WO2022265264A1 - B형 간염바이러스 pres1항원의 간세포 수용체 결합부위에 특이적으로 결합하는 인간 항체 및 이의 용도 - Google Patents

B형 간염바이러스 pres1항원의 간세포 수용체 결합부위에 특이적으로 결합하는 인간 항체 및 이의 용도 Download PDF

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WO2022265264A1
WO2022265264A1 PCT/KR2022/007679 KR2022007679W WO2022265264A1 WO 2022265264 A1 WO2022265264 A1 WO 2022265264A1 KR 2022007679 W KR2022007679 W KR 2022007679W WO 2022265264 A1 WO2022265264 A1 WO 2022265264A1
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antibody
seq
hbv
pres1
antigen
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French (fr)
Korean (ko)
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홍효정
조경희
홍지수
최영진
이지우
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Apitbio Inc
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Priority to JP2023577967A priority Critical patent/JP2024525184A/ja
Priority to EP22825190.6A priority patent/EP4357360A4/en
Priority to CN202280056403.0A priority patent/CN117858896A/zh
Publication of WO2022265264A1 publication Critical patent/WO2022265264A1/ko
Priority to US18/541,241 priority patent/US20240254200A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—DNA viruses
    • C07K16/082—Hepadnaviridae (F), e.g. hepatitis B virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5761—Hepatitis B
    • G01N33/5764—Hepatitis B surface antigen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to a human antibody that specifically binds to a hepatocyte receptor binding site of hepatitis B virus preS1 antigen and uses thereof.
  • HBV infection is one of the most serious and widespread public health problems. In 2016, there were about 290 million people chronically infected with HBV worldwide.
  • Genotypes A and D are distributed in Africa and Europe, genotypes B and C are mainly distributed in Asia, and genotypes E-J often appear in Europe, America and Asia.
  • the most prevalent genotypes are A-D, accounting for about 90% of hepatitis B cases.
  • the HBV envelope contains three structurally related proteins: S (small), M (middle) and L (Large).
  • S protein is the most abundant protein among these envelope proteins.
  • M protein is composed of preS2 and S
  • L protein is composed of preS1, preS2, and S.
  • Hepatocytes infected with HBV produce infectious HBV particles as well as non-infectious spherical and filamentous subviral particles. These quasiviral particles are composed mostly of the S protein and are typically produced 1000 to 10000 times more than virions. These subviral particles are presumed to reduce the virus-specific immune response.
  • the preS1 of the L protein is mainly present in infectious virions.
  • amino acids 20-26 of preS1 (A-C, F genotype; 9-15 for D genotype; 19-25 for E genotype) bind to the hepatocyte receptor NTCP (sodium taurocholate cotransporting polypeptide) to prevent HBV infection.
  • NTCP sodium taurocholate cotransporting polypeptide
  • Hepatitis B immune globulin is used for preventive medical treatment after accidental or perinatal exposure to HBV.
  • HBIG is prepared by collecting serum with high anti-S protein antibody titers.
  • mAb monoclonal antibody
  • A-C human monoclonal antibody
  • A-C F genotypes
  • amino acids 31-38 A-C, F genotypes
  • amino acids 20-26, A-C, F genotypes amino acids 20-26, A-C, F genotypes
  • mice immunized with HBV particles or recombinant preS1 antigen have been prepared 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 Kim JH, Gripon P, Bouezzedine F, Jeong MS, Chi SW, Ryu SE, Hong HJ.
  • the present inventors have made diligent efforts to provide a monoclonal antibody that can bind to various genotypes of HBV and is specific to the hepatocyte receptor binding site of preS1 for more efficient prevention or treatment, and as a result, most of the hepatitis B virus
  • the present invention was completed by developing a human monoclonal antibody that binds to all of the genotypes A to F occupied and binds to the hepatocyte receptor binding site to exhibit the efficacy of inhibiting HBV infection.
  • 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.
  • Another object of the present invention is to provide a method for producing the antibody.
  • 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 is introduced.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating HBV infection comprising the antibody.
  • Another object of the present invention is to provide a method for preventing or treating HBV infection using the antibody.
  • Another object of the present invention is a method for providing information for diagnosis of HBV infection, comprising the step of detecting preS1 present in a biological sample isolated from an individual suspected of HBV infection through an antigen-antibody reaction using the antibody. is to provide
  • Another object of the present invention is to provide a composition for detecting HBV, comprising the antibody.
  • Another object of the present invention is to provide a kit for detecting HBV comprising the antibody.
  • the antibody of the present invention targets sequences shared by various genotypes of HBV, can bind to various genotypes of HBV, and has excellent binding ability with HBV, so it can be useful not only for detection and diagnosis of HBV infection but also for fields requiring treatment.
  • HBV preS1 N-terminal amino acid sequences (genotypes A-F, numbered according to HBV genotype A).
  • Amino acids 20-26 are essential receptor-binding motifs of preS1 and are indicated in gray shading. Sequences included in the Bio-preS1-L peptide are underlined.
  • Figure 2 shows the experimental results of isolating the anti-preS1 monoclonal Fab from the human Fab synthetic library.
  • Figure 2A shows the input and output titers of phage for each round of panning. pfu represents a plaque-forming unit.
  • Figure 2B shows positive polyclonal phage amplification determined by indirect ELISA. For the isolation of monoclonal Fabs, the 4th round of panning with the most amplification for preS1 was selected.
