WO2018184593A1 - 用于***感染及相关疾病的抗体 - Google Patents

用于***感染及相关疾病的抗体 Download PDF

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WO2018184593A1
WO2018184593A1 PCT/CN2018/082157 CN2018082157W WO2018184593A1 WO 2018184593 A1 WO2018184593 A1 WO 2018184593A1 CN 2018082157 W CN2018082157 W CN 2018082157W WO 2018184593 A1 WO2018184593 A1 WO 2018184593A1
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seq
antibody
amino acid
sequence
antigen
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PCT/CN2018/082157
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English (en)
French (fr)
Chinese (zh)
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罗文新
康赐明
王一文
袁权
张天英
夏宁邵
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厦门大学
养生堂有限公司
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Priority to RU2019135404A priority Critical patent/RU2765878C2/ru
Priority to KR1020227030102A priority patent/KR102561555B1/ko
Priority to KR1020197033034A priority patent/KR20200010217A/ko
Publication of WO2018184593A1 publication Critical patent/WO2018184593A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C07K16/082—Hepadnaviridae, e.g. hepatitis B virus
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • 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
    • 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
    • C07K2317/50—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
    • C07K2317/567—Framework region [FR]
    • 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

Definitions

  • the present invention relates to the field of molecular virology and immunology, particularly in the field of treatment of hepatitis B virus (HBV) infection.
  • the present invention relates to antibodies against hepatitis B surface antigen (HBsAg), nucleic acid molecules encoding the same, methods of preparing the same, and pharmaceutical compositions containing the same.
  • the pharmaceutical composition can be used for preventing and/or treating HBV infection or a disease associated with HBV infection (eg, hepatitis B), for neutralizing the virulence of HBV in a subject (eg, a human), or for use in a test
  • the serum levels of HBV DNA and/or HBsAg are reduced in vivo.
  • the invention further relates to the use of said antibodies, in particular humanized antibodies, and variants thereof, in the preparation of a pharmaceutical composition for the prevention and/or treatment of HBV infection or associated with HBV infection
  • the disease eg, hepatitis B
  • a subject eg, a human
  • HBsAg serum levels of HBV DNA and/or HBsAg in a subject.
  • Hepatitis B virus infection is one of the most important public health problems in the world (Divag JL. Hepatitis B virus infection. N Engl J Med 2008 Oct 2; 359(14): 1486-1500).
  • Chronic HBV infection can lead to a series of liver diseases such as Chronic hepatitis B (CHB), Liver cirrhosis (LC) and Hepatocellular carcinoma (HCC) (Liaw YF, Chu CM. Hepatitis B virus infection. Lancet 2009 Feb 14; 373 (9663): 582-592).
  • CHB Chronic hepatitis B
  • LC Liver cirrhosis
  • HCC Hepatocellular carcinoma
  • IFNs interferon
  • NAs nucleoside or nucleotide
  • the former includes common interferon (IFN) and peg-interferon (Peg-interferon, also known as long-acting interferon), mainly through the overall enhancement of the patient's immune ability to achieve the effect of inhibiting HBV and treating CHB;
  • the latter mainly include lamivudine (LMV), adefovir dipivoxil (ADV), Entecavir (ETV), Telbivudine (LdT), tenofovir (Tenofovir).
  • LMV lamivudine
  • ADV adefovir dipivoxil
  • ETV Entecavir
  • Telbivudine LdT
  • Tenofovir tenofovir
  • HBV-infected patients such as CHB patients
  • the above drugs alone or in combination therapy have been more effective in inhibiting viral replication in vivo and significantly reducing HBV DNA levels; in particular, after 52 weeks or prolonged treatment, patients
  • the response rate of HBV DNA levels in vivo below the lower limit of detection (virological response) can reach 40-80% (Kwon H et al., supra).
  • the above-mentioned drugs alone or in combination therapy can not completely eliminate the HBV virus in the infected person, and the response rate of HBsAg negative conversion or HBsAg seroconversion (a sign of complete clearance of HBV virus in the infected person) is usually less than 5% ( Kwon H et al., ibid.). Therefore, it is urgent and necessary to develop innovative therapeutic methods and drugs for HBV-infected people that can more effectively eliminate HBV virus, especially HBsAg.
  • Immunotherapy for chronic HBV infection is usually carried out by active immunotherapy (which corresponds to a drug form such as a vaccine, etc.) and passive immunotherapy (which corresponds to a drug form such as an antibody, etc.).
  • Active immunotherapy refers to stimulating the body's cellular immune response (CTL effect, etc.) or/and humoral immune response against chronic HBV infection by administering therapeutic vaccines (including protein vaccines, peptide vaccines, and nucleic acid vaccines). (antibody, etc.) to achieve the purpose of inhibiting or eliminating HBV.
  • therapeutic vaccines including protein vaccines, peptide vaccines, and nucleic acid vaccines.
  • antibody, etc. antibody, etc.
  • Passive immunotherapy refers to the passive administration of antibodies with therapeutic properties to HBV-infected individuals, and the use of antibody-mediated viral neutralization to block HBV infection of newborn hepatocytes, or by antibody-mediated immune clearance. It acts to remove the virus from the body and infected liver cells, thus acting as a treatment.
  • polyclonal antibodies against Anti-HBs purified from the serum/plasma of preventive hepatitis B vaccine immune responders or HBV infection restorers namely high-efficiency hepatitis B immunoglobulin (HBIG), have been widely used to block HBV.
  • HBIG high-efficiency hepatitis B immunoglobulin
  • HBV-infected patients eg, CHB patients
  • HBV-infected patients eg, CHB patients
  • the invention provides an antibody or antigen-binding fragment thereof, which is capable of specifically binding to HBsAg, the antibody or antigen-binding fragment thereof comprising:
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • VH CDR1 which consists of the sequence of SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 18 or has one or more amino acid substitutions, deletions or additions thereto (eg, 1 a sequence of 2, or 3 amino acid substitutions, deletions or additions;
  • VH CDR2 consisting of the sequence of SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 19 or having one or more amino acid substitutions, deletions or additions thereto (eg, 1 a sequence of 2, or 3 amino acid substitutions, deletions or additions, and
  • VH CDR3 consisting of the sequence of SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 20, or having one or several amino acid substitutions, deletions or additions thereto (eg, 1 a sequence of one, two or three amino acid substitutions, deletions or additions;
  • VL light chain variable region
  • VL CDR1 which consists of SEQ ID NO: 9, SEQ ID NO: 15 or has one or several amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3) a sequence of amino acid substitutions, deletions or additions,
  • VL CDR2 which consists of SEQ ID NO: 10, SEQ ID NO: 16 or has one or several amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3) a sequence of amino acid substitutions, deletions or additions, and
  • VL CDR3 which consists of SEQ ID NO: 11, SEQ ID NO: 17 or has one or several amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3) Sequence of amino acid substitutions, deletions or additions).
  • an antibody or antigen-binding fragment thereof of the invention comprises a VH CDR1, VH CDR2 and VH CDR3 as defined above. In certain preferred embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a VL CDR1, a VL CDR2 and a VL CDR3 as defined above. In certain preferred embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 as defined above.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH Heavy chain variable region
  • VH CDR2 which consists of the sequence: SEQ ID NO: 7, or has one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3 amino acid substitutions, deletions or Add) the sequence,
  • VH CDR3 consisting of the sequence of SEQ ID NO: 8, or having one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3 amino acid substitutions, deletions or Add) the sequence,
  • VH CDR1 consisting of the sequence of the VH CDR1 contained in the heavy chain variable region of any immunoglobulin
  • VL light chain variable region
  • VL CDR1 consisting of the sequence of SEQ ID NO: 9, or having one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3 amino acid substitutions, deletions or Add) the sequence,
  • VL CDR3 consisting of the sequence: SEQ ID NO: 11, or having one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3 amino acid substitutions, deletions or Add) the sequence,
  • VL CDR2 which consists of the sequence of the VL CDR2 contained in the light chain variable region of any immunoglobulin (eg, the kappa light chain variable region).
  • the VH CDR1 is the sequence of a VH CDR1 comprised in a heavy chain variable region of a human immunoglobulin.
  • the VH CDR1 consists of: (a) SEQ ID NO: 6, or one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2) a sequence of one or three amino acid substitutions, deletions or additions; or, (b) a sequence of the VH CDR1 contained in the amino acid sequence encoded by the human heavy chain germline gene.
  • the human heavy chain germline gene is selected from the group consisting of IGHV4-4*08 and IGHV4-61*01.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: a VH CDR2 as set forth in SEQ ID NO: 7; a VH CDR3 as set forth in SEQ ID NO: 8; VH CDR1 from the following amino acid sequence: SEQ ID NO: 6, SEQ ID NO: 137 or SEQ ID NO: 138.
  • the VL CDR2 is the sequence of a VL CDR2 comprised in a light chain variable region of a human immunoglobulin (eg, a kappa light chain variable region).
  • the VL CDR2 consists of: (a) SEQ ID NO: 10, or one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2) a sequence of one or three amino acid substitutions, deletions or additions; or, (b) a sequence of the VL CDR2 contained in the amino acid sequence encoded by the human light chain germline gene.
  • the human light chain germline gene is selected from the group consisting of IGKV1-39*01 and IGKV1-5*03.
  • the VL of an antibody or antigen-binding fragment thereof of the invention comprises: a VL CDR1 as set forth in SEQ ID NO: 9; a VL CDR3 as set forth in SEQ ID NO: 11; VL CDR2 from the following amino acid sequence: SEQ ID NO: 10, SEQ ID NO: 139 or SEQ ID NO: 140.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: a VH CDR2 as set forth in SEQ ID NO: 7, a VH CDR3 as set forth in SEQ ID NO: 8, and having a selected from VH CDR1 of the following amino acid sequence: SEQ ID NO: 6, 137 or 138; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as set forth in SEQ ID NO: 9, as set forth in SEQ ID NO: VL CDR3, and VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 139 or 140.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH Heavy chain variable region
  • VH CDR1 which consists of the sequence: SEQ ID NO: 12, or has one or several amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3 amino acid substitutions, deletions or Add) the sequence,
  • VH CDR3 consisting of the sequence of SEQ ID NO: 14, or having one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3 amino acid substitutions, deletions or Add) the sequence, and
  • VH CDR2 consisting of the sequence of the VH CDR2 contained in the heavy chain variable region of any immunoglobulin
  • VL light chain variable region
  • VL CDR1 consisting of the sequence of SEQ ID NO: 15, or having one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3 amino acid substitutions, deletions or Add) the sequence,
  • VL CDR3 consisting of the sequence of SEQ ID NO: 17, or having one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3 amino acid substitutions, deletions or Add) the sequence, and
  • VL CDR2 which consists of the sequence of the VL CDR2 contained in the light chain variable region of any immunoglobulin (eg, the kappa light chain variable region).
  • the VH CDR2 is the sequence of a VH CDR2 comprised in the heavy chain variable region of a human immunoglobulin.
  • the VH CDR2 consists of: (a) SEQ ID NO: 13, or one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2) a sequence of one or three amino acid substitutions, deletions or additions; or, (b) a sequence of the VH CDR2 contained in the amino acid sequence encoded by the human heavy chain germline gene.
  • the human heavy chain germline gene is selected from the group consisting of IGHV4-30-4*07 and IGHV4-4*01.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: (a) a VH CDR1 as set forth in SEQ ID NO: 12; a VH CDR3 as set forth in SEQ ID NO: 14; , VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 141 or SEQ ID NO: 142.
  • the VL CDR2 is the sequence of a VL CDR2 comprised in a light chain variable region of a human immunoglobulin (eg, a kappa light chain variable region).
  • the VL CDR2 consists of: (a) SEQ ID NO: 16, or one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2) a sequence of one or three amino acid substitutions, deletions or additions; or, (b) a sequence of the VL CDR2 contained in the amino acid sequence encoded by the human light chain germline gene.
  • the human light chain germline gene is selected from the group consisting of IGKV2-28*01 and IGKV3-15*01.
  • the VL of an antibody or antigen-binding fragment thereof of the invention comprises: a VL CDR1 as set forth in SEQ ID NO: 15; a VL CDR3 as set forth in SEQ ID NO: 17; VLCDR2 of the amino acid sequence: SEQ ID NO: 16, SEQ ID NO: 143 or SEQ ID NO: 144.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: a VH CDR1 as set forth in SEQ ID NO: 12, a VH CDR3 as set forth in SEQ ID NO: 14, and having a selected from VH CDR2 of the following amino acid sequence: SEQ ID NO: 13, SEQ ID NO: 141 or SEQ ID NO: 142; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as set forth in SEQ ID NO: VL CDR3 as set forth in SEQ ID NO: 17, and VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 143 or SEQ ID NO: 144.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH Heavy chain variable region
  • VH CDR1 which consists of the sequence: SEQ ID NO: 18, or has one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3 amino acid substitutions, deletions or Add) sequence;
  • VH CDR2 consisting of the sequence of SEQ ID NO: 19, or having one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3 amino acid substitutions, deletions or Add) the sequence,
  • VH CDR3 consisting of the sequence: SEQ ID NO: 20, or having one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3 amino acid substitutions, deletions or Add) sequence;
  • VL light chain variable region
  • VL CDR1 consisting of the sequence of SEQ ID NO: 15, or having one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3 amino acid substitutions, deletions or Add) the sequence,
  • VL CDR2 which consists of the sequence of SEQ ID NO: 16, or has one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3 amino acid substitutions, deletions or Add) the sequence,
  • VL CDR3 consisting of the sequence of SEQ ID NO: 17, or having one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3 amino acid substitutions, deletions or Add) the sequence.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: VH CDR1 as set forth in SEQ ID NO: 18, VH CDR2 as set forth in SEQ ID NO: 19, and SEQ ID NO: The VH CDR3 shown by 20; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 15, VL CDR2 as shown in SEQ ID NO: 16, and SEQ ID NO: 17 VL CDR3.
  • the antibodies or antigen-binding fragments thereof of the invention are humanized. In certain preferred embodiments, the antibody or antigen-binding fragment thereof of the invention has a degree of humanization of at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95. %, at least 96%, at least 97% or at least 98%.
  • the non-CDR regions of the antibodies or antigen-binding fragments thereof of the invention comprise no more than 19, no more than 15, no more than 14, no more than 13, no more than 12, no more than 11 non-human sources (no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4 or no more than 3) Crab monkey source) amino acid residues.
  • an antibody or antigen-binding fragment thereof of the invention further comprises a framework region (FR).
  • FR framework region
  • an antibody or antigen-binding fragment thereof of the invention comprises:
  • VH heavy chain variable region
  • FR framework regions
  • VH FR1 which consists of the sequence: SEQ ID NO: 21, SEQ ID NO: 29, SEQ ID NO: 37, SEQ ID NO: 44, SEQ ID NO: 50, or has one or a sequence of amino acid substitutions, deletions or additions (eg, 1, 2, 3 or 4 amino acid substitutions, deletions or additions);
  • VH FR2 which consists of the sequence: SEQ ID NO: 22, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 51, or one or more amino acid substitutions, deletions thereto Or adding a sequence (eg, 1, 2, 3 or 4 amino acid substitutions, deletions or additions);
  • VH FR3 which consists of the sequence: SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 39, SEQ ID NO: 45, SEQ ID NO: 52, or has one or a sequence of amino acid substitutions, deletions or additions (eg, 1, 2, 3 or 4 amino acid substitutions, deletions or additions);
  • VH FR4 which consists of the sequence: SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 46, SEQ ID NO: 53, or one or more amino acid substitutions, deletions thereto Or adding a sequence (eg, 1, 2 or 3 amino acid substitutions, deletions or additions);
  • VL light chain variable region
  • FR framework regions
  • VL FR1 which consists of the sequence of SEQ ID NO: 25, SEQ ID NO: 33, SEQ ID NO: 47, SEQ ID NO: 54, or one or more amino acid substitutions, deletions thereto Or adding a sequence (eg, 1, 2, 3 or 4 amino acid substitutions, deletions or additions);
  • VL FR2 which consists of the sequence of SEQ ID NO: 26, SEQ ID NO: 34, SEQ ID NO: 48, SEQ ID NO: 55, or one or more amino acid substitutions, deletions thereto Or adding a sequence (eg, 1, 2, 3 or 4 amino acid substitutions, deletions or additions);
  • VL FR3 which consists of the sequence: SEQ ID NO:27, SEQ ID NO:35, SEQ ID NO:49, SEQ ID NO:56, or one or more amino acid substitutions, deletions thereto Or adding (eg, 1, 2, 3 or 4 amino acid substitutions, deletions or additions); and
  • VL FR4 which consists of the sequence of SEQ ID NO: 28, SEQ ID NO: 36, or one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2, 3) Sequence of one or four amino acid substitutions, deletions or additions).
  • an antibody or antigen-binding fragment thereof of the invention comprises VH FR1, VH FR2, VH FR3 and VH FR4 as defined above. In certain preferred embodiments, an antibody or antigen-binding fragment thereof of the invention comprises VL FR1, VL FR2, VL FR3 and VL FR4 as defined above. In certain preferred embodiments, an antibody or antigen-binding fragment thereof of the invention comprises VH FR1, VH FR2, VH FR3, VH FR4, VL FR1, VL FR2, VL FR3 and VL FR4 as defined above.
