID NO: 8 is alanine or serine.

5. The antibody is (a) a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 3, a light chain CDR2 of SEQ ID NO: 4, and a light chain CDR3 of SEQ ID NO: 5; and (b) a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO:7, a heavy chain CDR2 of SEQ ID NO:8, and a heavy chain CDR3 of SEQ ID NO:9; The antibody of claim 1 , comprising:

6. The antibody is (a) a light chain variable region comprising a light chain CDR1 of SEQ ID NO:3, a light chain CDR2 of SEQ ID NO:4, and a light chain CDR3 of SEQ ID NO:6; and (b) a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 10, a heavy chain CDR2 of SEQ ID NO: 11 or 12, and a heavy chain CDR3 of SEQ ID NO: 13; The antibody of claim 1 , comprising:

7. The antibody of claim 1 , which specifically binds to one or more genotypes selected from HBV genotypes A, B, C, D, E and F.

8. The antibody of claim 1 , wherein the light chain variable region of the antibody is composed of SEQ ID NO:

1.

9. The antibody of claim 1 , wherein the heavy chain variable region of the antibody is composed of SEQ ID NO:

2.

10. The antibody of claim 1 , comprising a light chain variable region set forth in SEQ ID NO: 14; and a heavy chain variable region set forth in SEQ ID NO: 15 or 16.

11. A polynucleotide encoding the antibody of any one of claims 1 to 10.

12. An expression vector comprising the polynucleotide of claim 11.

13. A host cell comprising the expression vector of claim 12.

14. A pharmaceutical composition for preventing or treating HBV infection, comprising the antibody according to any one of claims 1 to 10.

15. The composition of claim 14, wherein the composition is for the prevention or treatment of Hepatitis B.

16. A method for diagnosing HBV infection, comprising the step of detecting preS1 present in a biological sample isolated from an individual suspected of being infected with HBV through an antigen-antibody reaction using the antibody according to any one of claims 1 to 10.

17. The method of claim 12, wherein the method is for diagnosing Hepatitis B.

18. A composition for detecting HBV, comprising the antibody according to any one of claims 1 to 10.

19. The composition of claim 18, wherein the composition is for diagnosing hepatitis B.

20. A kit for detecting HBV, comprising the composition of claim 18.

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