  • Figure 2C shows the results of indirect ELISA of AbSO Fab phage having the highest antigen-binding activity against the bio-preS1-L peptide among the positive Fab clones selected from the output of the fourth panning.
  • Figure 3 is an analysis of the antigen-binding activity of AbSO mAb converted and purified from AbSO Fab to IgG1.
  • 3A shows SDS-PAGE of AbSO purified under non-reducing (NR, 6%) and reducing (R, 10%) conditions.
  • M represents a protein marker
  • HC represents a heavy chain
  • LC represents a light chain.
  • 3B shows the antigen-binding activity of purified AbSO mAb against preS1 of HBV genotypes (A-D) was evaluated by ELISA.
  • 3C shows the results of affinity analysis of AbSO by BLI using Octet RED384.
  • Fig. 3D shows the result of analysis by FACS that the AbSO antibody does not bind to the human ovarian cancer cell line SKOV3, which expresses L1CAM but does not express preS1, in order to exclude the possibility of non-specific binding of the AbSO antibody to preS1.
  • Figure 4 is the result of evaluating the virion binding activity and in-vitro HBV neutralization activity of AbSO for HBV genotype D.
  • 4A is immunoprecipitation of HBV genotype D particles by AbSO.
  • 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 represents relaxed circular DNA
  • DSL DNA represents double-stranded linear DNA.
  • Figure 4B shows HBV genotype D particles pre-incubated with AbS0 (10, 1, 0.1 ⁇ g) or mouse IgG (10 ⁇ g) and then added to cultured HepG2-NTCP cells, followed by medium every 2 days. was replaced and cells were harvested 7 days after infection. This is the result of Southern blot hybridization by extracting HBV DNA from the harvested cells.
  • 5 shows the results of expression, purification, and SDS-PAGE of an alanine substitution variant of amino acids 19-34 of GST-preS1 (aa 1-56)-strep for epitope mapping of AbSO antibody.
  • Figure 6 shows the results of analyzing the binding ability of the AbSO antibody to the alanine substitution variant of amino acids 19-34 of GST-preS1 (aa 1-56)-strep by indirect ELISA.
  • FIG. 8 shows antibody variants in which Ala33 of HCDR1 (FIGS. 8A-C) and Ser50 of HCDR2 (FIG. 8D) are substituted with other residues in order to analyze the binding site (paratope) of AbS0 antibody to the preS1 antigen. This is the result of analyzing the antigen binding ability by indirect ELISA.
  • Figure 9 is the antigen binding ability of the GST-preS1 (aa 1-56) -strep protein of Genotype A having a receptor binding motif of conservative (NPLGFFP; WT) and other sequences (NPLGFLP; preS1-L25) of AbS1 and AbS0 antibodies. This is the result of analysis by indirect ELISA.
  • FIG. 10 is a result of analyzing the antigen-binding ability of AbS1 antibodies to GST-preS1 (aa 1-56)-strep protein of genotypes E and F by quantitative ELISA (FIG. 10A) and indirect ELISA (FIG. 10B).
  • One aspect of the present invention for achieving the object of the present invention provides a novel antibody that specifically binds to the hepatitis B virus preS1 antigen.
  • antibody refers to a protein molecule that serves as a receptor that specifically recognizes an antigen, including an immunoglobulin molecule that is immunologically reactive with a specific antigen, and includes polyclonal antibodies, monoclonal antibodies, Both whole antibodies and antibody fragments are included.
  • the term also includes chimeric antibodies, humanized antibodies, human antibodies and bivalent or bispecific molecules (eg bispecific antibodies), diabodies, triabodies and tetrabodies.
  • the term further includes single-chain antibodies, scabs, derivatives of antibody constant regions and artificial antibodies based on protein scaffolds that have a binding function to FcRn.
  • a full antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected to the heavy chain by a disulfide bond.
  • the total antibody includes IgA, IgD, IgE, IgM, and IgG, and IgG, and IgG includes IgG1, IgG2, IgG3, and IgG4 as subtypes.
  • the antibody fragment refers to a fragment having an antigen-binding function, and includes Fd, Fab, Fab', F(ab') 2 and Fv.
  • the Fd refers to the heavy chain part included in the Fab fragment.
  • the Fab has a structure having light and heavy chain variable regions, a light chain constant region, and a first constant region (CH1 domain) of a heavy chain, 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.
  • An F(ab') 2 antibody is produced by forming a disulfide bond between cysteine residues in the hinge region of Fab'.
  • Fv means a minimum antibody fragment having only the heavy chain variable region and the light chain variable region.
  • double disulfide Fv dsFv
  • the heavy chain variable region and the light chain variable region are linked by a disulfide bond
  • scFv short chain Fv
  • the heavy chain variable region and the light chain variable region are generally covalently linked through a peptide linker.
  • Such antibody fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by restriction digestion of whole antibodies with papain, and F(ab') 2 fragments can be obtained by digestion with pepsin), specifically It can be produced through genetic recombination technology.
  • Antibodies of the present invention may be monoclonal antibodies.
  • the term "monoclonal antibody” refers to an antibody molecule of a single molecular composition obtained from substantially the same antibody population, and such a monoclonal antibody exhibits a single binding specificity and affinity for a specific epitope.
  • an immunoglobulin has a heavy chain and a light chain, each comprising a constant region and a variable region (the regions are also known as domains).
  • the variable regions of the light and heavy chains include three variable regions called complementarity-determining regions (hereinafter referred to as "CDRs") and four framework regions.
  • CDRs complementarity-determining regions
  • the CDR mainly serves to bind to an antigenic epitope.