  • the antibody or antigen-binding fragment thereof comprises:
  • a heavy chain framework region having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, compared to a heavy chain framework region comprised in a heavy chain variable region selected from At least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity: SEQ ID NOs: 1, 3, 5, 57, 59 Heavy chain variable regions shown in 157, 158, 163 and 164;
  • a light chain framework region having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93 of the light chain framework regions comprised in the light chain variable region selected from the group consisting of %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity: SEQ ID NOs: 2, 4, 58, 60, 159, 160 The light chain variable region shown in any of 165 and 166.
  • the antibody or antigen-binding fragment thereof comprises:
  • a heavy chain variable region as set forth in any one of SEQ ID NOs: 1, 3, 5, 57, 59, 157, 158, 163 and 164;
  • a light chain variable region as set forth in any one of SEQ ID NOs: 2, 4, 58, 60, 159, 160, 165 and 166.
  • the VH of the antibody or antigen-binding fragment thereof comprises:
  • VH FR1 as shown in SEQ ID NO: 21, VH FR2 as shown in SEQ ID NO: 22, VH FR3 as shown in SEQ ID NO: 23, and VH as shown in SEQ ID NO: FR4;
  • VH FR1 as shown in SEQ ID NO: 29, VH FR2 as shown in SEQ ID NO: 30, VH FR3 as shown in SEQ ID NO: 31, and VH as shown in SEQ ID NO: FR4;
  • VH FR1 as shown in SEQ ID NO: 37, VH FR2 as shown in SEQ ID NO: 38, VH FR3 as shown in SEQ ID NO: 39, and VH as shown in SEQ ID NO: FR4;
  • VH FR1 as shown in SEQ ID NO: 44, VH FR2 as shown in SEQ ID NO: 22, VH FR3 as shown in SEQ ID NO: 45, and VH as shown in SEQ ID NO: 46 FR4; or
  • VH FR1 as shown in SEQ ID NO: 50
  • VH FR2 as shown in SEQ ID NO: 51
  • VH FR3 as shown in SEQ ID NO: 52
  • VH as shown in SEQ ID NO: FR4;
  • the VL of the antibody or antigen-binding fragment thereof comprises:
  • VL FR1 as shown in SEQ ID NO: 25, VL FR2 as shown in SEQ ID NO: 26, VL FR3 as shown in SEQ ID NO: 27, and VL as shown in SEQ ID NO: FR4;
  • VL FR1 as shown in SEQ ID NO: 33, VL FR2 as shown in SEQ ID NO: 34, VL FR3 as shown in SEQ ID NO: 35, and VL as shown in SEQ ID NO: FR4;
  • VL FR1 as shown in SEQ ID NO: 47
  • VL FR2 as shown in SEQ ID NO: 48
  • VL FR3 as shown in SEQ ID NO: 49
  • VL as shown in SEQ ID NO: FR4;
  • VL FR1 as shown in SEQ ID NO: 54
  • VL FR2 as shown in SEQ ID NO: 55
  • VL FR3 as shown in SEQ ID NO: 56
  • VL as shown in SEQ ID NO: 36 FR4.
  • the antibody or antigen-binding fragment thereof comprises a framework region of a human immunoglobulin (eg, a framework region included in an amino acid sequence encoded by a human germline antibody gene), the framework region
  • a framework region of a human immunoglobulin eg, a framework region included in an amino acid sequence encoded by a human germline antibody gene
  • one or more back mutations from a human residue to a cynomolgus source residue are included.
  • the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region comprised in an amino acid sequence encoded by a human heavy chain germline gene, and/or a human light chain germline gene encoding Light chain framework regions contained in the amino acid sequence.
  • the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region encompassed by an amino acid sequence encoded by IGHV4-4*08, and an amino acid sequence encoded by IGKV1-39*01
  • the light chain framework regions comprised, the heavy chain framework regions and/or the light chain framework regions optionally comprise one or more back mutations from a human source residue to a cynomolgus monkey source residue.
  • the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region encompassed by an amino acid sequence encoded by IGHV4-4*02, and an amino acid sequence encoded by IGKV4-1*01
  • the light chain framework regions comprised, the heavy chain framework regions and/or the light chain framework regions optionally comprise one or more back mutations from a human source residue to a cynomolgus monkey source residue.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH FR1 as shown in SEQ ID NO: 21, VH FR2 as shown in SEQ ID NO: 22, VH FR3 as shown in SEQ ID NO: 23, and VH as shown in SEQ ID NO: FR4; and VL FR1 as shown in SEQ ID NO: 25, VL FR2 as shown in SEQ ID NO: 26, VL FR3 as shown in SEQ ID NO: 27, and SEQ ID NO: 28 VL FR4;
  • the amino acid sequence of the heavy chain variable region of an antibody or antigen-binding fragment thereof of the invention has at least 80%, at least 85%, at least an amino acid sequence selected from the following heavy chain variable regions 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity: as SEQ ID NOs: heavy chain variable regions shown in 1, 3, 5, 57, 59, 157, 158, 163 and 164.
  • the heavy chain variable region of the antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NOs: 1, 3, 5, 57, 59, 157, 158, 163 and 164. The heavy chain variable region is shown.
  • the amino acid sequence of the light chain variable region of an antibody or antigen-binding fragment thereof of the invention has at least 80%, at least 85%, at least an amino acid sequence selected from the following light chain variable regions 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity: as SEQ ID NOs: a light chain variable region as shown in any one of 2, 4, 58, 60, 159, 160, 165 and 166.
  • the light chain variable region of the antibody or antigen-binding fragment thereof is selected from the group consisting of any one of SEQ ID NOs: 2, 4, 58, 60, 159, 160, 165 and 166 Light chain variable region.
  • an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region as defined above and a light chain variable region as defined above.
  • the heavy chain variable region of an antibody or antigen-binding fragment thereof of the invention is selected from the heavy chain variable region set forth in any one of SEQ ID NOs: 1, 57, 157 and 158;
  • the light chain variable region of the antibody or antigen-binding fragment thereof is selected from the light chain variable region set forth in any one of SEQ ID NOs: 2, 58, 159, and 160;
  • the heavy chain variable region of the antibody or antigen-binding fragment thereof of the present invention is selected from the heavy chain variable region shown in any one of SEQ ID NOs: 3, 59, 163 and 164; and the antibody or antigen-binding fragment thereof
  • the light chain variable region is selected from the light chain variable regions set forth in any one of SEQ ID NOs: 4, 60, 165 and 166.
  • an antibody or antigen-binding fragment thereof of the invention comprises:
  • VH as shown in SEQ ID NO: 1 and VL as shown in SEQ ID NO: 2;
  • VH as shown in SEQ ID NO: 59 and VL as shown in SEQ ID NO: 60;
  • VH as shown in SEQ ID NO: 59 and VL as shown in SEQ ID NO: 165;
  • the antibody or antigen-binding fragment thereof of the invention is selected from the group consisting of scFv, Fab, Fab', (Fab') 2 , Fv fragment, diabody, bispecific antibody, multispecific Antibody, chimeric antibody or humanized antibody.
  • the antibody is a chimeric or humanized antibody.
  • the antibodies or antigen-binding fragments thereof of the invention are of the IgG class (eg, the IgGl, IgG2, IgG3 or IgG4 class).
  • an antibody or antigen-binding fragment thereof of the invention is capable of specifically binding to HBsAg, neutralizing the virulence of HBV, and/or reducing serum levels of HBV DNA and/or HBsAg in a subject.
  • an antibody or antigen-binding fragment thereof of the invention is labeled.
  • the antibody or antigen-binding fragment thereof carries a detectable label, such as a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme (e.g., horseradish peroxidase).
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof of the invention, or a heavy chain variable region thereof and/or a light chain variable region thereof.
  • the invention provides a vector (eg, a cloning vector or an expression vector) comprising an isolated nucleic acid molecule according to the invention.
  • a vector eg, a cloning vector or an expression vector
  • the invention provides a host cell comprising an isolated nucleic acid molecule according to the invention or a vector according to the invention.
  • a method of making an antibody or antigen-binding fragment thereof according to the invention comprising culturing a host cell according to the invention under conditions permitting expression of the antibody or antigen-binding fragment thereof, and from culturing The antibody or antigen-binding fragment thereof is recovered from the host cell culture.
  • the invention provides a kit comprising an antibody of the invention or an antigen binding fragment thereof.
  • the antibody or antigen-binding fragment thereof of the invention further comprises a detectable label.
  • the kit further comprises a second antibody that specifically recognizes an antibody or antigen-binding fragment thereof of the invention.
  • the second antibody further comprises a detectable label.
  • detectable labels are well known to those skilled in the art and include, but are not limited to, radioisotopes, fluorescent materials, luminescent materials, colored materials and enzymes (e.g., horseradish peroxidase), and the like.
  • the invention provides a method of detecting the presence or level of an HBsAg protein in a sample comprising the use of an antibody of the invention or an antigen binding fragment thereof.
  • the antibody or antigen-binding fragment thereof of the invention further comprises a detectable label.
  • the method further comprises detecting the antibody or antigen-binding fragment thereof of the invention using a second antibody carrying a detectable label. The method can be used for diagnostic purposes, or for non-diagnostic purposes (eg, the sample is a cell sample, not a sample from a patient).
  • the invention provides a method of diagnosing whether a subject is infected with HBV, comprising: detecting the presence of an HBsAg protein in a sample from the subject using an antibody of the invention or an antigen binding fragment thereof.
  • the antibody or antigen-binding fragment thereof of the invention further comprises a detectable label.
  • the method further comprises detecting the antibody or antigen-binding fragment thereof of the invention using a second antibody carrying a detectable label.
  • an antibody or antigen-binding fragment thereof of the invention in the preparation of a kit for detecting the presence or level of an HBsAg protein in a sample, or for diagnosing whether a subject is Infected with HBV.
  • the antibody or antigen-binding fragment thereof of the present invention can be used for preventing or treating a HBV infection of a subject (for example, a human) or a disease associated with HBV infection (for example, hepatitis B), for use in vitro or in a subject (for example, a human). And the virulence of HBV, as well as serum levels for reducing HBV DNA and/or HBsAg in a subject (eg, a human).
  • the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to the invention, and a pharmaceutically acceptable carrier and/or excipient.
  • the pharmaceutical compositions of the invention may further comprise additional pharmaceutically active agents.
  • the additional pharmaceutically active agent is a drug for preventing or treating a HBV infection or a disease associated with HBV infection, such as hepatitis B, such as an interferon drug, such as interferon or polyethylene. Alcohol interferon.
  • an antibody or antigen-binding fragment thereof according to the invention or a pharmaceutical composition according to the invention for the preparation of a medicament for the prevention or treatment of a HBV infection in a subject, such as a human Or a disease associated with HBV infection (eg, hepatitis B), used to neutralize the virulence of HBV in vitro or in a subject (eg, a human), and/or to reduce HBV DNA and in a subject (eg, a human) / or serum levels of HBsAg.
  • a human Or a disease associated with HBV infection eg, hepatitis B
  • the present invention provides a method for preventing or treating a HBV infection of a subject (eg, a human) or a disease associated with HBV infection (eg, hepatitis B) for use in a subject (eg, a human)
  • a subject eg, a human
  • a disease associated with HBV infection eg, hepatitis B
  • the method comprising administering an effective amount to a subject in need thereof
  • compositions provided by the present invention may be used alone or in combination, or may be combined with another pharmaceutically active agent (for example, other antiviral agents such as interferon drugs such as interferon or peginterferon). use.
  • another pharmaceutically active agent for example, other antiviral agents such as interferon drugs such as interferon or peginterferon.
  • Figure 1 shows the results of ELISA assay for determining the binding activity of three cynomolgus monkey-human chimeric monoclonal antibodies M1-23, M3-23, M3-13 to antigen HBsAg in Example 2, wherein the abscissa indicates the antibody concentration. (Log 10 ng/ml), the ordinate indicates the OD value. The results showed that all three cynomolgus monkey-human chimeric monoclonal antibodies have good antigen binding activity.
  • Figure 2 shows the results of a competitive ELISA assay for the cross-blocking of 3 cynomolgus-human chimeric monoclonal antibodies M1-23, M3-23, M3-13 with antigen HBsAg by 6D11 murine antibody in Example 2. .
  • the results showed that the binding of M1-23, M3-23, M3-13 to the antigen HBsAg was significantly inhibited by 6D11.
  • FIG. 3A-3B show the results of evaluation of the efficacy of cynomolgus monkey-human chimeric monoclonal antibodies M1-23, M3-23 in HBV transgenic mice in Example 2.
  • Figure 3A Changes in HBsAg levels in mouse serum, where the abscissa indicates the number of days after injection of the chimeric antibody and the ordinate indicates the relative decrease in HBsAg in the serum of the mouse (Log 10 IU/ml);
  • Figure 3B small The change in HBV DNA levels in rat serum, wherein the abscissa indicates the number of days after injection of the chimeric antibody, and the ordinate indicates the relative decrease in HBV DNA in the serum of the mouse (Log 10 IU/ml). The results showed that both M1-23 and M3-23 significantly cleared HBsAg and HBV DNA in animals.
  • Fig. 4 shows the results of ELISA detection of the binding activity of the humanized antibodies M1D, M3D and the antigen HBsAg in Example 4, wherein the abscissa indicates the antibody concentration (Log 10 ng/ml) and the ordinate indicates the OD value.
  • the results showed that both humanized antibodies had good antigen-binding activity, and their affinity for HBsAg was better than that of reference antibody 162.
  • Figure 5 shows the results of a competitive ELISA assay for the binding of 6D11 murine antibody cross-blocking humanized antibodies M1D, M3D to antigen HBsAg in Example 4. The results showed that the binding of the humanized antibodies M1D, M3D to the antigen HBsAg was significantly inhibited by 6D11.
  • FIG. 6A-6B show the results of measurement of the neutralizing activity of the humanized antibody M1D against HBV virus in Example 4, wherein FIG. 6A is a standard curve prepared using HBIG as a standard, and FIG. 6B is a neutralization activity of the sample. As a result of the measurement, the ordinate indicates how many mIU standard units of HBIG neutralizing activity per mg of antibody. The results showed that the neutralizing activity of M1D on HBV was comparable to that of reference antibody 162.
  • Figure 7 shows the results of evaluation of the efficacy of the humanized antibodies M1D, M3D and chimeric antibody M3-13 in Example 5 in HBV transgenic mice.
  • Figure 7A Changes in HBsAg levels in mouse serum, where the abscissa indicates the number of days after injection of the humanized antibody and the ordinate indicates the relative decrease in HBsAg in the serum of the mouse (Log 10 IU/ml);
  • Figure 7B The change in the level of HBV DNA in the serum of mice, wherein the abscissa indicates the number of days after injection of the chimeric antibody, and the ordinate indicates the relative decrease in HBV DNA in the serum of the mouse (Log 10 IU/ml). The results showed that all three antibodies could significantly eliminate HBsAg and HBV DNA in animals, which was better than reference antibody 162.
  • Figure 10 shows the results of western blot detection of the binding of the antibodies M1D, M3D, 162 to various single-point mutant HBsAg aa113-135 polypeptide fragments in Example 8.
  • the results showed that antibody 162 lost binding activity to HBsAg after a mutation of aa120, aa122 and aa123 to alanine, and its core epitope was CKTC (aa121-124); M1D and aa118-124 were mutated to alanine.
  • the acid-bearing HBsAg loses its binding activity, and its binding core epitope TGPCKTCT (aa118-124) is longer than the reference antibody 162 and has a position ratio of 162; M3D and after a mutation at aa121-125 to alanine
  • the HBsAg loses binding activity binding, its core epitope is CKTCT (aa121-125), and the core epitope position is later than 162.
  • 11A-11B show the results of ELISA detection of the binding activity of humanized antibodies M1D, M3D to HBsAg-aa113-135 corresponding to different subtypes in Example 9, wherein the abscissa indicates the antibody concentration (Log10 ng/ml), vertical The coordinates represent the OD value. The results show that both M1D and M3D can bind to most HBV subtypes.
  • 12A-12B show the blood concentration of the humanized antibody M1D in each cynomolgus monkey serum (Group 1) after a single intravenous injection of 20 mg/kg of the humanized antibody M1D, M3D in Example 11.
  • FIGS 13A-13B show the Tm values of the humanized antibodies M1D, M3D in Example 12 in three different buffers.
  • the abscissa indicates temperature (°C) and the ordinate indicates CP (cal/°C).
  • the results showed that the Tm onset of the humanized antibody M1D was above 61.7 ° C, and the Tm onset value of M3D was greater than 61.6 ° C, indicating that M1D and M3D have good thermal stability.
  • antibody refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having a "light” (L) chain and a “heavy” (H) chain.
  • Antibody light chains can be classified as kappa and lambda light chains.
  • Heavy chains can be classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain further comprises a "D" region of about 3 or more amino acids.
  • Each heavy chain is comprised of a heavy chain variable region (V H) and a heavy chain constant region (C H) composition.
  • the heavy chain constant region is comprised of three domains (C H 1, C H 2 and C H 3) components.
  • Each light chain is comprised of a light chain variable region (V L) and a light chain constant region (C L) components.
  • the light chain constant region is comprised of one domain, C L composition.
  • the constant region of the antibody mediates binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.
  • V H regions may be subdivided into hypervariability regions (termed complementarity determining regions (CDR)), interspersed with regions are more conserved, termed framework regions (FR) of.
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L the following order: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4 from the amino terminus to the carboxy terminus arranged three four FR and CDR components.
  • the assignment of amino acids to regions or domains follows Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917. ; Chothia et al. (1989) Nature 342: 878-883.