  • the CDRs of each chain are typically called CDR1, CDR2, CDR3 sequentially starting from the N-terminus, and are also identified by the chain in which a particular CDR is located.
  • Antibodies of the present invention may be human antibodies.
  • human antibody is a molecule derived from human immunoglobulin, and means that all amino acid sequences constituting the antibody, including complementarity determining regions and structural regions, are composed of human immunoglobulin amino acid sequences.
  • Human antibodies are commonly used in the treatment of human diseases, which may have at least three potential advantages. First, it interacts better with the human immune system, eg complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC) As a result, target cells can be destroyed more efficiently. Second, there is an advantage that the human immune system does not recognize the antibody as foreign. Thirdly, there is an advantage that the half-life in the human circulation is similar to that of naturally occurring antibodies even when the drug is administered in a smaller amount and less frequently.
  • the antibody of the present invention as described above includes a constant region, it may include a constant region derived from IgG, IgA, IgD, IgE, IgM, a combination thereof, or a hybrid thereof. .
  • dimers or multimers may be formed from two or more constant regions selected from the group consisting of IgG, IgA, IgD, IgE and IgM constant regions.
  • hybrid means that sequences corresponding to immunoglobulin heavy chain constant regions of two or more different origins exist within a single chain immunoglobulin heavy chain constant region, and examples include IgG, IgA, IgD, and IgE. And hybrids of domains consisting of 1 to 4 domains selected from the group consisting of CH1, CH2, CH3 and CH4 of IgM are possible.
  • hepatitis B virus is a term used interchangeably with HBV, and is a virus that can cause hepatitis B due to an immune response to it when infected.
  • preS1 antigen refers to a protein encoded by one S gene among the four S, C, X, and P genes of HBV DNA.
  • the S gene is a gene that encodes a surface antigen, and a Pre-S region exists in front of it, and Pre-S is divided into a PreS1 region and a PreS2 region.
  • Surface proteins such as S (small) protein, M (middle) protein, and L (large) protein are coded by these.
  • the S protein is encoded by the S gene, the M protein is composed of preS2 and S proteins, and the L protein is composed of preS1, preS2, and S proteins and is mainly present in infectious virus particles (virions). Therefore, preS1 constitutes a viral surface protein and is mainly present in infectious virions.
  • Antibodies provided in the present invention can bind to HBV of various genotypes, specifically, may be capable of binding to all of genotypes A, B, C, D, E and F.
  • the antibody provided in the present invention may bind to a receptor binding motif sequence shared by genotypes A, B, C, D, E and F of preS1, but is not limited thereto.
  • antibody that binds to the hepatitis B virus preS1 antigen refers to an antibody that can exhibit HBV neutralization activity and/or HBV infection inhibitory activity by binding to preS1, a surface antigen of HBV.
  • the antibody provided in the present invention may bind to an amino acid at a specific position of the preS1 antigen, and the position may be a position corresponding to positions 22, 23, and 25 based on SEQ ID NO: 19 .
  • SEQ ID NO: 19 is only a reference sequence for specifying the binding position of the antibody of the present invention in any preS1 antigen, and the preS1 antigen of the present invention is not limited to SEQ ID NO: 19, and any preS1 antigen protein
  • positions corresponding to positions 22, 23, and 25 based on SEQ ID NO: 19 can be identified through sequence alignment.
  • sequence alignment a Needleman-Wunsch algorithm known in the art, a Needle program of the EMBOSS package, multiple sequence alignment, etc. may be used, but is not limited thereto.
  • the amino acid sequence of the preS1 antigen can be obtained from a known database, and may include, for example, SEQ ID NO: 17 or 18.
  • the amino acids at positions 22, 23, and 25 may be amino acids represented by positions 3, 4, and 6, respectively, in the amino acid sequence of SEQ ID NO: 17 or 18. However, it is not limited thereto.
  • amino acids 22, 23, and 25 are binding sites of the antibody of the present invention, and the "epitope" of the preS1 antigen recognized by the antibody of the present invention is based on SEQ ID NO: 19. It can be expressed as positions corresponding to No. 22, No. 23 and No. 25 as . Meanwhile, positions corresponding to positions 22, 23, and 25 based on SEQ ID NO: 19 may be included in the site where the preS1 antigen binds to the hepatocyte receptor. Therefore, the antibodies of the present invention can inhibit the action of preS1 antigen binding to hepatocyte receptors.
  • the antibody is leucine (Leu22) corresponding to position 22 based on SEQ ID NO: 19, glycine (Gly23) corresponding to position 23 and position corresponding to position 25 It may bind to phenylalanine (Phe25) or leucine (Leu25).
  • the antibody may be an antibody having tyrosine 8 of the LCDR3 sequence of SEQ ID NO: 5 and leucine 6 of the HCDR3 sequence of SEQ ID NO: 9 as a paratope.
  • the antibody may be an antibody having amino acid No. 1 of the HCDR2 sequence represented by SEQ ID NO: 8 as a paratope.
  • Amino acid No. 1 of SEQ ID NO: 8 may be alanine or leucine.
  • paratope refers to a region or region on an antibody or antibody fragment where an antibody or antibody fragment specifically binds to an antigen, that is, where the antibody or antibody fragment physically contacts the antigen.
  • the antibody comprises a light chain comprising a light chain CDR1 of SEQ ID NO: 3 (LCDR1), a light chain CDR2 of SEQ ID NO: 4 (LCDR2), and a light chain CDR3 of SEQ ID NO: 5 (LCDR3) variable region; and a heavy chain variable region comprising heavy chain CDR1 (HCDR1) of SEQ ID NO: 7, heavy chain CDR2 (HCDR2) of SEQ ID NO: 8, and heavy chain CDR3 (HCDR3) of SEQ ID NO: 9, an antibody that specifically binds to preS1.
  • LCDR1 light chain CDR1 of SEQ ID NO: 3
  • LCDR2 light chain CDR2 of SEQ ID NO: 4
  • LCDR3 light chain CDR3 of SEQ ID NO: 5
  • the antibody comprises a light chain comprising a light chain CDR1 of SEQ ID NO: 3 (LCDR1), a light chain CDR2 of SEQ ID NO: 4 (LCDR2), and a light chain CDR3 of SEQ ID NO: 6 (LCDR3).
  • variable region; And a heavy chain variable region comprising the heavy chain CDR1 (HCDR1) of SEQ ID NO: 10, the heavy chain CDR2 (HCDR2) of SEQ ID NO: 11 or 12, and the heavy chain CDR3 (HCDR3) of SEQ ID NO: 13, which binds specifically to preS1 may be an antibody.
  • the light chain variable region of the antibody may be composed of the amino acid sequence described 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.
  • the heavy chain variable region of the antibody may be composed of the amino acid sequence described in SEQ ID NO: 2. Specifically, it may be composed of the amino acid sequence described in SEQ ID NO: 15 or 16, but is not limited thereto.
  • the antibody may include a light chain variable region described in SEQ ID NO: 1 and a heavy chain variable region described in SEQ ID NO: 2, but is not limited thereto.
  • an antibody that specifically binds to the preS1 region including the light chain variable region of SEQ ID NO: 14 and the heavy chain variable region of SEQ ID NO: 15, was named AbS0, and AbS0 is HBV genotypes A, B, and C And it was confirmed that it exhibits excellent binding ability for all of D.
  • the human antibody binding to preS1 of the present invention provides high affinity and low immunogenicity compared to conventional antibodies and exhibits excellent neutralization and infection inhibitory activity against HBV, the antibody of the present invention recognizes the antigen against preS1. Or, it can be used for any application that can be useful for neutralization of HBV, inhibition of infection.
  • Another aspect of the present invention provides a method for producing the antibody.
  • Antibodies of the present invention can be easily produced by known antibody production techniques. For example, methods for preparing monoclonal antibodies can be performed by preparing hybridomas using B lymphocytes obtained from immunized animals (Koeher and Milstein, 1976, Nature, 256:495), or phage display (phage display). display) technology, etc., but is not limited thereto, and can be easily prepared using other known antibody manufacturing techniques.
  • methods for preparing monoclonal antibodies can be performed by preparing hybridomas using B lymphocytes obtained from immunized animals (Koeher and Milstein, 1976, Nature, 256:495), or phage display (phage display). display) technology, etc., but is not limited thereto, and can be easily prepared using other known antibody manufacturing techniques.
  • An antibody library using phage display technology is a method of directly obtaining antibody genes from B lymphocytes and expressing antibodies on the surface of phage without preparing hybridomas. Using phage display technology, many existing difficulties associated with producing monoclonal antibodies by B-cell immortalization can be overcome.
  • phage display technology includes: 1) inserting an oligonucleotide of random sequence into a gene region corresponding to the N-terminus of the phage coat protein pIII (or pIV); 2) expressing a fusion protein of a portion of the native envelope protein and a polypeptide encoded by the oligonucleotide of 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 material using low pH or molecules with binding competence; 5) amplifying phages eluted by panning in host cells; 6) repeating the method to obtain the desired amount; and 7) determining the sequence of an antibody having activity from the DNA sequences of the phage clones selected by panning.
  • the method for producing a monoclonal antibody of the present invention can be performed using phage display technology.
  • phage display technology Those skilled in the art are familiar with known phage display techniques, for example 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.
  • Phages that can be used to construct an antibody library include, for example, filamentous phages, such as fd, M13, f1, If1, Ike, Zj/Z, Ff, Xf, Pf1 or Pf3 phages, but are limited thereto it is not going to be
  • vectors that can be used for the expression of heterologous genes on the surface of the filamentous phage include, for example, phage vectors such as fUSE5, fAFF1, fd-CAT1 or fdtetDOG or phagemids such as pHEN1, pComb3, pComb8 or pSEX Vectors are, but are not limited to.
  • helper phages that can be used to provide wild-type envelope proteins required for successful reinfection of recombinant phages for amplification include, but are not limited to, M13K07 or VSCM13.
  • Polynucleotides encoding the monoclonal antibodies or phagedisplay clones of the present invention can be readily isolated and sequenced using conventional procedures. For example, oligonucleotide primers designed to specifically amplify the heavy and light chain coding regions from the phage template DNA can be used. Once the polynucleotide has been isolated, it 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). .
  • the method for producing the human monoclonal antibody of the present invention comprises the step of amplifying the polynucleotide encoding the human monoclonal antibody in an expression vector containing the polynucleotide encoding the human monoclonal antibody. It may be a manufacturing method of, but is not limited thereto.
  • Another aspect of the present invention provides a polynucleotide encoding the antibody, an expression vector containing the polynucleotide, and a host cell into which the vector is introduced.
  • the antibody is as described above.