  • CDR complementarity determining region
  • LCDR1 ⁇ , 50-56 ⁇ LCDR2 ⁇ , 89-97 ⁇ LCDR3 ⁇ , and residues 31-35 ⁇ HCDR1 ⁇ , 50-65 ⁇ HCDR2 ⁇ , 95-102 ⁇ HCDR3 ⁇ in the heavy chain variable region see, for example, Kabat et al, Sequences of Proteins of lmmunological lnterest, Fifth Edition, Public Health Service, National Institutes of Health, Bethesda, Maryland (1991), or residues in the light chain variable region 26-32 ⁇ L1 ⁇ , 50-52 ⁇ L2 ⁇ , 91-96 ⁇ L3 ⁇ and residues 26-32 ⁇ H1 ⁇ , 53-55 ⁇ H2 ⁇ , 96-101 ⁇ H3 ⁇ in the heavy chain variable region (see, Chothia) And Lesk J. Mol. Bio
  • framework region or "FR” residue refers to those amino acid residues in the variable regions of an antibody other than the CDR residues as defined above.
  • antibody is not limited by any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
  • the antibodies may be antibodies of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • an antigen-binding fragment of an antibody refers to a polypeptide comprising a fragment of a full length antibody that retains the ability to specifically bind to the same antigen to which the full length antibody binds, and/or compete with the full length antibody.
  • Specific binding to an antigen which is also referred to as an "antigen-binding portion.” See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes.
  • an antigen-binding fragment of an antibody is produced by enzymatic or chemical cleavage of an intact antibody.
  • the antigen-binding fragment includes Fab, Fab', F(ab') 2 , Fd, Fv, dAb and complementarity determining regions (CDRs). Fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding ability to the polypeptide.
  • Fd fragment means an antibody fragment consisting of V H and C H 1 domains
  • dAb fragment means an antibody fragment consisting of a VH domain (Ward et al., Nature 341 :544 546 (1989))
  • Fab fragment means an antibody fragment consisting of VL, VH, CL and CH1 domains
  • F(ab')2 fragment means comprising disulfide through the hinge region An antibody fragment of two Fab fragments joined by a bridge.
  • Fv fragment means a single arm of an antibody fragment consisting of V L and V H domain of an antibody composition.
  • An Fv fragment is generally considered to be the smallest antibody fragment that forms a complete antigen binding site. It is believed that the six CDRs confer antigen binding specificity to the antibody. However, even a variable region (eg, an Fd fragment that contains only three CDRs specific for an antigen) recognizes and binds to the antigen, although its affinity is lower than the intact binding site.
  • the antigen-binding fragment is a single chain antibody (e.g., the scFv), wherein V L and V H domains are paired to form so that it can be produced by a linker to a single polypeptide chain monovalent molecules (see, e.g., Bird Et al, Science 242: 423-426 (1988); Huston et al, Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Edited by Moore, Springer-Verlag, New York, pp. 269-315 (1994)).
  • the scFv single chain antibody
  • Such scFv molecules can have the general structure: NH 2 -V L - linker -V H -COOH or NH 2 -V H - linker -V L -COOH.
  • Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or variants thereof.
  • a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • Other linkers useful in the present invention are by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol.
  • single-chain antibody-Fc or “scFv-Fc” means an engineered antibody formed by ligating an scFv to an Fc fragment of an antibody.
  • Fc fragment means that a second, third constant region of a first heavy chain of an antibody and a second, third constant region of a second heavy chain are formed by disulfide bonding.
  • Antibody fragment The Fc fragment of an antibody has many different functions but does not participate in antigen binding.
  • the antigen-binding fragments are diabodies, i.e., bivalent antibodies in which V H and V L, domains are expressed on a single polypeptide chain, but using a linker that is too short to not allow the same chain in two Pairing between domains forces the domain to pair with the complementary domain of another strand and create two antigen binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444 -6448 (1993), and Poljak RJ et al., Structure 2: 1121-1123 (1994)).
  • An antigen-binding fragment of an antibody can be obtained from a given antibody (eg, a monoclonal antibody provided herein) using conventional techniques known to those skilled in the art (eg, recombinant DNA techniques or enzymatic or chemical cleavage methods) (eg, The antibody fragment), and specifically screens the antigen-binding fragment of the antibody in the same manner as used for the intact antibody.
  • a given antibody eg, a monoclonal antibody provided herein
  • conventional techniques known to those skilled in the art eg, recombinant DNA techniques or enzymatic or chemical cleavage methods
  • antibody As used herein, unless the context clearly dictates otherwise, when referring to the term “antibody”, it includes not only intact antibodies, but also antigen-binding fragments of antibodies.
  • the terms “monoclonal antibody” and “monoclonal antibody” refer to a fragment of an antibody or antibody from a population of highly homologous antibody molecules, ie, in addition to a natural mutation that may occur spontaneously. , a group of identical antibody molecules.
  • Monoclonal antibodies are highly specific for a single epitope on the antigen.
  • Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least two or more different antibodies, which typically recognize different epitopes on the antigen.
  • Monoclonal antibodies are typically obtained using hybridoma technology first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA techniques (see, for example, U.S. Patent 4,816,567).
  • monoclonal antibodies can be prepared as follows.
  • the non-human primate e.g., cynomolgus monkey
  • an immunogen additional of an adjuvant if necessary.
  • the method of injection of the immunogen or adjuvant is usually subcutaneous injection or intraperitoneal injection.
  • the immunogen can be pre-coupled to certain known proteins, such as serum albumin or soybean trypsin inhibitors, to enhance the immunogenicity of the antigen in the host.
  • the adjuvant may be Freund's adjuvant or MPL-TDM or the like. After the animal is immunized, the body will produce lymphocytes that secrete antibodies that specifically bind to the immunogen.
  • lymphocytes can also be obtained by in vitro immunization.
  • the lymphocytes of interest are collected and fused with myeloma cells using a suitable fusing agent, such as PEG, to obtain hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996).
  • the hybridoma cells prepared above may be inoculated into a suitable culture medium, and the culture solution preferably contains one or more substances capable of inhibiting the growth of unfused, parental myeloma cells.
  • HGPRT hypoxanthine guanine phosphotransferase
  • HAT medium hypoxanthine, aminoguanidine, and thymine
  • the culture medium for growing hybridoma cells is used to detect the production of monoclonal antibodies against specific antigens.
  • Methods for determining the binding specificity of monoclonal antibodies produced by hybridoma cells include, for example, immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • the affinity of the monoclonal antibody can be determined using the Scatchard assay described by Munson et al., Anal. Biochem. 107: 220 (1980).
  • the target cell strain can pass the standard described by (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996). The dilution method was subcloned.
  • a suitable culture solution may be DMEM or RPMI-1640 or the like.
  • hybridoma cells can also be grown in animals in the form of ascites tumors.
  • immunoglobulin purification methods such as protein A agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography, the monoclonal antibodies secreted by the subcloned cells can be cultured from the cell culture medium, Separated from ascites or serum.
  • Monoclonal antibodies can also be obtained by genetic engineering recombinant techniques.
  • a DNA molecule encoding a monoclonal antibody heavy chain and a light chain gene can be isolated from a hybridoma cell by PCR amplification using a nucleic acid primer that specifically binds to the monoclonal antibody heavy chain and light chain genes.
  • the obtained DNA molecule is inserted into an expression vector, and then transfected into a host cell (such as E. coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulin), and cultured under appropriate conditions.
  • the recombinant antibody expressed can be obtained.
  • epitope and epitopope refer to a site on an antigen that is specifically bound by an immunoglobulin or antibody. "Epitope” is also referred to in the art as an "antigenic determinant.”
  • An epitope or antigenic determinant typically consists of a chemically active surface group of a molecule, such as an amino acid or a carbohydrate or sugar side chain, and typically has specific three dimensional structural characteristics as well as specific charge characteristics.
  • an epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation, which may be "linear" "or” conformational.
  • germline antibody gene or “germline antibody gene segment” refers to a sequence encoding an immunoglobulin present in the genome of an organism. It has not experienced a maturation process that leads to genetic rearrangements and mutations that express specific immunoglobulins.
  • the expression “heavy chain germline gene” refers to a germline antibody gene or gene fragment encoding an immunoglobulin heavy chain, which includes a V gene (variable), a D gene (diversity), and a J gene (joining).
  • germline gene refers to a germline antibody gene or gene fragment encoding an immunoglobulin light chain, which includes a V gene (variable), a J gene (joining), and C gene (constant).
  • amino acid sequence encoded by the germline antibody gene or the germline antibody gene fragment is also referred to as a "germline sequence”.
  • Germline antibody gene or germline antibody gene fragments and their corresponding germline sequences are well known to those skilled in the art and can be obtained or queried from a professional database (eg, IMGT, UNSWIg, NCBI or VBASE2).
  • an antibody that specifically binds to an antigen means that the antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, Affinity (K D ) of 10 -8 M, 10 -9 M or 10 -10 M or less binds to the antigen.
  • K D refers to a particular antibody - antigen interaction dissociation equilibrium constant, which is used to describe the binding affinity between antibody and antigen.
  • the antibody e.g., an antibody of the invention
  • the dissociation equilibrium constant (K D ) binds to an antigen (eg, HBsAg), for example, as determined using surface plasmon resonance (SPR) in a BIACORE instrument.
  • the term "immunogenicity” refers to the ability to stimulate the body to form specific antibodies or sensitize lymphocytes. It means that the antigen can stimulate specific immune cells, activate, proliferate and differentiate immune cells, and finally produce the characteristics of immune effector substances such as antibodies and sensitized lymphocytes. It also means that after the antigen stimulates the body, the body's immune system can form antibodies or A specific immune response to sensitized T lymphocytes.
  • the immunogenicity of the heterologous antibody in the subject is undesirable because such immunogenicity will result in the immunological/immune cell of the subject against the heterologous antibody.
  • heterologous antibody eg, a cynomolgus monkey-derived antibody
  • chimeric antibody refers to an antibody whose light chain or/and a portion of a heavy chain is derived from an antibody (which may be derived from a particular species or belong to a particular antibody class or Subclass), and another portion of the light or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belonging to the same or different antibody class or subclass), but in any case, it remains Binding activity to the antigen of interest (USP 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)).
  • chimeric antibody can include an antibody (eg, a human monkey chimeric antibody) wherein the heavy and light chain variable regions of the antibody are from a first antibody (eg, a cynomolgus monkey-derived antibody), while the antibody is heavy
  • the chain and light chain variable regions are derived from a second antibody (eg, a human antibody).
  • humanized antibody refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase homology to the sequence of a human antibody.
  • CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (eg, variable region FR and/or constant region) are derived from a human source.
  • Immunoglobulin (receptor antibody) Humanized antibodies typically retain the desired properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, ability to neutralize the virus, and/or ability to eliminate the virus, and the like.
  • a donor antibody can be a mouse, rat, rabbit, or non-human primate having desirable properties (eg, antigen specificity, affinity, reactivity, ability to neutralize the virus, and/or ability to clear the virus) (eg, , cynomolgus monkey) antibodies.
  • desirable properties eg, antigen specificity, affinity, reactivity, ability to neutralize the virus, and/or ability to clear the virus
  • a humanized antibody is capable of retaining both the expected properties of a non-human donor antibody (eg, a cynomolgus monkey-derived antibody) and an effective reduction of a non-human donor antibody (eg, a cynomolgus monkey-derived antibody) in a human subject. Immunogenicity, therefore, is particularly advantageous.
  • the expected properties of the humanized antibody eg, antigen specificity, affinity, reactivity, neutralizing viral ability, and/or clearance of the virus) Ability
  • non-human donor antibodies eg, cynomolgus monkey-derived antibodies.
  • the skilled person needs to explore, explore and modify specific donor antibodies, and it is possible to obtain a large amount of creative labor, which has a high degree of humanization (for example, at least 89%, at least 90%, at least 91%, at least 92%, a human source that is at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% humanized), yet retains the expected properties of a particular donor antibody Antibody.
  • a high degree of humanization for example, at least 89%, at least 90%, at least 91%, at least 92%, a human source that is at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% humanized
  • the human source of the present invention may comprise both the amino acid residue of the human receptor antibody and the corresponding amino acid residue of the non-human donor antibody.
  • a partial amino acid derived from a CDR region of a donor antibody in a humanized antibody can be used.
  • the residue is replaced, for example, by the amino acid residue of the corresponding human immunoglobulin CDR region, or other amino acid residue.
  • M1-23, M3-23, M3-13 cynomolgus monkey-human chimeric monoclonal antibodies having excellent properties
  • M1-23, M3-23, M3-13 its heavy chain and The light chain variable regions are shown in SEQ ID NOs: 1-5, respectively: three antibodies not only specifically recognize/bind HBsAg, but also neutralize the virulence of HBV and reduce HBV DNA and/or in the subject. Or the serum level of HBsAg can effectively eliminate HBV and HBV-infected cells in the body.
  • M1-23, M3-23 and M3-13 antibodies have the potential to prevent and treat HBV infection as well as diseases associated with HBV infection, such as hepatitis B.
  • Humanized antibodies The humanized antibodies of the invention not only have a very high degree of humanization (humanization can be as high as 98%), but also have substantially the same (or even better) expectations as the parent antibody. Properties (including but not limited to, HBsAg binding activity, neutralization of HBV activity, clearance of HBV DNA or HBsAg activity in vivo, or clearance of HBV and HBV-infected cells in vivo, etc.).
  • the antibody of the present invention (particularly a humanized antibody) is extremely advantageous, which not only retains the function and properties of the parent antibody, but also has a disease for preventing and treating HBV infection and associated with HBV infection (for example, hepatitis B).
  • HBV infection and associated with HBV infection for example, hepatitis B.
  • the potential and has a very high degree of humanization (humanization can be as high as 98%), so that it can be safely administered to human subjects without eliciting an immunogenic response.
  • the antibodies of the invention, particularly humanized antibodies have significant clinical value.
  • the expected properties of the antibodies of the present invention include, the specific binding to HBsAg activity, the neutralization of HBV activity, the clearance of HBV DNA or HBsAg activity in vivo, and/or the elimination of HBV and HBV-infected cells in vivo.
  • the humanized antibody according to the invention retains one or more of the above-mentioned expected properties of the parent antibody (cynomolgus-human chimeric antibody), preferably retaining the parent antibody (cynomolgus-human chimeric antibody) All of the above expected properties.
  • humanization of the present invention is achieved by engineering a parent antibody (cynomolgus-human chimeric antibody) of the present invention, for example, by replacing a part of the amino acid residues in the FR (for example, conservative substitution). antibody.
  • substitutions may, for example, (1) reduce the sensitivity of the antibody to proteolysis; (2) reduce the susceptibility of the antibody to oxidation; (3) alter (eg, enhance) the binding affinity of the antibody to the antigen; (4) alter (eg, Enhancing) the activity of the antibody to neutralize HBV; (5) altering (eg, enhancing) the activity of the antibody to clear HBV; (6) further increasing the degree of humanization of the antibody, reducing the immunogenicity of the antibody; or (7) altering the antibody's other Biochemical or functional properties; while still retaining the expected properties of the antibody.
  • Such substitutions may be present in the CDR regions and/or FR regions and may be a single amino acid substitution or multiple amino acid substitutions.
  • degree of humanization is used to refer to an indicator of the amount of non-human amino acid residues in a humanized antibody.
  • neutralizing antibody refers to an antibody or antigen-binding fragment thereof that is capable of significantly reducing or completely inhibiting the virulence of a target virus (eg, the ability to infect a cell).
  • a neutralizing antibody is capable of recognizing and binding to a target virus and preventing the target virus from entering/infecting the cells of the subject.
  • the antibody of the present invention is a neutralizing antibody.
  • neutralizing virus refers to the process of neutralizing the virulence of a target virus by inhibiting the process by which the target virus enters/infects the cells of the subject (ie, significantly reduces or completely inhibits the toxicity of the target virus). force).
  • nuclearing the virus means that the target virus in the body (whether or not it is infected with the cells) is eliminated from the body, so that the body changes toward a state before the virus is infected (for example, the serum of the virus) The test results turned negative.
  • neutralizing antibodies do not necessarily have the ability to eliminate viruses.
  • the inventors have unexpectedly discovered that the antibody of the present invention not only has the ability to neutralize HBV, but also has the ability to scavenge viruses (i.e., is capable of scavenging HBV DNA and/or HBsAg in vivo, and eliminating HBV and The cells infected with HBV) have great clinical value.
  • the term "isolated” refers to artificially obtained from a natural state. If a certain "separated” substance or ingredient appears in nature, it may be that the natural environment in which it is located has changed or that it has been separated from the natural environment, or both. For example, for a non-isolated polynucleotide or polypeptide naturally occurring in a living animal, the same high-purity polynucleotide or polypeptide isolated from this natural state is called It is "separated.”
  • isolated does not exclude the presence of artificial or synthetic materials, nor does it exclude the presence of other impure substances that do not affect the activity of the material.
  • vector refers to a nucleic acid vehicle into which a polynucleotide can be inserted.
  • a vector is referred to as an expression vector when the vector enables expression of the protein encoded by the inserted polynucleotide.
  • the vector can be introduced into the host cell by transformation, transduction or transfection, and the genetic material element carried thereby can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 derived artificial chromosomes (PAC).
  • Phage such as lambda phage or M13 phage and animal virus.
  • Animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, nipples Multi-tumor vacuolar virus (such as SV40).
  • a vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication.