  • the expression vector containing the polynucleotide encoding the antibody provided in the present invention is not particularly limited thereto, but is mammalian cell (eg, human, monkey, rabbit, rat, hamster, mouse cell, etc.), plant cell, yeast It can be a vector capable of replicating and / or expressing the polynucleotide in eukaryotic or prokaryotic cells, including cells, insect cells, or bacterial cells (eg, E. coli, etc.), specifically, the nucleotide in the host cell It may be a vector operably linked to an appropriate promoter for expression and containing at least one selectable marker.
  • the polynucleotide may be introduced into a phage, plasmid, cosmid, mini-chromosome, virus or retroviral vector.
  • the expression vector containing the polynucleotide encoding the antibody may be an expression vector containing polynucleotides encoding the heavy chain or light chain of the antibody, respectively, or an expression vector including both polynucleotides encoding the heavy chain or light chain of the antibody.
  • the host cell into which the expression vector provided in the present invention is introduced is not particularly limited thereto, but is transformed into bacterial cells such as Escherichia coli, Streptomyces, and Salmonella typhimurium; yeast cells; fungal cells such as Pichia pastoris; Insect cells such as Drozophila and Spodoptera Sf9 cells; CHO (Chinese hamster ovary cells, chinese hamster ovary cells), SP2/0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), Bowes melanoma cells, HT-1080 , animal cells such as BHK (baby hamster kidney cells), HEK (human embryonic kidney cells), and PERC.6 (human retinal cells); or plant cells.
  • bacterial cells such as Escherichia coli, Streptomyces, and Salmonella typhimurium
  • yeast cells fungal cells such as Pichia pastoris
  • transduction refers to a method of delivering a vector containing a polynucleotide encoding the antibody to a host cell.
  • introduction is performed by calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofectamine and protoplast fusion, etc. It can be performed by several methods known in the art.
  • transduction means the transfer of a target into a cell using virus particles as a means of infection.
  • vectors can be introduced into host cells by gene gun bombardment or the like. In the present invention, introduction may be used in combination with transformation.
  • compositions for preventing or treating HBV infection comprising the antibody.
  • the composition may be for preventing or treating hepatitis B.
  • hepatitis B refers to a disease in which liver inflammation occurs due to an immune response caused by infection with HBV. Since the antibody of the present invention exhibits excellent neutralization and infection inhibitory effects against HBV, it can be used for the prevention or treatment of hepatitis B.
  • prevention may refer to any action that suppresses or delays the onset of HBV infection by administration of the composition
  • treatment means that symptoms caused by HBV infection are improved by administration of the composition. It can mean any action that becomes or is advantageously changed.
  • the pharmaceutical composition may further include a pharmaceutically acceptable carrier.
  • the term "pharmaceutically acceptable carrier” refers to a carrier or diluent that does not stimulate organisms and does not inhibit the biological activity and properties of the administered compound.
  • Acceptable pharmaceutical carriers for compositions formulated as liquid solutions are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added if necessary.
  • diluents such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.
  • the pharmaceutical composition may be in various oral or parenteral formulations. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in one or more compounds, for example, starch, calcium carbonate, sucrose or lactose ( lactose) and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.
  • Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc.
  • Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories.
  • Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents.
  • a base for the suppository witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.
  • the pharmaceutical composition is any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. It can have one formulation.
  • composition of the present invention is administered in a pharmaceutically effective amount.
  • the term "pharmaceutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is dependent on the type and severity of the subject, age, sex, and cancer. It may be determined according to factors including type, activity of drug, sensitivity to drug, administration time, route of administration and excretion rate, duration of treatment, drugs used concurrently, 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. And it can be single or multiple administration. It is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects in consideration of all the above factors, and can be easily determined by those skilled in the art.
  • Another aspect of the present invention provides a method for preventing or treating HBV infection using the antibody.
  • the method may include administering the antibody to a subject suspected of being infected with HBV.
  • the method may be a method for preventing or treating HBV infection comprising administering a pharmaceutical composition further comprising an antibody and a pharmaceutically acceptable carrier to an HBV-infected or infected subject, wherein the pharmaceutically acceptable Possible carriers are the same as described above.
  • the subject includes mammals including cows, pigs, sheep, chickens, dogs, humans, and the like, birds, and the like, and includes, without limitation, subjects whose HBV infection is treated by administration of the composition of the present invention.
  • the antibody may be administered single or multiple times in a pharmaceutically effective amount.
  • the antibody may be administered in the form of a solution, powder, aerosol, capsule, enteric-coated tablet or capsule, or suppository.
  • Routes of administration include, but are not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, endothelial administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration, and the like.
  • the method may also be used for the prevention or treatment of hepatitis B, and specifically for the treatment of any one or more HBV infections selected from HBV genotypes A, B, C, D, E and F, but is not limited thereto. .
  • Another aspect of the present invention is the use of the antibody for the manufacture of a pharmaceutical for the prevention or treatment of HBV infection.
  • the medicine may also be used for the prevention or treatment of hepatitis B and for the treatment of any one or more HBV infections 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.
  • Another aspect of the present invention is a method for providing information for diagnosis of HBV infection, comprising the step of detecting preS1 present in a biological sample isolated from an individual suspected of HBV infection through an antigen-antibody reaction using the antibody provides
  • the method may be a method for diagnosing HBV infection. Also, the method can be used for diagnosis of hepatitis B.
  • the antibody and HBV were as described above.
  • the method for providing information for diagnosis of HBV may detect the preS1 protein by reacting the preS1-specific antibody of the present invention with a biological sample isolated from an individual suspected of having HBV infection and detecting the formation of an antigen-antibody complex, Through this, it is possible to provide information for diagnosis of HBV infection.
  • detecting the preS1 protein through an antigen-antibody reaction by treating a biological sample isolated from an individual suspected of being infected with HBV with the antibody; (b) comparing the level of preS1 protein detected in (a) with a control group, and determining that the patient is HBV-infected if the level of preS1 protein is higher than that of the control group.
  • biological sample includes tissues, cells, whole blood, serum, plasma, tissue autopsy samples (brain, skin, lymph nodes, spinal cord, etc.), cell culture supernatants, disrupted eukaryotic cells, bacterial expression systems, and the like. However, it is not limited thereto.
  • the term "antigen-antibody complex” refers to a combination of a preS1 protein antigen in a sample and an antibody according to the present invention recognizing it, and the formation of such an antigen-antibody complex is performed by a colorimetric method or an electrochemical method. Detection by any method selected from the group consisting of electrochemical method, fluorescence method, luminometry, particle counting method, visual assessment, and scintillation counting method can do. However, it is not necessarily limited only to these, and various applications and applications are possible.
  • markers can be used to detect antigen-antibody complexes.
  • Specific examples may be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, microparticles, and radioactive isotopes, but are not necessarily limited thereto.
  • Enzymes used as detection markers include acetylcholinesterase, alkaline phosphatase, ⁇ -D-galactosidase, horseradish peroxidase, ⁇ -latamase, and the like, and fluorescent substances include fluorescein , Eu 3+ , Eu 3+ chelate or cryptate, etc., including biotin derivatives as ligands, acridinium esters, isoluminol derivatives, etc. as light-emitting substances, colloidal gold as microparticles, It includes colored latex and the like, and radioactive isotopes include, but are not limited to, 57 Co, 3 H, 125 I, 125 I-Bolton Hunter reagent, and the like.
  • the detection method may be an antigen-antibody complex using enzyme immunosorbent assay (ELISA).
  • Enzyme immunosorbent assay includes direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, and indirect ELISA using a labeled secondary antibody that recognizes a capture antibody in a complex of antibodies that recognize an antigen attached to a solid support.
  • direct sandwich ELISA using another labeled antibody that recognizes an antigen in a complex of antibody and antigen attached to a solid support, followed by reaction with another antibody that recognizes an antigen in a complex of antibody and antigen attached to a solid support. It includes various ELISA methods such as indirect sandwich ELISA using a labeled secondary antibody that recognizes the antibody.
  • the antibody may have a detection label, and when it does not have a detection label, these monoclonal antibodies can be captured and identified by treatment with another antibody having a detection label.
  • Another aspect of the present invention provides a composition for detecting HBV, comprising the antibody provided in the present invention.
  • the antibody is as described above.
  • the HBV detection may be to detect the preS1 protein of HBV.
  • the composition is a conventional method for confirming the presence or absence of an antigen protein from a biological sample using an antibody, such as a tool for collecting a test sample or applying a detection reagent, a reagent or device for confirming antigen-antibody binding, and the like. Tools, devices, or reagents included in the composition for detection of may be further included.
  • kits for detecting HBV comprising the antibody.
  • the HBV detection is as described above.
  • the kit may also be in the form of a hepatitis diagnosis kit.
  • the composition, hepatitis B, and diagnosis are as described above.
  • Another aspect of the present invention is the use of the antibody for the preparation of a composition for detecting HBV.
  • the antibody and HBV were as described above.
  • the thus prepared recombinant preS1 antigen and Bio-preS1-L peptide were used as antigens for antibody library panning.
  • the GST-preS1(1-56)-strep tag of HBV genotype AF was expressed and purified in E. coli.
  • the sequence of preS1(1-56) of genotype AF is shown in FIG. 1 .
  • preS1 antigens GST-preS1 (1-119), GST-preS1 (1-56), Bio-preS1-L peptide and preS1 (1-119) of Example 1, respectively, 1, 2, and 3 and 4 rounds of panning of the human synthetic Fab library (diversity 1.35 ⁇ 10 9 ) according to the standard panning procedure.
  • Each preS1 antigen was coated on an immunotube (streptavidin-coated plate in the case of Bio-preS1-L peptide) and stored at 4°C overnight.
  • Antibody library phages were incubated with antigen and unbound phages were washed with 0.1% PBST (0.1% Tween 20 in PBS).
  • Bound phages were eluted using 10 ⁇ g/ml trypsin solution for 30 minutes at 37°C.
  • E. coli TG1 cells were infected with the eluted phage, grown at 37°C for 1 hour, and incubated at 37°C overnight.
  • the next round of panning was performed with the amplified phage, and the degree of washing was gradually intensified in each round.
  • the input and output of phage counts for each round were calculated as dilution and CFU (Fig. 2A).
  • the AbS0 Fab phage showed the highest antigen-binding ability to the Bio-PreS1-L peptide, and also showed a high binding ability to the preS1 (1-119) antigen of Example 1 (Fig. 2C) .
  • AbSO Fab was converted to human IgG1 and expressed in HEK293F cells, and protein purification was performed on the culture supernatant.
  • VH and VK sequences were amplified by PCR, and then combined with IgG heavy chain and light chain leader sequences, respectively, using recombinant PCR.
  • the resulting VH and VK sequences were subcloned into the Eco RI- Apa I and HindIII- Bsi WI sites of the pdCMV- dhfr C expression plasmid containing human C ⁇ 1 and C ⁇ genes to construct the pdCMV- dhfr C-AbS0 expression plasmid. .