  • the term "host cell” refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as Escherichia coli or Bacillus subtilis, such as a fungal cell such as a yeast cell or an Aspergillus.
  • a prokaryotic cell such as Escherichia coli or Bacillus subtilis
  • a fungal cell such as a yeast cell or an Aspergillus.
  • S2 Drosophila cells or insect cells such as Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • identity is used to mean the matching of sequences between two polypeptides or between two nucleic acids.
  • a position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by adenine, or two
  • Each position in each of the polypeptides is occupied by lysine, and then each molecule is identical at that position.
  • the "percent identity" between the two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 of the 10 positions of the two sequences match, then the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match).
  • the comparison is made when the two sequences are aligned to produce maximum identity.
  • Such alignment can be achieved by, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). It is also possible to use the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)) integrated into the ALIGN program (version 2.0), using the PAM 120 weight residue table.
  • the gap length penalty of 12 and the gap penalty of 4 were used to determine the percent identity between the two amino acid sequences.
  • the Needleman and Wunsch (J MoI Biol. 48: 444-453 (1970)) algorithms in the GAP program integrated into the GCG software package can be used, using the Blossum 62 matrix or The PAM250 matrix and the gap weight of 16, 14, 12, 10, 8, 6 or 4 and the length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences .
  • conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include substitutions of amino acid residues with similar side chains in place of amino acid residues, for example, physically or functionally similar to corresponding amino acid residues (eg, having similar size, shape, charge, chemical properties, including Substitution of residues by formation of a covalent bond or a hydrogen bond, etc.).
  • a family of amino acid residues having similar side chains has been defined in the art.
  • These families include basic side chains (eg, lysine, arginine, and histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine) , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg alanine, valine, leucine, isoluminescence) Acid, valine, phenylalanine, methionine), beta branch side chains (eg, threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, Amino acids of phenylalanine, tryptophan, histidine).
  • basic side chains eg, lysine, arginine, and histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged polar side chains eg, glycine
  • amino acids are generally represented by single letter and three letter abbreviations as are known in the art.
  • alanine can be represented by A or Ala.
  • the terms “monoclonal antibody” and “monoclonal antibody” have the same meaning and are used interchangeably; the terms “polyclonal antibody” and “polyclonal antibody” have the same meaning and are used interchangeably. .
  • pharmaceutically acceptable carrier and/or excipient refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, It is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ions. Strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, preservatives.
  • pH adjusting agents include, but are not limited to, phosphate buffers.
  • Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80.
  • Ionic strength enhancers include, but are not limited to, sodium chloride.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like.
  • Agents that delay absorption include, but are not limited to, monostearate and gelatin.
  • adjuvant refers to a non-specific immunopotentiator that, when brought together with an antigen or pre-delivered into the body, enhances the body's immune response to the antigen or alters the type of immune response.
  • adjuvants including but not limited to aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvant (such as complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, etc. .
  • Freund's adjuvant is the most commonly used adjuvant in animal testing.
  • Aluminum hydroxide adjuvant is used more in clinical trials.
  • prevention refers to a method performed to prevent or delay the onset of a disease or condition or symptom (eg, an HBV infection or a disease associated with HBV infection) in a subject.
  • treatment refers to a method performed to obtain a beneficial or desired clinical result.
  • beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, narrowing the extent of the disease, stabilizing (i.e., not worsening) the state of the disease, delaying or slowing the progression of the disease, ameliorating or ameliorating the disease The state, and alleviating symptoms (whether part or all), whether detectable or undetectable.
  • the antibody of the present invention has the ability to neutralize HBV, and thus can be used for preventing/preventing infection of HBV by an unaffected subject or a cell thereof. Furthermore, the antibodies of the present invention have the ability to scavenge HBV (i.e., are capable of removing HBV DNA and/or HBsAg in vivo, removing HBV from the body and cells infected with HBV), thereby being useful for treating HBV infection in a diseased subject or A disease associated with HBV infection.
  • the term "subject" refers to a mammal, such as a primate mammal, such as a human.
  • an effective amount refers to an amount sufficient to achieve, or at least partially achieve, a desired effect.
  • an effective amount to prevent a disease eg, HBV infection or a disease associated with HBV infection
  • an amount sufficient to prevent, prevent, or delay the onset of a disease eg, HBV infection or a disease associated with HBV infection
  • the amount effective for therapeutic use will depend on the severity of the condition to be treated, the overall condition of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments for simultaneous administration. and many more.
  • M1-23, M3-23, M3-13 the heavy and light chains are variable.
  • the regions are shown in SEQ ID NOs: 1-5, respectively: three antibodies can not only specifically recognize/bind HBsAg, but also neutralize the virulence of HBV, and can reduce the serum of HBV DNA and/or HBsAg in the subject. Levels are effective in removing HBV from the body and cells infected with HBV.
  • M1-23, M3-23 and M3-13 antibodies have the potential to prevent and treat HBV infection as well as diseases associated with HBV infection, such as hepatitis B.
  • humanization of the present invention The antibody not only has a very high degree of humanization (humanization can be as high as 98%), but also has substantially the same (or even better) expected properties (including but not limited to, HBsAg binding activity, in the parental antibody). And the activity of HBV, remove the activity of HBV DNA or HBsAg in the body, or remove the activity of HBV and HBV-infected cells in the body, etc.).
  • the antibody of the present invention (particularly a humanized antibody) is extremely advantageous, which not only retains the function and properties of the parent antibody, but also has a disease for preventing and treating HBV infection and associated with HBV infection (for example, hepatitis B).
  • HBV infection and associated with HBV infection for example, hepatitis B.
  • the potential and has a very high degree of humanization (humanization can be as high as 98%), so that it can be safely administered to human subjects without eliciting an immunogenic response.
  • the antibodies of the invention, particularly humanized antibodies have significant clinical value.
  • the invention provides an antibody or antigen-binding fragment thereof, which is capable of specifically binding to HBsAg, the antibody or antigen-binding fragment thereof comprising:
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • VH CDR1 which consists of the sequence of SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 18 or has one or several amino acid substitutions, deletions or additions thereto (eg, 1 a sequence of 2, or 3 amino acid substitutions, deletions or additions;
  • VH CDR2 consisting of the sequence of SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 19 or having one or more amino acid substitutions, deletions or additions thereto (eg, 1 a sequence of 2, or 3 amino acid substitutions, deletions or additions, and
  • VH CDR3 consisting of the sequence of SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 20, or having one or several amino acid substitutions, deletions or additions thereto (eg, 1 a sequence of one, two or three amino acid substitutions, deletions or additions;
  • VL light chain variable region
  • VL CDR1 which consists of SEQ ID NO: 9, SEQ ID NO: 15 or has one or several amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3) a sequence of amino acid substitutions, deletions or additions,
  • VL CDR2 which consists of SEQ ID NO: 10, SEQ ID NO: 16 or has one or several amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3) a sequence of amino acid substitutions, deletions or additions, and
  • VL CDR3 which consists of SEQ ID NO: 11, SEQ ID NO: 17 or has one or several amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3) Sequence of amino acid substitutions, deletions or additions).
  • an antibody or antigen-binding fragment thereof of the invention comprises a VH CDR1, VH CDR2 and VH CDR3 as defined above. In certain preferred embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a VL CDR1, a VL CDR2 and a VL CDR3 as defined above. In certain preferred embodiments, an antibody or antigen-binding fragment thereof of the invention comprises a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 as defined above.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH Heavy chain variable region
  • VH CDR2 which consists of the sequence: SEQ ID NO: 7, or has one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3 amino acid substitutions, deletions or Add) the sequence,
  • VH CDR3 consisting of the sequence of SEQ ID NO: 8, or having one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3 amino acid substitutions, deletions or Add) the sequence,
  • VH CDR1 consisting of the sequence of the VH CDR1 contained in the heavy chain variable region of any immunoglobulin
  • VL light chain variable region
  • VL CDR1 consisting of the sequence of SEQ ID NO: 9, or having one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3 amino acid substitutions, deletions or Add) the sequence,
  • VL CDR3 consisting of the sequence: SEQ ID NO: 11, or having one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3 amino acid substitutions, deletions or Add) the sequence,
  • VL CDR2 which consists of the sequence of the VL CDR2 contained in the light chain variable region of any immunoglobulin (eg, the kappa light chain variable region).
  • the VH CDR1 is the sequence of a VH CDR1 comprised in a heavy chain variable region of a human immunoglobulin.
  • the VH CDR1 consists of: (a) SEQ ID NO: 6, or one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2) a sequence of one or three amino acid substitutions, deletions or additions; or, (b) a sequence of the VH CDR1 contained in the amino acid sequence encoded by the human heavy chain germline gene.
  • the human heavy chain germline gene is selected from the group consisting of IGHV4-4*08 and IGHV4-61*01.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: a VH CDR2 as set forth in SEQ ID NO: 7; a VH CDR3 as set forth in SEQ ID NO: 8; VH CDR1 from the following amino acid sequence: SEQ ID NO: 6, SEQ ID NO: 137 or SEQ ID NO: 138.
  • the VL CDR2 is the sequence of a VL CDR2 comprised in a light chain variable region of a human immunoglobulin (eg, a kappa light chain variable region).
  • the VL CDR2 consists of: (a) SEQ ID NO: 10, or one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2) a sequence of one or three amino acid substitutions, deletions or additions; or, (b) a sequence of the VL CDR2 contained in the amino acid sequence encoded by the human light chain germline gene.
  • the human light chain germline gene is selected from the group consisting of IGKV1-39*01 and IGKV1-5*03.
  • the VL of an antibody or antigen-binding fragment thereof of the invention comprises: a VL CDR1 as set forth in SEQ ID NO: 9; a VL CDR3 as set forth in SEQ ID NO: 11; VL CDR2 from the following amino acid sequence: SEQ ID NO: 10, SEQ ID NO: 139 or SEQ ID NO: 140.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: a VH CDR2 as set forth in SEQ ID NO: 7; a VH CDR3 as set forth in SEQ ID NO: 8; and a VH CDR1, It consists of the sequence of the VH CDR1 contained in the heavy chain variable region of any immunoglobulin (for example, SEQ ID NO: 6, 137 or 138);
  • VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 9, VL CDR3 as shown in SEQ ID NO: 11, and VL CDR2 as shown in SEQ ID NO: .
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: a VH CDR2 as set forth in SEQ ID NO: 7; a VH CDR3 as set forth in SEQ ID NO: 8; NO: 6 shows the VH CDR1;
  • VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 9, VL CDR3 as shown in SEQ ID NO: 11, and VL CDR2, which consists of the following sequence: The sequence of the VL CDR2 contained in the light chain variable region of the immunoglobulin (eg, the kappa light chain variable region) (eg, SEQ ID NO: 10, 139 or 140).
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: a VH CDR2 as set forth in SEQ ID NO: 7, a VH CDR3 as set forth in SEQ ID NO: 8, and having a selected from VH CDR1 of the following amino acid sequence: SEQ ID NO: 6, 137 or 138; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as set forth in SEQ ID NO: 9, as set forth in SEQ ID NO: VL CDR3, and VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 139 or 140.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: a VH CDR1 as set forth in SEQ ID NO: 6, a VH CDR2 as set forth in SEQ ID NO: 7, and a SEQ ID NO: the VH CDR3 shown by 8; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 9, VL CDR2 as shown in SEQ ID NO: 10, and SEQ ID NO: VL CDR3 shown by 11.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: a VH CDR1 as set forth in SEQ ID NO: 137, a VH CDR2 as set forth in SEQ ID NO: 7, and a SEQ ID NO: the VH CDR3 shown by 8; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 9, VL CDR2 as shown in SEQ ID NO: 10, and SEQ ID NO: VL CDR3 shown by 11.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: a VH CDR1 as set forth in SEQ ID NO: 138, a VH CDR2 as set forth in SEQ ID NO: 7, and a SEQ ID NO: the VH CDR3 shown by 8; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 9, VL CDR2 as shown in SEQ ID NO: 10, and SEQ ID NO: VL CDR3 shown by 11.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: a VH CDR1 as set forth in SEQ ID NO: 6, a VH CDR2 as set forth in SEQ ID NO: 7, and a SEQ ID NO: the VH CDR3 shown by 8; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 9, VL CDR2 as shown in SEQ ID NO: 139, and SEQ ID NO: VL CDR3 shown by 11.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: a VH CDR1 as set forth in SEQ ID NO: 6, a VH CDR2 as set forth in SEQ ID NO: 7, and a SEQ ID NO: The VH CDR3 shown by 8; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 9, VL CDR2 as shown in SEQ ID NO: 140, and SEQ ID NO: VL CDR3 shown by 11.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH Heavy chain variable region
  • VH CDR1 which consists of the sequence: SEQ ID NO: 12, or has one or several amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3 amino acid substitutions, deletions or Add) the sequence,
  • VH CDR3 consisting of the sequence of SEQ ID NO: 14, or having one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3 amino acid substitutions, deletions or Add) the sequence, and
  • VH CDR2 consisting of the sequence of the VH CDR2 contained in the heavy chain variable region of any immunoglobulin
  • VL light chain variable region
  • VL CDR1 consisting of the sequence of SEQ ID NO: 15, or having one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3 amino acid substitutions, deletions or Add) the sequence,
  • VL CDR3 consisting of the sequence of SEQ ID NO: 17, or having one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3 amino acid substitutions, deletions or Add) the sequence,
  • VL CDR2 which consists of the sequence of the VL CDR2 contained in the light chain variable region of any immunoglobulin (eg, the kappa light chain variable region).
  • the VH CDR2 is the sequence of a VH CDR2 comprised in the heavy chain variable region of a human immunoglobulin.
  • the VH CDR2 consists of: (a) SEQ ID NO: 13, or one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2) a sequence of one or three amino acid substitutions, deletions or additions; or, (b) a sequence of the VH CDR2 contained in the amino acid sequence encoded by the human heavy chain germline gene.
  • the human heavy chain germline gene is selected from the group consisting of IGHV4-30-4*07 and IGHV4-4*01.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: (a) a VH CDR1 as set forth in SEQ ID NO: 12; a VH CDR3 as set forth in SEQ ID NO: 14; , VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 141 or SEQ ID NO: 142.
  • the VL CDR2 is the sequence of a VL CDR2 comprised in a light chain variable region of a human immunoglobulin (eg, a kappa light chain variable region).
  • the VL CDR2 consists of: (a) SEQ ID NO: 16, or one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2) a sequence of one or three amino acid substitutions, deletions or additions; or, (b) a sequence of the VL CDR2 contained in the amino acid sequence encoded by the human light chain germline gene.
  • the human light chain germline gene is selected from the group consisting of IGKV2-28*01 and IGKV3-15*01.
  • the VL of an antibody or antigen-binding fragment thereof of the invention comprises: a VL CDR1 as set forth in SEQ ID NO: 15; a VL CDR3 as set forth in SEQ ID NO: 17; VL CDR2 from the following amino acid sequence: SEQ ID NO: 16, SEQ ID NO: 143 or SEQ ID NO: 144.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: a VH CDR1 as set forth in SEQ ID NO: 12, a VH CDR3 as set forth in SEQ ID NO: 14, and a VH CDR2, It consists of the sequence of the VH CDR2 contained in the heavy chain variable region of any immunoglobulin (for example, SEQ ID NO: 13, SEQ ID NO: 141 or SEQ ID NO: 142);
  • VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 15, VL CDR3 as shown in SEQ ID NO: 17, and VL CDR2 as shown in SEQ ID NO: .
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: a VH CDR1 as set forth in SEQ ID NO: 12; a VH CDR3 as set forth in SEQ ID NO: 14; NO: 13 VH CDR2;
  • the VL of the antibody or antigen-binding fragment thereof comprises: a VL CDR1 as shown in SEQ ID NO: 15, a VL CDR3 as shown in SEQ ID NO: 17, and a VL CDR2, which consists of the following sequence:
  • the sequence of the VL CDR2 contained in the light chain variable region of the immunoglobulin eg, the kappa light chain variable region
  • SEQ ID NO: 16 SEQ ID NO: 143, or SEQ ID NO: 144.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: a VH CDR1 as set forth in SEQ ID NO: 12, a VH CDR3 as set forth in SEQ ID NO: 14, and a VH CDR2,
  • the VH CDR2 has a sequence selected from the group consisting of SEQ ID NO: 13, 141 or 142; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as set forth in SEQ ID NO: 15, such as SEQ ID NO VL CDR3 as shown in 17 and VL CDR2 having a sequence selected from the group consisting of SEQ ID NO: 16, 143 or 144.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: a VH CDR1 as set forth in SEQ ID NO: 12, a VH CDR2 as set forth in SEQ ID NO: 13, and SEQ ID NO: 14 is a VH CDR3; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 15, VL CDR2 as shown in SEQ ID NO: 16, and SEQ ID NO: 17 VL CDR3.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: a VH CDR1 as set forth in SEQ ID NO: 12, a VH CDR2 as set forth in SEQ ID NO: 141, and a SEQ ID NO: 14 is a VH CDR3; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 15, VL CDR2 as shown in SEQ ID NO: 16, and SEQ ID NO: 17 VL CDR3.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: a VH CDR1 as set forth in SEQ ID NO: 12, a VH CDR2 as set forth in SEQ ID NO: 142, and SEQ ID NO: 14 is a VH CDR3; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 15, VL CDR2 as shown in SEQ ID NO: 16, and SEQ ID NO: 17 VL CDR3.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: a VH CDR1 as set forth in SEQ ID NO: 12, a VH CDR2 as set forth in SEQ ID NO: 13, and SEQ ID NO: 14 VH CDR3; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 15, VL CDR2 as shown in SEQ ID NO: 143, and SEQ ID NO: 17 VL CDR3.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: a VH CDR1 as set forth in SEQ ID NO: 12, a VH CDR2 as set forth in SEQ ID NO: 13, and SEQ ID NO: 14 is a VH CDR3; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 15, VL CDR2 as shown in SEQ ID NO: 144, and SEQ ID NO: 17 VL CDR3.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH Heavy chain variable region
  • VH CDR1 which consists of the sequence: SEQ ID NO: 18, or has one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3 amino acid substitutions, deletions or Add) sequence;
  • VH CDR2 consisting of the sequence of SEQ ID NO: 19, or having one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3 amino acid substitutions, deletions or Add) the sequence,
  • VH CDR3 consisting of the sequence: SEQ ID NO: 20, or having one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3 amino acid substitutions, deletions or Add) sequence;
  • VL light chain variable region
  • VL CDR1 consisting of the sequence of SEQ ID NO: 15, or having one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3 amino acid substitutions, deletions or Add) the sequence,
  • VL CDR2 which consists of the sequence of SEQ ID NO: 16, or has one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3 amino acid substitutions, deletions or Add) the sequence,
  • VL CDR3 consisting of the sequence of SEQ ID NO: 17, or having one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2 or 3 amino acid substitutions, deletions or Add) the sequence.