  • the expression plasmid prepared in 3-1 was introduced into 30 mL of HEK293F cells at a ratio of 1:4 (30 ⁇ g:120 ⁇ g) using polyethyleneimine (PEI).
  • PEI polyethyleneimine
  • the transformed cells were cultured for 7 days, affinity chromatography was performed using Protein A in the culture supernatant, the concentration of the antibody was determined using a UV-Vis spectrophotometer, and the purity of the antibody purified for SDS-PAGE was determined. verified (Fig. 3A).
  • each well was coated with GST-preS1 (1-56)-strep (30 nM/well) of genotypes A-D (Fig. 1). Then, it was reacted with serially diluted AbSO antibody. Bound AbSO antibody was detected using anti-human IgG Fc-HRP (1:10,000 v/v, Jackson) as a secondary antibody.
  • the affinity of the antibody 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 minutes in 0.1% PBA (0.1% BSA in PBS). . AbSO antibody (1 ⁇ g/ml, 200 ⁇ l) was added, captured for 10 minutes, and washed with 0.1% PBA for 2 minutes.
  • GST-preS1(1-56)-strep antigen of HBV genotype A was subjected to 2-fold serial dilution (25, 12.5, 6.25, 3.125, 1.5625 nM) with 0.1% PBA, followed by assotiation with the antibody bound to the tip for 5 minutes, respectively. step and dissociation step was performed for 10 minutes. For baseline drift, the measured value was corrected by subtracting the value of the control sensor (antibody-captured AHC sensor) exposed only to the running buffer without antibody. The operating temperature was maintained at 30°C. Data were analyzed using ForteBio data analysis software version 7.1 using a 1:1 interaction model (fitting global, Rmax unlinked by sensor).
  • the affinity of the AbSO antibody was 0.5 nM.
  • the human ovarian cancer cell line SKOV3 expressing L1CAM but not preS1 was used to perform flow cytometry.
  • the AbS0 antibody specifically binds to the preS1 antigen.
  • Immunoprecipitation assay was performed using HBV genotype D to confirm whether AbSO antibody binds to HBV virions.
  • Anti-preS1 humanized antibody HzKR127-3.2 which exhibits conventional HBV-neutralizing efficacy, 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.
  • HBV particles of HBV genotype D HepG2 cells were seeded in a 6-well plate at a density of 6 ⁇ 10 5 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 supernatant was harvested and incubated with 1 ⁇ g/ml of AbS0, HzKR127-3.2 (positive control), or mouse IgG (negative control) overnight at 4°C, followed by immunoprecipitation with 20 ⁇ l of protein A beads at 4°C for 6 hours. was performed. After the immunoprecipitated complex was washed three times with PBS, viral DNA was detected as follows.
  • the immunocomplexes were treated with DNase I (Sigma) and mung bean nuclease (Takara, Kusatsu, Shiga, Japan) at 37°C for 20 minutes.
  • DNase I Sigma
  • mung bean nuclease Takara, Kusatsu, Shiga, Japan
  • HBV DNA was extracted and purified using ethanol and 3M sodium acetate. Purified DNA was separated on a 1% agarose gel and HBV DNA was detected by Southern blotting.
  • HBV particles of genotype D were pre-incubated with different concentrations of antibody and added to HepG2-NTCP cells overexpressing the cellular receptor NTCP.
  • HepG2-NTCP HepG2 cell line overexpressing NTCP.
  • HepG2-NTCP cells were seeded in a 6-well plate at a density of 6 ⁇ 10 5 and cultured at 37°C. The next day, HepG2-NTCP cells were infected with HBV particles of genotype D ( ⁇ 2000 viral genome equivalents per cell) in primary hepatocyte maintenance medium (PMM) medium containing 4% polyethylene glycol (PEG) and 2.5% DMSO.
  • PMM primary hepatocyte maintenance medium
  • PEG polyethylene glycol
  • HBV particles were pre-incubated with diluted AbS0 antibody (10, 1, 0.1 ⁇ g) or mouse IgG (10 ⁇ g) for 1 hour at room temperature, respectively, and then added to cultured HepG2-NTCP cells, followed by 2.5 Cells were cultured for 7 days, replacing every 2 hours with PMM medium containing % DMSO. Intracellular HBV DNA was extracted from infected HepG2-NTCP cells and Southern blotting was performed.
  • AbS0 has the ability to neutralize HBV infection.
  • the novel antibody of the present invention can bind to both HBV genotypes A-D and can effectively neutralize viruses of the genotype, preventing viral infection of the genotype or causing viral infection. This suggests that it is effective in treating hepatitis patients. In addition, this suggests that the HBV genotype A-D infection can also be specifically detected.
  • 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.
  • DNA in which each residue of preS1 (19-34) is substituted with alanine is synthesized by recombinant PCR using a primer containing a mutant sequence, digested with BamHI and EcoRI (NEB) restriction enzymes, and wild type
  • BamHI and EcoRI (NEB) restriction enzymes By subcloning each of the pGST-preS1(1-56)-strep expression plasmid containing the preS1 sequence into the BamHI-EcoRI site, an expression plasmid having an alanine substitution mutant instead of the wild type preS1(1-56) was constructed. Thereafter, in order to express each variant, the recombinant E. coli DH5 ⁇ was cultured for 19 hours at 18° C. in a medium containing 0.2 mM IPTG.
  • 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 determined by 12% SDS-PAGE. It was confirmed (FIG. 5).