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises: VH CDR1 as set forth in SEQ ID NO: 18, VH CDR2 as set forth in SEQ ID NO: 19, and SEQ ID NO: The VH CDR3 shown by 20; and the VL of the antibody or antigen-binding fragment thereof comprises: VL CDR1 as shown in SEQ ID NO: 15, VL CDR2 as shown in SEQ ID NO: 16, and SEQ ID NO: 17 VL CDR3.
  • an antibody or antigen-binding fragment thereof of the invention further comprises a framework region.
  • an antibody or antigen-binding fragment thereof of the invention comprises:
  • VH heavy chain variable region
  • FR framework regions
  • VH FR1 which consists of the sequence: SEQ ID NO: 21, SEQ ID NO: 29, SEQ ID NO: 37, SEQ ID NO: 44, SEQ ID NO: 50, or has one or a sequence of amino acid substitutions, deletions or additions (eg, 1, 2, 3 or 4 amino acid substitutions, deletions or additions);
  • VH FR2 which consists of the sequence: SEQ ID NO: 22, SEQ ID NO: 30, SEQ ID NO: 38, SEQ ID NO: 51, or one or more amino acid substitutions, deletions thereto Or adding a sequence (eg, 1, 2, 3 or 4 amino acid substitutions, deletions or additions);
  • VH FR3 which consists of the sequence: SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 39, SEQ ID NO: 45, SEQ ID NO: 52, or has one or a sequence of amino acid substitutions, deletions or additions (eg, 1, 2, 3 or 4 amino acid substitutions, deletions or additions);
  • VH FR4 which consists of the sequence: SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 46, SEQ ID NO: 53, or one or more amino acid substitutions, deletions thereto Or adding a sequence (eg, 1, 2, 3 or 4 amino acid substitutions, deletions or additions);
  • VL light chain variable region
  • FR framework regions
  • VL FR1 which consists of the sequence of SEQ ID NO: 25, SEQ ID NO: 33, SEQ ID NO: 47, SEQ ID NO: 54, or one or more amino acid substitutions, deletions thereto Or adding a sequence (eg, 1, 2, 3 or 4 amino acid substitutions, deletions or additions);
  • VL FR2 which consists of the sequence of SEQ ID NO: 26, SEQ ID NO: 34, SEQ ID NO: 48, SEQ ID NO: 55, or one or more amino acid substitutions, deletions thereto Or adding a sequence (eg, 1, 2, 3 or 4 amino acid substitutions, deletions or additions);
  • VL FR3 which consists of the sequence: SEQ ID NO:27, SEQ ID NO:35, SEQ ID NO:49, SEQ ID NO:56, or one or more amino acid substitutions, deletions thereto Or adding (eg, 1, 2, 3 or 4 amino acid substitutions, deletions or additions); and
  • VL FR4 which consists of the sequence of SEQ ID NO: 28, SEQ ID NO: 36, or one or more amino acid substitutions, deletions or additions thereto (eg, 1, 2, 3) Sequence of one or four amino acid substitutions, deletions or additions).
  • an antibody or antigen-binding fragment thereof of the invention comprises VH FR1, VH FR2, VH FR3 and VH FR4 as defined above. In certain preferred embodiments, an antibody or antigen-binding fragment thereof of the invention comprises VL FR1, VL FR2, VL FR3 and VL FR4 as defined above. In certain preferred embodiments, an antibody or antigen-binding fragment thereof of the invention comprises VH FR1, VH FR2, VH FR3, VH FR4, VL FR1, VL FR2, VL FR3 and VL FR4 as defined above.
  • the VH of the antibody or antigen-binding fragment thereof comprises:
  • VH FR1 as shown in SEQ ID NO: 21, VH FR2 as shown in SEQ ID NO: 22, VH FR3 as shown in SEQ ID NO: 23, and VH as shown in SEQ ID NO: FR4;
  • VH FR1 as shown in SEQ ID NO: 29, VH FR2 as shown in SEQ ID NO: 30, VH FR3 as shown in SEQ ID NO: 31, and VH as shown in SEQ ID NO: FR4; or
  • VH FR1 as shown in SEQ ID NO: 37
  • VH FR2 as shown in SEQ ID NO: 38
  • VH FR3 as shown in SEQ ID NO: 39
  • VH as shown in SEQ ID NO: FR4.
  • the VL of the antibody or antigen-binding fragment thereof comprises:
  • VL FR1 as shown in SEQ ID NO: 25, VL FR2 as shown in SEQ ID NO: 26, VL FR3 as shown in SEQ ID NO: 27, and VL as shown in SEQ ID NO: FR4; or
  • VL FR1 as shown in SEQ ID NO: 33
  • VL FR2 as shown in SEQ ID NO: 34
  • VL FR3 as shown in SEQ ID NO: 35
  • VL as shown in SEQ ID NO: FR4.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH FR1 as shown in SEQ ID NO: 21, VH FR2 as shown in SEQ ID NO: 22, VH FR3 as shown in SEQ ID NO: 23, and VH as shown in SEQ ID NO: FR4; and VL FR1 as shown in SEQ ID NO: 25, VL FR2 as shown in SEQ ID NO: 26, VL FR3 as shown in SEQ ID NO: 27, and SEQ ID NO: 28 VL FR4;
  • the antibodies or antigen-binding fragments thereof of the invention are humanized.
  • the antibody or antigen-binding fragment thereof of the invention has a degree of humanization of at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90 %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.
  • the non-CDR regions of the antibodies or antigen-binding fragments thereof of the invention comprise no more than 19, no more than 15, no more than 14, no more than 13, no more than 12, no more than 11, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no More than one non-human (eg, cynomolgus source) amino acid residue, or it does not contain non-human (eg, cynomolgus source) amino acid residues.
  • the FR region of an antibody or antigen-binding fragment thereof of the invention comprises no more than 19, no more than 15, no more than 14, no more than 13, no more than 12, no more than 11 No more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than One non-human (eg, cynomolgus source) amino acid residue, or it does not contain a non-human (eg, cynomolgus source) amino acid residue.
  • One non-human (eg, cynomolgus source) amino acid residue or it does not contain a non-human (eg, cynomolgus source) amino acid residue.
  • an antibody or antigen-binding fragment thereof of the invention comprises: a framework region of a human immunoglobulin, such as a framework region comprised in an amino acid sequence encoded by a human germline antibody gene.
  • the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region comprised in an amino acid sequence encoded by a human heavy chain germline gene, and/or a human light chain germline gene encoding Light chain framework regions contained in the amino acid sequence.
  • the heavy chain framework region and/or the light chain framework region of an antibody or antigen-binding fragment thereof of the invention may comprise one or more non-human (eg, cynomolgus source) amino acid residues.
  • the heavy chain framework region and/or the light chain framework region comprises one or more amino acid residues that are back-mutated to the corresponding cynomolgus monkey source residue or the corresponding cynomolgus monkey source Conservative amino acid amino acid substitutions of residues (such mutations are referred to as back mutations).
  • the VH of the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region comprised in an amino acid sequence encoded by the human heavy chain germline gene IGHV4-4*08, said heavy chain The framework region optionally comprises one or more back mutations from a human source residue to a cynomolgus monkey source residue.
  • the amino acid sequence encoded by IGHV4-4*08 is set forth in SEQ ID NO:40.
  • the VH of the antibody or antigen-binding fragment thereof comprises:
  • VH FR1 which is SEQ ID NO: 21, or differs from SEQ ID NO: 21 by one or several amino acid substitutions (eg, 1, 2 or 3 amino acid substitutions) selected from:
  • VH FR2 which is SEQ ID NO: 22;
  • VH FR3 which is SEQ ID NO: 23, or differs from SEQ ID NO: 23 by one or several amino acid substitutions (eg, 1, 2 or 3 amino acid substitutions) selected from:
  • VH FR4 which is SEQ ID NO: 24, or differs from SEQ ID NO: 24 by one or several amino acid substitutions (eg, 1 or 2 amino acid substitutions) selected from:
  • the VH of the antibody or antigen-binding fragment thereof comprises: VH FR1 as set forth in SEQ ID NO: 44, VH FR2 as set forth in SEQ ID NO: 22, and SEQ ID NO: VH FR3 shown at 45, and VH FR4 as shown in SEQ ID NO: 46.
  • the VH of the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region comprised in an amino acid sequence encoded by the human heavy chain germline gene IGHV4-4*02, said heavy chain The framework region optionally comprises one or more back mutations from a human source residue to a cynomolgus monkey source residue.
  • the amino acid sequence encoded by IGHV4-4*02 is set forth in SEQ ID NO:42.
  • the VH of the antibody or antigen-binding fragment thereof comprises:
  • VH FR1 which is SEQ ID NO: 29, or differs from SEQ ID NO: 29 by one or several amino acid substitutions (eg, 1 or 2) selected from the group consisting of:
  • VH FR2 which is SEQ ID NO: 30, or differs from SEQ ID NO: 30 by the following amino acid substitutions: V at H37;
  • VH FR3 which is SEQ ID NO: 31, or differs from SEQ ID NO: 31 by one or several amino acid substitutions selected from the group consisting of (eg, 1, 2, 3 or 4 amino acid substitutions) ):
  • VH FR4 which is SEQ ID NO: 32, or differs from SEQ ID NO: 32 by one of the following amino acid substitutions: T at H107;
  • the VH of the antibody or antigen-binding fragment thereof comprises: VH FR1 as set forth in SEQ ID NO: 50, VH FR2 as set forth in SEQ ID NO: 51, and SEQ ID NO: VH FR3 shown in 52, and VH FR4 as shown in SEQ ID NO: 53.
  • the VL of the antibody or antigen-binding fragment thereof comprises: a light chain framework region comprised in an amino acid sequence encoded by the human light chain germline gene IGKV1-39*01, the light chain The framework region optionally comprises one or more back mutations from a human source residue to a cynomolgus monkey source residue.
  • the amino acid sequence encoded by IGKV1-39*01 is set forth in SEQ ID NO:41.
  • the VL of the antibody or antigen-binding fragment thereof comprises:
  • VL FR1 which is SEQ ID NO: 25, or differs from SEQ ID NO: 25 by one or several amino acid substitutions (eg, one, or two amino acid substitutions) selected from the group consisting of:
  • VL FR2 which is SEQ ID NO: 26, or differs from SEQ ID NO: 26 by the following amino acid substitutions: K at L45;
  • VL FR3 which is SEQ ID NO: 27, or differs from SEQ ID NO: 27 by one or several amino acid substitutions selected from the group consisting of (eg, 1, 2, 3 or 4 amino acid substitutions) ):
  • VL FR4 which is SEQ ID NO: 28;
  • the VL of the antibody or antigen-binding fragment thereof comprises: VL FR1 as set forth in SEQ ID NO: 47, VL FR2 as set forth in SEQ ID NO: 48, and SEQ ID NO: VL FR3 shown as 49, and VL FR4 as shown in SEQ ID NO: 28.
  • the VL of the antibody or antigen-binding fragment thereof comprises: a light chain framework region comprised in an amino acid sequence encoded by the human light chain germline gene IGKV4-1*01, said light chain The framework region optionally comprises one or more back mutations from a human source residue to a cynomolgus monkey source residue.
  • the amino acid sequence encoded by IGKV4-1*01 is set forth in SEQ ID NO:43.
  • the VL of the antibody or antigen-binding fragment thereof comprises:
  • VL FR1 which is SEQ ID NO: 33, or differs from SEQ ID NO: 33 by one or several amino acid substitutions (eg, 1, 2 or 3 amino acid substitutions) selected from:
  • VL FR2 which is SEQ ID NO: 34, or differs from SEQ ID NO: 34 by the following amino acid substitutions: V at L43;
  • VL FR3 which is SEQ ID NO: 35, or differs from SEQ ID NO: 35 by one or more amino acid substitutions (eg, 1, 2 or 3 amino acid substitutions) selected from:
  • VL FR4 which is SEQ ID NO: 36;
  • amino acid positions mentioned above are based on the position of the Kabat numbering system.
  • the VL of the antibody or antigen-binding fragment thereof comprises: VL FR1 as set forth in SEQ ID NO: 54, VL FR2 as set forth in SEQ ID NO: 55, and SEQ ID NO: VL FR3 shown in 56, and VL FR4 as shown in SEQ ID NO: 36.
  • an antibody or antigen-binding fragment thereof of the invention comprises:
  • the antibody or antigen-binding fragment thereof comprises:
  • VH FR1 as shown in SEQ ID NO: 44 VH FR2 as shown in SEQ ID NO: 22, VH FR3 as shown in SEQ ID NO: 45, VH FR4 as shown in SEQ ID NO: 46
  • the VH of an antibody or antigen-binding fragment thereof of the invention comprises:
  • VH FR1 as shown in SEQ ID NO: 21, VH FR2 as shown in SEQ ID NO: 22, VH FR3 as shown in SEQ ID NO: 23, and VH as shown in SEQ ID NO: FR4;
  • VH FR1 as shown in SEQ ID NO: 29, VH FR2 as shown in SEQ ID NO: 30, VH FR3 as shown in SEQ ID NO: 31, and VH as shown in SEQ ID NO: FR4;
  • VH FR1 as shown in SEQ ID NO: 37, VH FR2 as shown in SEQ ID NO: 38, VH FR3 as shown in SEQ ID NO: 39, and VH as shown in SEQ ID NO: FR4;
  • VH FR1 as shown in SEQ ID NO: 44, VH FR2 as shown in SEQ ID NO: 22, VH FR3 as shown in SEQ ID NO: 45, and VH as shown in SEQ ID NO: 46 FR4; or
  • VH FR1 as shown in SEQ ID NO: 50
  • VH FR2 as shown in SEQ ID NO: 51
  • VH FR3 as shown in SEQ ID NO: 52
  • VH as shown in SEQ ID NO: FR4.
  • the VL of an antibody or antigen-binding fragment thereof of the invention comprises:
  • VL FR1 as shown in SEQ ID NO: 25, VL FR2 as shown in SEQ ID NO: 26, VL FR3 as shown in SEQ ID NO: 27, and VL as shown in SEQ ID NO: FR4;
  • VL FR1 as shown in SEQ ID NO: 33, VL FR2 as shown in SEQ ID NO: 34, VL FR3 as shown in SEQ ID NO: 35, and VL as shown in SEQ ID NO: FR4;
  • VL FR1 as shown in SEQ ID NO: 47
  • VL FR2 as shown in SEQ ID NO: 48
  • VL FR3 as shown in SEQ ID NO: 49
  • VL as shown in SEQ ID NO: FR4;
  • VL FR1 as shown in SEQ ID NO: 54
  • VL FR2 as shown in SEQ ID NO: 55
  • VL FR3 as shown in SEQ ID NO: 56
  • VL as shown in SEQ ID NO: 36 FR4.
  • the heavy chain variable region of an antibody or antigen-binding fragment thereof of the invention comprises VH FR1, VH CDR1, VH FR2, VH CDR2, VH FR3, VH CDR3 and VH FR4 as defined above .
  • the light chain variable region of an antibody or antigen-binding fragment thereof of the invention comprises VL FR1, VL CDR1, VL FR2, VL CDR2, VL FR3, VL CDR3 and VL FR4 as defined above .
  • the heavy chain variable region of an antibody or antigen-binding fragment thereof of the invention comprises VH FR1, VH CDR1, VH FR2, VH CDR2, VH FR3, VH CDR3 and VH FR4 as defined above
  • the light chain variable region comprises VL FR1, VL CDR1, VL FR2, VL CDR2, VL FR3, VL CDR3 and VL FR4 as defined above.
  • the amino acid sequence of the heavy chain variable region of an antibody or antigen-binding fragment thereof of the invention has at least 80%, at least 85%, at least an amino acid sequence selected from the following heavy chain variable regions 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity: as SEQ ID NOs: a heavy chain variable region as shown in any one of 1, 3, 5, 57, 59, 157, 158, 163 and 164.
  • the heavy chain variable region of an antibody or antigen-binding fragment thereof of the invention is selected from the group consisting of SEQ ID NOs: 1, 3, 5, 57, 59, 157, 158, 163 and 164 The heavy chain variable region is shown.
  • the amino acid sequence of the light chain variable region of an antibody or antigen-binding fragment thereof of the invention has at least 80%, at least 85%, at least an amino acid sequence selected from the following light chain variable regions 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity: as SEQ ID NOs: a light chain variable region as shown in any one of 2, 4, 58, 60, 159, 160, 165 and 166.
  • the light chain variable region of an antibody or antigen-binding fragment thereof of the invention is selected from the group consisting of any one of SEQ ID NOs: 2, 4, 58, 60, 159, 160, 165 and 166 Light chain variable region.
  • an antibody of the invention comprises a heavy chain variable region as defined above and a light chain variable region as defined above.
  • the heavy chain variable region of an antibody or antigen-binding fragment thereof of the invention is selected from the heavy chain variable region set forth in any one of SEQ ID NOs: 1, 57, 157 and 158;
  • the light chain variable region of the antibody or antigen-binding fragment thereof is selected from the light chain variable regions set forth in any one of SEQ ID NOs: 2, 58, 159 and 160.
  • the heavy chain variable region of an antibody or antigen-binding fragment thereof of the invention is selected from the heavy chain variable region set forth in any one of SEQ ID NOs: 3, 59, 163 and 164;
  • the light chain variable region of the antibody or antigen-binding fragment thereof is selected from the light chain variable regions set forth in any one of SEQ ID NOs: 4, 60, 165 and 166.
  • the antibodies of the invention comprise:
  • VH as shown in SEQ ID NO: 1 and VL as shown in SEQ ID NO: 2;
  • VH as shown in SEQ ID NO: 59 and VL as shown in SEQ ID NO: 60;
  • VH as shown in SEQ ID NO: 59 and VL as shown in SEQ ID NO: 165;
  • the antibodies of the invention can be obtained by genetic engineering recombination techniques.
  • a DNA molecule encoding a heavy chain and a light chain gene of an antibody of the present invention is obtained by chemical synthesis or PCR amplification.
  • the resulting DNA molecule is inserted into an expression vector and then transfected into a host cell, such as an E. coli cell, a simian COS, a CHO cell, or other myeloma cell that does not produce an immunoglobulin.
  • the transfected host cells are then cultured under specific conditions and the antibodies of the invention are expressed.
  • the antibodies of the invention have high specificity and high affinity for the HBsAg protein.
  • the antibody of the invention may have a KD value in combination with HBsAg of less than 1 x 10 -5 M; preferably, the KD value is less than 1 x 10 -6 M; more preferably, the KD value is less than 1 x 10 -7 M; most preferably, the KD value Less than 1x 10 -8 M.
  • the antibody of the present invention may be an antibody having a conventional "Y" type structure comprising two heavy chains and two light chains.
  • the antibody of the present invention may also be a Fab fragment, Fab', F(ab) 2 , Fv, or other type of fragment of an antibody having a conventional "Y" type structure, which retains affinity for the HBsAg protein, and Its affinity for binding to HBsAg protein can be higher or lower than antibodies with a traditional "Y" type structure.
  • the antigen-binding fragment of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24: 107-117 (1992) and Brennan et al., Science 229: 81 (1985)) .
  • these antigen-binding fragments can also be directly produced by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000) )).
  • Fab' fragments can be obtained directly from E.
  • Fv, Fab or F(ab') 2 fragments can be isolated directly from recombinant host cell culture media. Other techniques for preparing these antigen-binding fragments are well known to those of ordinary skill in the art.
  • the antibody or antigen-binding fragment thereof of the invention is selected from the group consisting of scFv, Fab, Fab', (Fab') 2 , Fv fragment, diabody, bispecific antibody, multiple Specific antibodies, chimeric antibodies or humanized antibodies.
  • the antibody or antigen-binding fragment thereof of the invention is a chimeric antibody or a humanized antibody.
  • an antibody or antigen-binding fragment thereof of the invention is capable of specifically binding to HBsAg, neutralizing the virulence of HBV, and/or reducing serum levels of HBV DNA and/or HBsAg in a subject.
  • the antibodies or antigen-binding fragments thereof of the invention are of the IgG class.
  • an antibody of the invention or antigen-binding fragment thereof can be of the IgGl or IgG2 or IgG3 or IgG4 class.
  • a fusion antibody or immunoadhesin can be prepared, for example, an antibody of the invention or antigen-binding fragment thereof can be linked to another polypeptide.
  • the fusion antibody comprises a heavy chain variable region and a light chain variable region against an antibody of the invention.
  • the fusion antibody comprises a VH domain and a VL domain of an antibody of the invention; wherein the VH domain is linked to a first polypeptide and the VL domain is linked to a second polypeptide .
  • An antibody or antigen-binding fragment thereof of the invention can be derivatized, for example, linked to another molecule (e.g., another polypeptide or protein).
  • derivatization e.g, labeling
  • an antibody or antigen-binding fragment thereof of the invention is also intended to include such derivatized forms.
  • an antibody or antigen-binding fragment thereof of the invention can be functionally linked (by chemical coupling, gene fusion, non-covalent attachment or otherwise) to one or more other molecular groups, such as another antibody (eg, formed) A bispecific antibody), a detection reagent, a pharmaceutical agent, and/or a protein or polypeptide (eg, an avidin or a polyhistidine tag) capable of mediating binding of an antibody or antigen-binding fragment to another molecule.
  • another antibody eg, formed
  • a bispecific antibody e.g, a detection reagent, a pharmaceutical agent, and/or a protein or polypeptide (eg, an avidin or a polyhistidine tag) capable of mediating binding of an antibody or antigen-binding fragment to another molecule.
  • a protein or polypeptide eg, an avidin or a polyhistidine tag
  • One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or different types).
  • Suitable crosslinking agents include, for example, heterobifunctional difunctional agents containing two different reactive groups (eg, m-butyramide benzoic acid-N-hydroxysuccinimide ester) separated by appropriate spacers; And, a homobifunctional (bissuccinimide suberate).
  • Such crosslinkers are commercially available from Pierce Chemical Company, Rockford, II.
  • an antibody of the invention or antigen-binding fragment thereof can be ligated to a useful detection reagent.
  • detection reagents include, for example, fluorescent compounds such as fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamino-1-casulfonyl chloride, phycoerythrin, strontium phosphor, and the like.
  • the antibody may also be labeled with an enzyme such as horseradish peroxidase, ⁇ -galactosidase, luciferase, alkaline phosphatase, glucose oxidase or the like.
  • the labeled antibody When the antibody is labeled with an enzyme, the labeled antibody can be detected by the addition of a reagent that can be utilized by the enzyme and produces an identifiable signal or reaction product.
  • a reagent that can be utilized by the enzyme and produces an identifiable signal or reaction product.
  • hydrogen peroxide and diaminobiphenyl can be added to produce a detectable colored reaction product to detect the presence or amount of labeled antibody.
  • antibodies can also be labeled with biotin. In this case, the presence or amount of the labeled antibody can be detected by indirectly measuring the binding of avidin.
  • antibodies can also be labeled with a tag that is recognized by a second reporter molecule (eg, a leucine zipper complementary sequence, a metal binding domain, an epitope tag, etc.).
  • a second reporter molecule eg, a leucine zipper complementary sequence, a metal binding domain, an epitope tag, etc.
  • the tag is attached to the antibody by spacer arms of different lengths to reduce potential steric hindrance.
  • the antibodies or antigen-binding fragments thereof of the invention may also be derivatized with a chemical group, such as polyethylene glycol (PEG), methyl or ethyl, or a glycosyl group. These groups can be used to improve the biological properties of the antibody, such as increasing serum half-life.
  • a chemical group such as polyethylene glycol (PEG), methyl or ethyl, or a glycosyl group.
  • Nucleic acid molecule Nucleic acid molecule, vector and host cell
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof of the invention, or a heavy chain variable region thereof and/or a light chain variable region thereof.
  • an isolated nucleic acid molecule of the invention encodes an antibody or antigen-binding fragment thereof according to the invention, or a heavy chain variable region thereof and/or a light chain variable region thereof.
  • the invention provides a vector (eg, a cloning vector or an expression vector) comprising an isolated nucleic acid molecule according to the invention.
  • the vectors of the invention are, for example, plasmids, cosmids, phages, and the like.
  • the vector is capable of expressing an antibody or antigen-binding fragment thereof of the invention in a subject, such as a mammal, such as a human.
  • the invention provides a host cell comprising an isolated nucleic acid molecule according to the invention or a vector according to the invention.
  • host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (eg, mammalian cells, such as mouse cells, human cells, etc.).
  • the cells of the invention may also be cell lines, such as 293T cells.
  • a method of making an antibody or antigen-binding fragment thereof according to the invention comprising culturing a host cell according to the invention under conditions permitting expression of the antibody or antigen-binding fragment thereof, and from culturing The antibody or antigen-binding fragment thereof is recovered from the host cell culture.
  • the antibody or antigen-binding fragment thereof of the present invention is capable of specifically binding to HBsAg, and thus can be used for detecting the presence or level of HBsAg protein in a sample, and can be used for diagnosing whether a subject is infected with HBV.
  • the invention provides a kit comprising an antibody of the invention or an antigen binding fragment thereof.
  • the antibody or antigen-binding fragment thereof of the invention further comprises a detectable label.
  • the kit further comprises a second antibody that specifically recognizes an antibody or antigen-binding fragment thereof of the invention.
  • the second antibody further comprises a detectable label.
  • the antibodies of the invention, or antigen-binding fragments thereof, or secondary antibodies can be labeled according to the methods detailed above.
  • an antibody or antigen-binding fragment thereof of the invention can be ligated to a detectable label.
  • detectable labels are well known to those skilled in the art and include, but are not limited to, radioisotopes, fluorescent materials, luminescent materials, colored materials and enzymes (e.g., horseradish peroxidase), and the like.
  • detectable labels also include, for example, radioisotopes such as 125 iodine; fluorescent substances such as fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamino-1-casulfonyl chloride, phycoerythrin , ⁇ phosphorescent agents, and the like; enzymes capable of producing an identifiable signal or reaction product, such as horseradish peroxidase, ⁇ -galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like; A tag that is recognized by a second reporter molecule, such as biotin, avidin, a leucine zipper complementary sequence, a metal binding domain, an epitope tag, and the like.
  • a detection reagent eg, a tag
  • a linker of varying length to reduce potential steric hindrance.
  • the invention provides a method of detecting the presence or level of an HBsAg protein in a sample comprising the use of an antibody of the invention or an antigen binding fragment thereof.
  • the antibody or antigen-binding fragment thereof of the invention further comprises a detectable label.
  • the method further comprises detecting the antibody or antigen-binding fragment thereof of the invention using a second antibody carrying a detectable label. The method can be used for diagnostic purposes, or for non-diagnostic purposes (eg, the sample is a cell sample, not a sample from a patient).
  • the invention provides a method of diagnosing whether a subject is infected with HBV, comprising: detecting the presence of an HBsAg protein in a sample from the subject using an antibody of the invention or an antigen binding fragment thereof.
  • the antibody or antigen-binding fragment thereof of the invention further comprises a detectable label.
  • the method further comprises detecting the antibody or antigen-binding fragment thereof of the invention using a second antibody carrying a detectable label.
  • an antibody or antigen-binding fragment thereof of the invention in the preparation of a kit for detecting the presence or level of an HBsAg protein in a sample, or for diagnosing whether a subject is Infected with HBV.
  • the antibody or antigen-binding fragment thereof of the present invention can be used for preventing or treating a HBV infection of a subject (for example, a human) or a disease associated with HBV infection (for example, hepatitis B), for use in vitro or in a subject (for example, a human). And the virulence of HBV, as well as serum levels for reducing HBV DNA and/or HBsAg in a subject (eg, a human).
  • the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to the invention, and a pharmaceutically acceptable carrier and/or excipient.
  • the pharmaceutical compositions of the invention may further comprise additional pharmaceutically active agents.
  • the additional pharmaceutically active agent is a drug for preventing or treating a HBV infection or a disease associated with HBV infection, such as hepatitis B, such as other antiviral agents, such as interferon drugs, such as Interferon or peginterferon.
  • an antibody or antigen-binding fragment thereof according to the invention or a pharmaceutical composition according to the invention for the preparation of a medicament for the prevention or treatment of a HBV infection in a subject, such as a human Or a disease associated with HBV infection (eg, hepatitis B), used to neutralize the virulence of HBV in vitro or in a subject (eg, a human), and/or to reduce HBV DNA and in a subject (eg, a human) / or serum levels of HBsAg.
  • a human Or a disease associated with HBV infection eg, hepatitis B
  • the invention provides a method for preventing or treating a HBV infection or a disease associated with HBV infection (eg, hepatitis B) in a subject, for neutralizing HBV toxicity in a subject (eg, a human) And, or a method for reducing serum levels of HBV DNA and/or HBsAg in a subject (eg, a human), the method comprising administering to a subject in need thereof an effective amount of an antibody of the invention Or an antigen binding fragment thereof, or a pharmaceutical composition of the invention.
  • a HBV infection or a disease associated with HBV infection eg, hepatitis B
  • a method for reducing serum levels of HBV DNA and/or HBsAg in a subject eg, a human
  • the antibody or antigen-binding fragment thereof of the present invention or the pharmaceutical composition of the present invention may be administered by a conventional administration route including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, Intracytoplasmic stenosis, inguinal, intravesical, topical (eg, powder, ointment or drops), or nasal route.
  • the antibodies or antigen-binding fragments thereof of the invention can be administered by a variety of methods known in the art. However, for many therapeutic uses, the preferred route/mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). The skilled artisan will appreciate that the route and/or manner of administration will vary depending on the intended purpose.
  • the antibody or antigen-binding fragment thereof of the invention is administered by intravenous infusion or injection.
  • the antibody or antigen-binding fragment thereof of the present invention or the pharmaceutical composition of the present invention may be formulated into various dosage forms such as liquid, semi-solid and solid dosage forms, such as solutions (for example, injections), dispersing or suspending agents, tablets, powders. , granules, emulsions, pills, syrups, powders, liposomes, capsules and suppositories.
  • dosage forms such as liquid, semi-solid and solid dosage forms, such as solutions (for example, injections), dispersing or suspending agents, tablets, powders. , granules, emulsions, pills, syrups, powders, liposomes, capsules and suppositories.
  • the preferred dosage form will depend on the intended mode of administration and therapeutic use.
  • a preferred dosage form is an injectable.
  • Such an injection may be a sterile injectable solution.
  • a sterile injectable solution can be prepared by incorporating the required amount of the antibody or antigen-binding fragment thereof of the present invention in a suitable solvent, and optionally, incorporating other desired ingredients (including but not Limited to pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof, followed by filtration sterilization.
  • sterile injectable solutions can be prepared as a sterile lyophilized powder (for example, by vacuum drying or freeze drying) for ease of storage and use. Such sterile lyophilized powders can be dispersed in a suitable vehicle, such as sterile pyrogen-free water, before use.
  • Dispersing agents can be prepared by incorporating the antibody or antigen-binding fragment thereof of the invention into a sterile carrier containing a base dispersion medium and optionally other desired ingredients (including but not limited to, pH adjustment) Agents, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof.
  • agents which delay absorption may be incorporated into the dispersing agent, such as monostearate and gelatin, to achieve the desired pharmacokinetic profile.
  • Such solid phase dosage forms typically comprise at least one of the following: (a) an inert pharmaceutical excipient (or carrier) such as sodium citrate, calcium phosphate; (b) a filler such as starch, lactose, sucrose, nectar Sugar and silicic acid; (c) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (d) wetting agents such as glycerin; (e) fragmentation agents, Such as agar, calcium carbonate, potato flour or tapioca flour; (f) retarder, such as paraffin; (g) an absorbent, such as a tetraamino mixture; (h) a humectant, such as cetyl alcohol and monostearic acid a glyceride; (i) an adsorbent such as kaolin and bentonit
  • release rate modifying agents i.e., agents that alter the rate of drug release
  • release rate modifying agents include, but are not limited to, carboxypropyl methylcellulose, methylcellulose, sodium carbon methylcellulose, cellulose ethane, cellulose acetate, polyethylene oxide, xanthan gum, Aminoacrylic acid copolymer, hydrogenated flavoring oil, carnauba wax, paraffin wax, cellulose acetate phthalate, carboxypropyl methylcellulose phthalate, methacrylic acid copolymer, or any combination thereof.
  • Modified release and pulsed release dosage forms can contain one or a set of release rate modifying agents.
  • Another preferred dosage form is a liquid dosage form for oral administration including, for example, emulsions, solutions, suspensions, syrups and the like.
  • such oral liquid dosage forms may contain inert solutions such as water or other solvents commonly employed in the art, such as ethane alcohol, isopropyl alcohol, propylene glycol, 1,3-butyl Alkenyl glycol, oils (such as cottonseed oil, groundnut oil, corn oil, olive oil, flavoring oil and sesame oil), glycerin, polyethylene glycol and fatty acid sorbitol ester, and any combination thereof.
  • inert solutions such as water or other solvents commonly employed in the art, such as ethane alcohol, isopropyl alcohol, propylene glycol, 1,3-butyl Alkenyl glycol, oils (such as cottonseed oil, groundnut oil, corn oil, olive oil, flavoring oil and sesame oil), glycerin, polyethylene glycol and fatty acid sorbitol ester, and
  • the antibody or antigen-binding fragment thereof of the present invention may be present in a pharmaceutical composition in unit dosage form for ease of administration.