  • the antigen-binding ability of the AbSO antibody for each alanine substitution variant was analyzed by indirect ELISA (FIG. 6).
  • each well was coated with mutated GST-preS1(1-56)-strep antigen (200 ng) and then incubated with serially diluted AbSO antibody. Bound AbSO antibody was detected using anti-human IgG Fc-HRP (1:10,000 v/v, Thermo) as a secondary antibody.
  • Example 6 Determination of epitope binding site (paratope) of AbSO antibody and identification of variants with increased affinity
  • VH or VL genes were synthesized using a recombinant PCR method using primers containing mutant sequences.
  • each synthesized VH gene was digested with Eco RI and Apa I restriction enzymes, and then subcloned at the Eco RI -Apa I site of the expression vector (pCMV-dhfr-AbS0H) containing the heavy chain gene of AbS0 antibody
  • each synthesized VL gene was digested with HindIII and BsiWI restriction enzymes, and then HindIII-BsiWI of the expression vector (pCMV-dhfr- AbS0k ) containing the AbS0 antibody light chain gene.
  • the constructed expression plasmid was mixed with polyethyleneimine (PEI) at a ratio of 1:4 (30 ⁇ g:120 ⁇ g) and introduced into HEK293F cells. After culturing the transformed cells for 7 days to express the mutant antibody, the culture supernatant was obtained and their antigen-binding ability was analyzed by indirect ELISA. Specifically, each well was coated with recombinant GST-preS1(1-56)-strep antigen (100 ng) of HBV genotype C, and then serially diluted AbS0 antibody or AbS0 variant was conjugated and anti-human as a secondary antibody. IgG Fc-HRP (1:10,000 v/v, Thermo) was added and reacted.
  • PEI polyethyleneimine
  • the concentration of AbS0 antibody or AbS0 variant used in indirect ELISA was determined by quantitative ELISA. After coating each well with anti-human IgG kappa antibody (100 ng, Thermo), serially diluted AbSO antibodies or variants were bound, and as a secondary antibody, anti-human IgG Fc-HRP (1:10,000 v/v, Thermo) was added to react.
  • HCDR3 variant L100A and LCDR3 variant Y96A have no or very low antigen-binding activity, whereas the other 6 variants (V95A, S99A of HCDR3; S91A, Y92A, S93A, S94A of LCDR3) have the same antigen as wild type AbS0. binding capacity was shown (FIG. 7).
  • CDR3 variants I96A and Y97A were not expressed and could not be analyzed. From this result, it was confirmed that Leu100 of HCDR3 of AbS0 and Tyr96 of LCDR3 are very important in antigen binding.
  • amino acids 33 of HCDR1 and 50 amino acids of HCDR2 in antibodies vary widely from antibody to antibody and are known to be involved in antigen binding. Therefore, in the case of AbS0 antibodies, to analyze whether residues 33 and 50 are involved in antigen binding Site-directed mutagenesis was performed for (Positions 33 and 50 are positions described based on SEQ ID NO: 15)
  • amino acid 33 of HCDR1 is alanine.
  • alanine was converted to 8 different residues (S, P, V, T, G, D, L, N). Since amino acid number 50 is serine, it was substituted with 6 other residues (A, Y, E, F, V, P).
  • mutant gene recombinant PCR was performed using degenerate primers, and the synthesized VH gene was digested with Eco RI and Apa I restriction enzymes, and then subcloned at the Eco RI -Apa I site of pdCMV-dhfr-AbS0 to express variants Plasmids were obtained, and through sequencing, it was confirmed that a total of 14 variants were secured.
  • Each expression plasmid was introduced into HEK293F cells by the same method as in 6-1 above, and the culture supernatant obtained after culturing for 7 days was analyzed by indirect ELISA and quantitative ELISA as in 6-1 above.
  • the S50A variant showed slightly increased antigen-binding ability compared to the AbS0 antibody, and the antigen-binding activity of the S50Y variant was greatly reduced, while the S50F and S50E variants did not show antigen-binding activity ( Figure 8D).
  • the S50V and S50P variants expression was not detected, so antigen binding ability could not be analyzed. This result means that residue 50 of HCDR2 plays an important role in antigen binding, and alanine is more optimal for antigen binding than serine.
  • Example 6-2 the recombinant GST-preS1(1-56)-strep antigen of HBV genotype C was used to analyze the antigen-binding ability of the AbS0 antibody and its variants. In order to analyze whether the binding capacity is higher, indirect ELISA was performed on the recombinant GST-preS1(1-56)-strep antigen of genotype A. As a result, it was confirmed that AbS1 showed a higher antigen binding ability to preS1 of genotype A than AbS0 (FIG. 9).
  • the epitope of the AbS1 antibody (Leu22, Gly23, Phe25 of preS1) is contained within the HBV receptor-binding motif, in order to estimate whether the AbS1 antibody can broadly bind to HBV present on earth, the HBV database (https://hbvdb.
  • the HBV database https://hbvdb.
  • 96.6% of the preS1 sequences had NPLGFFP sequence as a receptor-binding motif, and 2.6% had NPLGFLP with leucine instead of phenylalanine at position 25 (Table 3).
  • genotypes E and F Quantitative ELISA (FIG. 10A) and indirect ELISA (FIG. 10B) were performed on the recombinant GST-preS1(1-56)-strep antigen.
  • the F25A mutant preS1 antigen of genotype C of Example 5 was used as a negative control.
  • AbS1 binds well to preS1 of genotypes E and F (FIG. 10).

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