  • the pharmaceutical compositions of the invention should be sterile and stable under the conditions of manufacture and storage.
  • the pharmaceutical and pharmaceutical compositions provided by the present invention may be used alone or in combination, or may be combined with another pharmaceutically active agent (for example, other antiviral agents such as interferon drugs such as interferon or peginterferon). use.
  • the antibodies or antigen-binding fragments thereof of the invention are used in combination with other antiviral agents to prevent and or treat diseases associated with hepatitis B virus infection.
  • the antibody or antigen-binding fragment thereof of the present invention can be administered simultaneously, separately or continuously with such an antiviral agent.
  • antiviral agents include, but are not limited to, interferons, ribavirin, adamantane, carboxyurea, IL-2, L-12, and pentacarboxylate cyano.
  • compositions of the invention may comprise a "therapeutically effective amount” or a “prophylactically effective amount” of an antibody or antigen-binding fragment of the invention.
  • prophylactically effective amount is meant an amount sufficient to prevent, arrest, or delay the onset of a disease, such as a HBV infection or a disease associated with HBV infection.
  • therapeutically effective amount is meant an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease.
  • the therapeutically effective amount of an antibody or antigen-binding fragment of the invention may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's own immune system, the general condition of the patient such as age, weight and sex, and the mode of administration of the drug. , as well as other treatments administered at the same time, and the like.
  • the dosage regimen can be adjusted to achieve the best purpose response (eg, a therapeutic or prophylactic response). For example, it may be administered in a single administration, may be administered multiple times over a period of time, or may be proportionally reduced or increased depending on the urgency of the treatment.
  • a therapeutic or prophylactic response e.g., a prophylactic response
  • a typical non-limiting range of therapeutically or prophylactically effective amounts of an antibody or antigen-binding fragment of the invention is from 0.025 to 50 mg/kg, more preferably from 0.1 to 50 mg/kg, more preferably from 0.1 to 25 mg/kg, from 0.1 to 10 mg/kg. It should be noted that the dosage may vary depending on the type and severity of the condition to be treated. Moreover, those skilled in the art understand that for any particular patient, the particular dosage regimen should be adjusted over time according to the needs of the patient and the professional evaluation of the physician; the dosage ranges given herein are for illustrative purposes only and are not limiting Use or range of the pharmaceutical compositions of the invention.
  • the antibody of the present invention can not only specifically recognize/bind HBsAg, but also neutralize the virulence of HBV, and can lower the serum level of HBV DNA and/or HBsAg in a subject, and can effectively remove HBV and the body in vivo. HBV-infected cells. Therefore, the antibody of the present invention has a potential for preventing and treating HBV infection and diseases associated with HBV infection such as hepatitis B.
  • the antibody (especially the humanized antibody) of the present invention not only retains the function and properties of the parent monkey-derived antibody, but has potential for preventing and treating HBV infection and diseases associated with HBV infection such as hepatitis B; Moreover, it has a very high degree of humanization (humanization can be as high as 98%), so that it can be safely administered to a human subject without eliciting an immunogenic reaction. Therefore, the antibodies (especially humanized antibodies) of the invention have great clinical value.
  • Example 1 Preparation of human-cynomolgus chimeric monoclonal antibody that specifically binds to HBsAg
  • the recombinant HBV vaccine developed in the laboratory was used for intramuscular immunization of cynomolgus monkeys with a body weight of 4 ⁇ 1 kg for more than 6 months at an injection dose of 20 ⁇ g/monkey/time.
  • the immunization time points were at 0, 2, 6, 10, 14, 18, 22, and 26 weeks, respectively.
  • cynomolgus monkey serum titer reached plateau, we produced a large number of memory B cells on the 13th, 17th, 25th, and 29th week (ie, the third week after each immunization).
  • the peripheral blood of cynomolgus monkeys was collected at 10 ml/monkey/time.
  • SepMate TM tubes for separating mononuclear cells (the MNCs) from human peripheral whole blood and cord blood samples by density gradient centrifugation. Make sure that the sample, PBS buffer (PBS + 2% FBS) containing 2% fetal bovine serum (FBS), density gradient centrifugation, and centrifuge are at room temperature (15-25 ° C) before separation. Specific steps are as follows:
  • PBMC cells isolated according to the method of 1.2 above were centrifuged at 300 g for 10 minutes, and then the cells were washed once with 10 mL of serum-free 1640 medium.
  • the irradiation dose is 40Gy.
  • specific operation steps refer to the "RS 2000 Biological X-ray Irradiation Instrument User's Manual”.
  • the cells resuspended in RPMI 1640 medium were mixed at 5 cells/well with the prepared 50 ng/mL IL21 and the irradiated HEK293-CD154 at 10 4 cells/well, and then incubated together in a 96-well plate.
  • the well is 250 ⁇ L.
  • the cells were placed in a CO 2 incubator at 37 ° C and stimulated for 14 days.
  • a commercial HBsAg plate (purchased from Beijing Wantai) was taken, 100 ⁇ L of the supernatant to be tested was added to each well, and incubated at 37 ° C for 1 h. Subsequently, the ELISA plate was washed 5 times with PBST, then 100 ⁇ L of GAH-HRP diluted 1:5000 was added, and incubated at 37 ° C for 30 min. Subsequently, the ELISA plate was washed 5 times with PBST and the substrate TMB solution was added. After 15 min of color development, the color reaction was stopped with H 2 SO 4 and the reading was determined at OD450/620. RPMI 1640 medium was used as a negative control.
  • the culture plate containing the positive B cell culture well was centrifuged at 300 ⁇ g for 5 min, and the supernatant of the cells was aspirated.
  • 150 ⁇ L of Lysis Buffer (purchased from Beijing GenMagBio) lysate was added, repeatedly pipetted several times with a pipette tip, transferred to a RNase-free 1.5 mL Eppendorf tube, and placed in a -80 ° C refrigerator for one year.
  • RNA extraction method is referred to the viral DNA/RNA co-extraction kit instructions (GenMagBio).
  • B cell antibody H chain, ⁇ light chain and ⁇ light chain variable region genes are amplified by nested PCR, amplification primer reference (Meng W, Li L, Xiong W, et al. Efficient generation of monoclonal antibodies from single rhesus macaque Antibody secreting cells [C]//mAbs.Taylor&Francis, 2015, 7(4): 707-718).
  • amplification primer reference Meng W, Li L, Xiong W, et al. Efficient generation of monoclonal antibodies from single rhesus macaque Antibody secreting cells [C]//mAbs.Taylor&Francis, 2015, 7(4): 707-718.
  • Replace the homologous sequence on the second round of primers with the vector into the eukaryotic expression vector A section on (see Example 1.5.5.1) is used as a homologous sequence.
  • the PCR reaction system was prepared according to Table 3, and each system was 25 ⁇ L
  • the first round of nested reaction uses reverse-transcribed cDNA as a template, and the reaction conditions are: 95 ° C, 5 min; 95 ° C, 30 s; 55 ° C, 60 s; 72 ° C, 90 s; 72 ° C, 7 min;
  • the second round of nested reaction uses the first round of amplification products as a template.
  • the reaction conditions are: 95 ° C, 5 min; 95 ° C, 30 s; 58 ° C, 60 s; 72 ° C, 90 s; 72 ° C, 7 min; The number is 35.
  • the amplified product was then analyzed by agarose gel electrophoresis, and the amplified product was recovered using a DNA purification recovery kit (TianGen, DP214-03).
  • the invention specially transforms the existing eukaryotic expression vector pTT5 in the laboratory, and constructs a recombinant vector for light and heavy chain for co-transfection of double plasmids.
  • the light and heavy chain signal peptide uses MGWSCIILFLVATATGVHS. Downstream of the signal peptide, the sequences encoding the light and heavy chain constant regions of human antibodies were respectively inserted into a set of pTT5-CH, pTT5-C ⁇ and pTT5-C ⁇ eukaryotic expression vectors for antibody recombination.
  • the constructed eukaryotic expression vector was ligated with the antibody variable region gene PCR product recovered in 1.5.4 (primer with vector homologous sequence) using a laboratory-made Gibson assembly solution to obtain a recombinant vector VH+pTT5. -CH, VH+pTT5-C ⁇ and VH+pTT5-C ⁇ .
  • the recombinant vector was transformed into Escherichia coli DH5 ⁇ strain, coated with LB plates, and cultured overnight in a 37 ° C incubator. Monoclonal colonies were picked from the plates and sequenced. The sequencing results were aligned using IgBlast to confirm the correctness of the genes and exclude the pseudogenes.
  • the constructed recombinant vector VH+pTT5-CH and VH+pTT5-C ⁇ and VH+pTT5-C ⁇ were co-transfected into HEK293- cells, and the small expression double plasmid was co-transfected into 500 ⁇ L 24-well plates per well, such as small.
  • the cell supernatant of the expressed amount has antigenic activity, and the transfection system is amplified to 100 mL (depending on the amount of antibody) of CHO-S suspension cells (cell density is about 2 x 10 6 cells/ml).
  • the transfected cells were cultured in a shake flask at 32 ° C in a 5% CO 2 incubator, and after 7 days of expression, the supernatant was collected.
  • Cell expression supernatants were collected and purified using a Protein A column according to the manufacturer's instructions. The specific procedure was as follows: The harvested cell culture supernatant was centrifuged at 8000 rpm for 10 min, and the supernatant was retained, and the pH was adjusted to 8.4 with dry powder Na 2 HPO 4 , followed by filtration through a 0.22 ⁇ m pore size filter. The 10 mL column of Sepharose 4B medium coupled with Protein A was connected to the AKTA Explorer 100 system, the A pump was connected to a 0.2 M disodium hydrogen phosphate solution, and the B pump was connected to a 0.2 M citric acid solution.
  • Example 2 Characterization of cynomolgus-human chimeric monoclonal antibodies that specifically bind to HBsAg
  • Three cynomolgus monkey-human chimeric monoclonal antibodies specific for binding to HBsAg were obtained by the method of Example 1, and were named M1-23, M3-23, M3-13, respectively.
  • the VH and VL amino acid sequences of the three antibodies are shown in the following table (SEQ ID NOS: 1-5).
  • the CDR sequences of the three antibodies were also determined using IMGT, and the amino acid sequences of the CDRs of the heavy chain variable region and the light chain variable region are shown in Table 5 (SEQ ID NO: 6-20).
  • the inventors performed a series of properties analysis on the purified three monoclonal antibodies. Firstly, the binding ability of M1-23, M3-23 and M3-13 to HBsAg was detected by ELISA. The concentration of the first well was 10 ⁇ g/mL, and the concentration gradient was diluted by 3 times to dilute 12 concentration gradients. Subsequently, the diluted antibody was added to a commercial HBsAg plate (purchased from Beijing Wantai), and incubated at 37 ° C for 1 h, washed with PBST 5 times, and dried. Subsequently, the GAH-HRP enzyme-labeled secondary antibody was added, incubated for 30 min, washed with PBST 5 times, and dried. Add the substrate TMB solution. After 15 min of color development, the color reaction was stopped with H 2 SO 4 and the reading was determined at OD450/630. As a result, as shown in Fig. 1, M1-23, M3-23 and M3-13 all had good antigen-binding activity.
  • HBsAg aa113-135 a murine monoclonal antibody 6D11 (described in Chinese Patent Application No. 201610879693.5) that recognizes an HBsAg SA type epitope (HBsAg aa113-135) happensing. See Example 4.2 for specific steps of the competitive ELISA method. As shown in Figure 2, the results showed that murine mAb 6D11 significantly blocked the binding of M1-23, M3-23, and M3-13 to HBsAg, indicating M1-23, M3-23, M3-13 and murine sources. Monoclonal antibody 6D11 recognizes the same HBsAg SA linear epitope.
  • the virus clearance ability of cynomolgus-human chimeric antibodies in animals was evaluated by HBV transgenic mice.
  • the cynomolgus monkey-human chimeric antibody was administered to HBV transgenic mice in a single dose tail vein injection at a dose of 10 mg/kg, each group of 6 HBV transgenic mice. Subsequently, blood was collected from the venous plexus of the eyelid, and changes in HBsAg levels and HBV DNA levels in the serum of the mice were examined. For the detection of HBsAg levels and HBV DNA levels in mouse serum, see Examples 5.1 and 5.2.
  • M1-23, M3-23 and M3-13 have been used to treat HBV.
  • the inventors In order to reduce the immunogenicity caused when a heterologous antibody is administered to a human subject, the inventors have humanized two antibodies, M1-23 and M3-23. Due to the high amino acid sequence between the cynomolgus monkey and the human immunoglobulin, it is only necessary for the two antibodies against M1-23 and M3-23 to be applied to human therapy. The region FR minority amino acid is replaced by the amino acid at the corresponding position in the human variable region FR sequence. However, although antibodies primarily contact and recognize antigens via CDRs, some of the residues in the FR region of an antibody may also be involved in antigen-antibody interactions, affecting the steric configuration of the CDRs, for example, by replacing the FR region of the murine antibody with a human.
  • the steric configuration of the CDR of the murine antibody tends to change, resulting in a significant decrease in the affinity of the humanized antibody to recognize/bind antigen, and even the ability of the humanized antibody to lose antigen binding (Ge Yan, human Development strategy analysis and application research of sourced antibodies [J]. Foreign Medical Medicine, Immunology, 2004, 27 (5): 271). Therefore, in the process of humanizing M1-23 and M3-23, the position of the point mutation in the variable region FR sequence and the selection of the amino acid after replacement are very important.
  • M1-23-H, M1-23-L, M3-23-H and M3-23-L is a template that mutates all of the original amino acids at the corresponding positions.
  • M1-23-H M1-23 heavy chain variable region
  • the oligonucleotide primers PTT5-1-23H-F, 1-23H-F1, 1-23H-F2, 1 were designed.
  • -23H-R1, 1-23H-F3 and PTT5-1-23H-R (the sequences are shown in the above table).
  • the specific protocol of PCR is as follows: using the coding gene of M1-23-H as a template, the primer pairs 1-23H-F1/1-23H-R1 and 1-23H-F3/PTT5-1-23H-R were amplified respectively. Fragment 1 and Fragment 2; using Fragment 1 as a template, amplifying fragment 3 with primer pair 1-23H-F2/1-23H-R1; using fragment 3 as a template, using primer pair PTT5-1-23H-F/1 Fragment 4 was amplified by -23H-R1; Fragment 4 and Fragment 2 were ligated together by overlap extension PCR, and then amplified with PTT5-1-23H-F/PTT5-1-23H-R as the upstream and downstream primers. Humanized gene fragment M1D-H.
  • the humanized gene fragment M1D-L of M1-23-L, the humanized gene fragment M3D-H of M3-23-H, and the humanized gene fragment M3D-L of M3-23-L were obtained.
  • the humanized gene fragments M1D-H and M3D-H obtained were ligated with the eukaryotic expression vector pTT5-CH, respectively, using the Cibson assembly liquid, and the M1D-L and M3D-L were respectively associated with the eukaryotic expression vector pTT5-C ⁇ . Connect to obtain a recombinant vector.
  • the recombinant vector was transformed into Escherichia coli DH5 ⁇ strain, coated with LB plates, and cultured overnight in a 37 ° C incubator. Monoclonal colonies were picked from the plates, sequenced, and the correct plasmids were sequenced, and the expression and purification of the antibodies were carried out according to the method of Example 1.
  • the amino acid sequences of the humanized antibodies M1D and M3D are shown in Table 11.
  • the degree of humanization of M1D and M3D was calculated by the following formula. As shown in Table 10 below, after humanization of the two antibodies, the degree of humanization increased from about 90% to about 98%.
  • Degree of humanization (number of amino acids in the FR region - number of amino acids retained in the FR region) / number of amino acids in the FR region ⁇ 100%
  • the binding activity of the humanized antibody M1D, M3D and the antigen HBsAg was examined by an ELISA method (enzyme-linked immunosorbent assay).
  • concentration of the first well was 10 ⁇ g/mL, and a 3-fold concentration gradient dilution was performed to dilute 12 concentration gradients, and humanized antibody 162 (described in detail in Chinese patent application CN201610879693.5) was used as a reference antibody.
  • the diluted antibody was added to a commercial HBsAg plate (purchased from Beijing Wantai), and incubated at 37 ° C for 1 h, washed with PBST 5 times, and dried.
  • the humanized antibodies M1D and M3D all had excellent antigen-binding activity, and their affinity for the antigen HBsAg was superior to that of the reference antibody 162.
  • the above results indicate that the humanized two antibodies not only have a very high degree of humanization (more than 97%), but also substantially retain the antigen-binding activity of the parent antibody, and even show better than the parent antibody. Antigen binding activity.
  • the inventors also performed a preliminary identification of the epitope of the antibody to determine whether the selected human monkey chimeric antibody and the humanized antibody recognize the SA epitope on HBsAg.
  • the high concentration of 6D11 recognizing the SA type epitope cross-blocks the binding of the antibody to be tested to HBsAg. If there is a significant blocking (competition) effect, the antibody to be tested recognizes the same epitope as 6D11.
  • the specific steps are as follows:
  • the antibody concentration was determined and added to a commercial HBsAg ELISA plate at a dose of 10 ⁇ g/well, and then the volume was made up to 50 ⁇ L with 20% NBS, and incubated at 37 ° C for 30 min.
  • the inventors used the laboratory-made cell model HepaG2-NTCP to assess the ability of antibodies to neutralize HBV infection (100 MOI HBV infection titer). A certain amount of HBV was co-incubated with the gradient-diluted antibody in HepaG2 cells, and HBV was able to invade the differentiated HepaG2 cells and replicate in the cells. With the addition of different antibodies and the amount of added antibody, HBV invasion and replication It will be attenuated or inhibited to varying degrees, so that different levels of HBV antigen (HBeAg) will appear in the cell supernatant. Therefore, by measuring the level of HBV antigen (HBeAg), the ability of different humanized antibodies to neutralize HBV can be judged.
  • HBV antigen HBV antigen
  • the humanized antibody was subjected to a two-fold gradient dilution, and 162 antibody was used as a positive control, starting from 10 ⁇ g/mL, and a total of 18 gradients were diluted.
  • the virus infection system was separately mixed with the antibody and pre-incubated for 1 h. Then, the cells were pre-plated with HepaG2, cultured at 37 ° C for 24 hours, and the cells were exchanged every two days. On the 7th day after infection, the cell culture supernatant was taken and the level of HBeAg was measured.
  • the detection method is as follows:
  • reaction plate The anti-HBeAg monoclonal antibody was diluted to 2 ⁇ g/mL with 20 mM PB buffer (Na 2 HPO 4 /NaH 2 PO 4 buffer, pH 7.4). Diluted anti-HBeAg monoclonal antibody (100 ⁇ L/well) was added to a 96-well chemical reflector and incubated at 37 ° C for 2 h at 4 ° C overnight. The raw materials used were purchased from Beijing Wantai Biological Pharmaceutical Co., Ltd. The 96-well chemical reflector was washed once with PBST and dried. A blocking solution was added to a 96-well chemical reflector, 200 ⁇ L per well, and blocked at 37 ° C for 2 h. Subsequently, the blocking solution was discarded, the plate was placed in a dry room and dried, and stored at 2-8 ° C until use.
  • PB buffer Na 2 HPO 4 /NaH 2 PO 4 buffer, pH 7.4
  • Reading plate The value was read using a chemiluminescent microplate reader.
  • Example 5 Determination of the therapeutic effect of humanized antibody M1D, M3D
  • the above 5 strains of the antibody were administered to a HBV transgenic mouse in a single dose of 10 mg/kg at a dose of 10 mg/kg, and each group of 6 HBV transgenic mice. Subsequently, blood was collected from the venous plexus of the eyelid, and changes in HBsAg levels and HBV DNA levels in the serum of the mice were examined.
  • Mouse monoclonal antibody HBs-45E9 was diluted to 2 ⁇ g/mL with 20 mM PB buffer (Na 2 HPO 4 /NaH 2 PO 4 buffer, pH 7.4), in a chemiluminescent plate. 100 ⁇ L of the coating solution was added to the wells, and coated at 6-8 ° C for 16-24 hours, followed by coating at 37 ° C for 2 hours, and the plate was washed once with PBST washing solution and dried. After washing, 200 ⁇ L of blocking solution was added to each well and blocked at 37 ° C for 2 h. Subsequently, the blocking solution was discarded, the plate was placed in a dry room and dried, and stored at 2-8 ° C until use.
  • PB buffer Na 2 HPO 4 /NaH 2 PO 4 buffer, pH 7.4
  • Enzyme label reaction The HBs-A6A7-HRP reaction solution was added to a chemiluminescent plate at 100 ⁇ L/well, and reacted at 37 ° C for 1 h. The plate was then washed 5 times with PBST and dried.
  • Luminescence reaction and measurement A luminescent liquid (100 ⁇ L/well) was added to the chemiluminescent panel, and light intensity detection was performed.
  • the quantitative detection of HBV DNA was carried out according to the instructions of the HBV DNA real-time fluorescence quantitative detection kit (the kit was purchased from Shanghai Kehua Bioengineering Co., Ltd.).
  • the humanized antibody of the present invention not only has a very high degree of humanization (up to 98%), but also reduces the possibility of immunological rejection, and exhibits superiority and superiority to monkey-human chimeric antibodies.
  • Example 6 Determination of non-critical CDR regions of humanized antibody M1D, M3D
  • the site directly binding to the antigen is mainly the CDR region, but many studies have shown that not all of the six CDRs of the antibody are critical regions for antigen-antibody binding, and the non-critical CDR regions can pass.
  • the method of CDR replacement is determined.
  • the invention selects the CDR replacement sequence according to the optimal matching principle, and inputs the VH and VL amino acid sequences of the humanized antibody M1D and M3D into the search box through the IMGT information system, and then searches the system for the corresponding antibody of the germline gene.
  • the chain amino acid sequence selects the humanized antibody CDR replacement sequence according to the principle that the FR region has the highest homology and the CDR region sequence has the largest difference, and each of the CDR regions except the heavy chain CDR3 finally selects two corresponding replacement sequences.
  • the replacement sequence is shown in the following table:
  • the relevant primers were designed according to the CDR replacement sequences described in the above table, and the corresponding CDR regions in the M1D and M3D antibodies were replaced by PCR.
  • the primer design is shown in the following table.
  • the oligonucleotide primers M1D-H-CDR1-A R and M1D-H-CDR1 were designed by taking the first substitution of M1D heavy chain variable region CDR1 (M1D-HCDR1) (M1D-H-CDR1-A) as an example.
  • -A F the sequence of which is shown in the table below
  • the antibody after replacement of the CDR is named M1D HCDR1-1, and the naming manner of other substituted antibodies is deduced by analogy.
  • the specific protocol of PCR is as follows: using the coding gene of M1D-H as a template, and using primer pairs pTT5-M1D-HF/M1D-H-CDR1-A R and M1D-H-CDR1-A F/pTT5-M1D-HR respectively.
  • the upstream and downstream fragments were added; the fragments were ligated together by overlap extension PCR, and then amplified with pTT5-M1D-HF/pTT5-M1D-HR as the upstream and downstream primers to obtain a gene fragment in which CDR substitution occurred in M1D-HCDR1.
  • the obtained gene fragment of the heavy chain CDR substitution was ligated to the eukaryotic expression vector pTT5-CH, respectively, and the HEK293-cell was transfected with a corresponding light chain expression vector without CDR replacement, and the detection was small. Whether the expressed cell supernatant has antigenic activity; likewise, the obtained light chain CDR replacement gene fragment is ligated to the eukaryotic expression vector pTT5-C ⁇ , respectively, and is associated with a corresponding heavy chain expression vector without CDR substitution. The amount of the antigen-binding activity of the transfected supernatant was transfected and detected.
  • the corresponding cell transfected supernatant has no antigen binding activity or a significant decrease in binding activity, indicating that the replaced CDR region is a key CDR region for antibody antigen binding. If the corresponding cell transfection supernatant antigen binding activity is substantially unchanged after replacement, the substituted CDR region is not a critical region for antibody antigen binding.
  • the M1D substitution results are shown in Figures 8A-8E. After substitution of the M1D heavy chain CDR1 (M1D-H-CDR1) and the light chain CDR2 (M1D-L-CDR2), the binding activity to the HBsAg antigen is substantially unchanged; and the heavy chain CDR2 and After the replacement of the light chain CDR1, the antigen binding activity was significantly decreased; while the light chain CDR3 was replaced, the antigen binding activity was completely lost.
  • the above results indicate that the HCDR1 and LCDR2 of the antibody M1D are not key CDR regions that bind to the antigen HBsAg, and their substitution does not affect the binding activity of the antibody to the antigen.
  • the M3D substitution results are shown in Figures 9A-9E. After the replacement of the M3D heavy chain CDR2 (HCDR2) and the light chain CDR2 (LCDR2), the antigen binding activity is substantially unchanged; while the light chain CDR1 is replaced, a replacement (M3D-LCDR1- 1) The antigen binding activity is markedly decreased, and the other substitution (M3D-LCDR1-2) has substantially no antigen binding activity; after replacement of the heavy chain CDR1 (HCDR1) and the light chain CDR3 (LCDR3), there is substantially no antigen binding activity.
  • the above results indicate that the HCDR2 and LCDR2 of the antibody M3D are not key CDR regions that bind to the antigen, and their substitution does not affect the binding activity of the antibody to the antigen.
  • Example 7 Determination of key amino acids in humanized antibody M1D, M3D binding to antigen
  • the mutated antibody was tested for binding activity to HBsAg by Elisa method and the rEC50 of the antibody after mutation was calculated (WT/Mutation, the relative antibody EC50 relative value was 1, and the binding activity was completely lost by 0), and the mutation was followed by rEC50 ⁇ 0.2. That is, the activity is reduced by 5 times and is indicated by blue, including amino acid residues such as D, E, R, H, L, W, and Y.
  • Table 14 in which the acidic amino acids D, E and the basic amino acids R and H form a salt bridge when the antibody antigen binds, thereby playing an important role in the binding reaction of the antibody antigen, and mutating these amino acids into C.
  • the aliphatic amino acid L which belongs to the hydrophobic amino acid W
  • Y belongs to the aromatic amino acid
  • M3D H33Y, H34D, H35W, H94R, H95D, H101E, L27dY, L32Y, L91H After mutating to alanine, the activity was significantly decreased, which was an immutable point.
  • Example 8 Identification of core epitopes recognized by M1D and M3D
  • M1D and M3D recognize SA linear epitopes.
  • a single point mutation of as113-135 (SEQ ID NO: 168) of HBsAg was synthesize 14 polypeptides in the following table.
  • the peptide was diluted with CB, 100 ng/mL coated plate, and the antibody was released to 1000 ng/mL with 20% NBS solution. Then, 12 gradients were diluted by 3 times, and the binding activity of the antibody to different single point mutant polypeptides was detected by Elisa method.
  • the software draws a binding curve. If the binding activity is lost after the mutation, the mutation site is a key amino acid on the SA linear epitope.
  • the EC50 values of the binding of M1D, M3D and reference antibody 162 to the above single point mutant polypeptide are summarized in Table 16 below (>500 with complete loss of binding activity). From the results, the core epitope of 162 binding is CKTC (aa121- 124), while the M1D-bound core epitope TGPCKTCT (aa118-124) is longer than the reference antibody 162 and is in front of the aa113-135; the core epitope of the M3D binding is CKTCT (aa121-125), the core table The bit position is later than 162.
  • Table 16 EC50 values for binding of M1D, M3D and HBsAg (aa113-135) single point mutant polypeptides
  • PBST is washed once every 5 minutes, for a total of 3 times;
  • the coated chip was run on a Biacore 3000 device with 5 ⁇ g/mL of HBsAg dissolved in sodium acetate (pH 4.5), and the HBsAg was coated onto a CM5 chip with a coating amount of 2400 RU. Seven concentrations of samples were prepared by serial dilution of the analyte from 100 nM.
  • the affinity assay procedure was run on a Biacore 3000 device with a flow rate of 50 ⁇ l/min, a binding time of 90 s, a dissociation time of 600 s, a sample chamber temperature of 10 ° C, a regenerant of 50 mM NaOH, and a regeneration flow rate of 50 ⁇ L/min. The regeneration time is 60s.
  • Table 19 shows the affinity of the M1D molecule and the M3D molecule to the antigen HBsAg, 1.06 nM and 1.12 nM, respectively.
  • Example 11 Evaluation of pharmacokinetics and preliminary toxicity of a single intravenous injection of antibodies M1D and M3D in cynomolgus monkeys
  • Table 21 Body weight of experimental animals after M1D administration
  • the concentrations of antibodies M1D and M3D in cynomolgus monkey serum were measured by chemiluminescence immunoassay (CLIA).
  • CLIA chemiluminescence immunoassay
  • LLOQ lower limit of quantitation
  • UEOQ upper limit of quantitation
  • Figure 12A is a graph showing the plasma concentration of humanized antibody M1D in each cynomolgus monkey serum (Group 1) as a function of time after a single intravenous injection of 20 mg/kg of humanized antibody M1D.
  • Figure 12B is a graph showing the plasma concentration of humanized antibody M3D in each cynomolgus monkey serum (Group 2) as a function of time after a single intravenous injection of 20 mg/kg of humanized antibody M3D.
  • the logarithmic linear trapezoidal method was used to calculate the following parameters: initial serum drug concentration (C0), last detectable time (T last ), elimination half-life (T 1/2 ), apparent volume of distribution (Vdss), overall clearance Rate (CL), the average residence time (MRT 0-last ) from the zero time point to the last time point at which the concentration can be detected, the average residence time from zero time point to infinity (MRT 0-inf ), from zero time The area under the serum concentration-time curve (AUC 0-last ) at the time point of the last detectable concentration, and the area under the serum concentration-time curve from zero time point to infinity (AUC 0-inf ).
  • the CL of the male cynomolgus monkey against the humanized antibody M3D was 0.0049 ⁇ 0.0025 mL/min/kg.
  • the average elimination half-life (t 1/2 ) of the humanized antibody M3D was 134 ⁇ 94.6 hours.
  • the Vdss and AUC 0-inf values of the humanized antibody M3D in cynomolgus monkey serum were 0.0497 ⁇ 0.0143 L/kg and 79419 ⁇ 33500 ⁇ g ⁇ h/mL, respectively.
  • Table 25 Main pharmacokinetic parameters of humanized antibody M1D in serum of group 1 male cynomolgus monkeys
  • Table 26 Main pharmacokinetic parameters of humanized antibody M3D in serum of group 2 male cynomolgus monkeys
  • the humanized antibody M1D or M3D of the invention can be administered to a subject (eg, a human) to prevent and/or treat a HBV infection or a disease associated with HBV infection (eg, hepatitis B).
  • a subject eg, a human
  • a disease associated with HBV infection eg, hepatitis B
  • the isoelectric points of the humanized antibodies M1D and M3D were measured by capillary isoelectric focusing electrophoresis (cIEF). The experimental results are shown in Table 27. The results showed that the humanized antibody M1D had a pl value of 8.8 and the M3D had a pl of 9.0.
  • the Tm value is typically used to describe the stability of an antibody molecule. The higher the Tm value, the better the thermal stability of the antibody molecule.
  • Humanized antibodies M1D and M3D were separately dissolved in three buffers, diluted to 1 mg/ml, and tested by differential scanning calorimetry (DSC). The start scan temperature is set to 10 ° C, the end scan temperature is set to 110 ° C, the scan speed is 200 ° C / hr, the cooling rate is set to Exp, the final instrument hold temperature is set to 25 ° C, the data acquisition frequency is set to 10 sec, the capillary before injection The temperature was set to 30 °C. The experimental results are shown in Figures 13A-13B and Tables 28-29. The results showed that the Tm onset of the humanized antibody M1D was above 61.7 ° C, and the Tmonset value of M3D was greater than 61.6 ° C, indicating that M1D and M3D have good thermal stability.
  • Buffer TM onset(°C) TM1 (°C) TM2 (°C) Acetate 5.0 61.6 68.8 82.2 Histidine 6.0 62.8 71.1 82.3 PBS7.2 65.7 78.5 /
  • the humanized antibodies M1D and M3D were dissolved in a histidine buffer of pH 6.0 at a concentration of 60 mg/ml, dissolved in a phosphate buffer of pH 7.2 at a concentration of 2 mg/ml, and then stored at 40 mg, respectively. Under the conditions of °C. The physical and chemical properties (including appearance, pH, protein concentration, etc.) of the samples were monitored at week 0 (T0), week 1 (T1W), and week 2 (T2W). The results are described in Tables 30 and 31.
  • the absorbance of the sample at A280 was measured and the protein concentration in the sample was calculated using Beer-Lambert law.
  • the pH of the sample was measured using a pH meter, and the measurement was repeated twice, and the average value was taken as the final result.
  • the experimental results are shown in Tables 30-31 above. The results showed that the humanized antibodies M1D and M3D did not change significantly in pH and protein concentration after storage for 2 weeks at 40 ° C in different buffers.
  • Humanized antibody M1D and M3D samples subjected to different storage conditions were analyzed by SEC-HPLC using an Agilent 1260 infinity, TSK G3000SWXL gel column (5 ⁇ m, 7.8 mm ⁇ 300 mm), wherein the mobile phase composition was 50 mM PB + 300 mM NaCl. , pH 7.0 ⁇ 0.2; flow rate of 1.0 mL / min; detection wavelength of 280 nm; sample concentration of 10 mg / mL, injection volume of 10 ⁇ L.
  • the experimental results are shown in Tables 30-31 above. The results showed that the main peaks of the samples containing the humanized antibodies M1D and M3D were greater than 95% under different storage conditions (different buffers, different storage times). This indicates that the humanized antibodies M1D and M3D are stable.
  • the sample was analyzed by capillary electrophoresis.
  • the results showed that the main peak of cIEF decreased by 6.0% after storage of M1D molecule in histidine buffer at 40 °C for 2 weeks, while the main peak decreased by 24.9% after storage for 2 weeks in PBS buffer.
  • the cIEF main peak decreased by 13.7% after storage for 2 weeks at 40 ° C in histidine buffer, while the main peak decreased by 30.6% after 2 weeks of storage in PBS buffer.
  • the humanized antibodies M1D and M3D were dissolved in a 25 mM histidine solution (pH 6.0) containing 5% sucrose and 0.02% PS80 at a concentration of 60 mg/mL, respectively, and concentrated by centrifugation ultrafiltration (centrifugation temperature was 5 ° C, The rotation speed was 4850 rpm) until the liquid level of the sample solution no longer decreased significantly with the increase of the centrifugation time. The sample was carefully collected with a pipette and the properties of the sample at this time were observed. The results showed that the solution sample was yellow.

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