WO2018184593A1 - 用于治疗乙肝感染及相关疾病的抗体 - Google Patents

用于治疗乙肝感染及相关疾病的抗体 Download PDF

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Publication number
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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English (en)
French (fr)
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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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Yang Sheng Tang Co Ltd
Xiamen University
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Yang Sheng Tang Co Ltd
Xiamen University
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Priority to KR1020227030102A priority Critical patent/KR102561555B1/ko
Priority to KR1020197033034A priority patent/KR20200010217A/ko
Priority to RU2019135404A priority patent/RU2765878C2/ru
Publication of WO2018184593A1 publication Critical patent/WO2018184593A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—DNA viruses
    • C07K16/082—Hepadnaviridae (F), e.g. hepatitis B virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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 [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IG], 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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Abstract

提供了抗乙肝表面抗原的抗体、其编码核酸、制备方法及包含所述抗体的药物组合物。还提供了所述抗体和药物组合物的用途。所述抗体和药物组合物可用于预防和/或治疗 HBV 感染或与 HBV 感染相关的疾病（如乙肝），可在受试者（如人）体内中和 HBV 的毒力，降低 HBV DNA 和/或HBsAg 的血清水平。

Description

用于治疗乙肝感染及相关疾病的抗体 技术领域
本发明涉及分子病毒学和免疫学领域，特别是治疗乙型肝炎病毒(Hepatitis B virus，HBV)感染的领域。具体而言，本发明涉及抗乙肝表面抗原(HBsAg)的抗体，编码它们的核酸分子，制备它们的方法，以及包含它们的药物组合物。所述药物组合物可用于预防和/或治疗HBV感染或与HBV感染相关的疾病(例如乙肝)，用于在受试者(例如人)体内中和HBV的毒力，或用于在受试者体内降低HBV DNA和/或HBsAg的血清水平。因此，本发明进一步涉及，所述抗体(特别是人源化抗体)及其变体在制备药物组合物中的用途，所述药物组合物用于预防和/或治疗HBV感染或与HBV感染相关的疾病(例如乙肝)，用于在受试者(例如人)体内中和HBV的毒力，或用于在受试者体内降低HBV DNA和/或HBsAg的血清水平。
背景技术
乙型肝炎病毒感染，尤其是慢性HBV感染是全球最为重要的公共卫生问题之一(Dienstag JL.Hepatitis B virus infection.N Engl J Med 2008 Oct 2；359(14)：1486-1500)。慢性HBV感染可导致慢性乙型病毒性肝炎(Chronic hepatitis B，CHB)、肝硬化(Liver cirrhosis，LC)和原发性肝细胞癌(Hepatocellular carcinoma，HCC)等一系列肝脏疾病(Liaw YF，Chu CM.Hepatitis B virus infection.Lancet 2009 Feb 14；373(9663)：582-592)。据报道，目前全球约有20亿人曾经感染过HBV，现约有3.5亿的慢性乙型肝炎病毒感染者，这些感染者最终死于HBV感染相关肝脏疾病的风险可达15％-25％，全球每年超过100万人死于此类疾病(Dienstag JL.，同上；和Liaw YF等，同上)。
当前针对慢性HBV感染的治疗药物主要可分为干扰素类(Interferon，IFNs)和核苷/核苷酸类似物(nucleoside or nucleotide，NAs)(Dienstag JL.，同上；Kwon H，Lok AS.Hepatitis B therapy.Nat Rev Gastroenterol Hepatol 2011 May；8(5)：275-284；和Liaw YF等，同上)。前者包括普通干扰素(IFN)和聚乙二醇干扰素(Peg-interferon，Peg-IFN，又称为长效干扰素)，主要通过整体增强患者免疫能力来达到抑制HBV和治疗CHB的效果；后者主要包括拉米夫定(lamivudine，LMV)、阿德福韦酯(adefovir dipivoxil，ADV)、恩替卡韦(Entecavir，ETV)、替比夫定(Telbivudine，LdT)、替诺福韦(Tenofovir)等5种，主要通过直接抑制HBV的聚合酶活性来抑制HBV复制。对于HBV感染者(例如CHB患者) 来说，上述药物的单独或联合治疗，已能较为有效地抑制体内病毒复制，大幅降低HBV DNA水平；特别地，52周或延长的此类治疗后，患者体内HBV DNA水平低于检测下限(病毒学应答)的应答率可达40-80％(Kwon H等，同上)。然而，上述药物的单独或联合治疗均不能完全清除感染者体内的HBV病毒，其导致的HBsAg阴转或HBsAg血清学转换(感染者体内HBV病毒彻底清除的标志)的应答率通常小于5％(Kwon H等，同上)。因此，针对HBV感染者发展创新的、能更为有效地清除HBV病毒、尤其是清除HBsAg的治疗方法和药物是迫切而必要的。
基于免疫学手段发展新的治疗慢性HBV感染的药物是此领域的重要研究方向之一。慢性HBV感染的免疫治疗通常通过主动免疫治疗(其对应药物形式如疫苗等)和被动免疫治疗(其对应药物形式如抗体等)等两种方式来进行。主动免疫治疗则是指，通过施用治疗性疫苗(包括蛋白疫苗、多肽疫苗和核酸疫苗等)，刺激慢性HBV感染者机体主动产生针对HBV的细胞免疫应答(CTL效应等)或/和体液免疫应答(抗体等)，从而达到抑制或清除HBV的目的。目前尚无明确显著有效的、可用于治疗慢性HBV感染的主动免疫治疗药物/疫苗。被动免疫治疗(以抗体为例)是指，向HBV感染者被动施用具治疗特性的抗体，并利用抗体介导的病毒中和作用阻断HBV感染新生肝细胞，或利用抗体介导的免疫清除作用清除体内病毒和被感染的肝细胞，从而起到治疗的效果。目前，从预防性乙型肝炎疫苗免疫应答者或HBV感染恢复者血清/血浆中纯化获得的Anti-HBs多克隆抗体，即高效价的乙肝免疫球蛋白(HBIG)已广泛应用于阻断HBV的母婴垂直传播、预防慢性HBV感染者肝移植后的HBV再感染、以及预防意外暴露于HBV的人群的感染。然而，将HBIG直接用于HBV感染者(例如CHB患者)的治疗无明显疗效，且其存在例如高效价血浆来源较少、价格昂贵、性质不稳定、潜在的安全性问题等诸多局限性。
因此，针对HBV感染者发展创新的、能更为有效地治疗HBV感染的治疗方法和药物是迫切而必要的。
发明概述
在一个方面，本发明提供了一种能够特异性结合HBsAg的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
(a)选自下列的重链可变区(VH)互补决定区(CDR)中的一个或多个(例如1个，2个或3个)：
(i)VH CDR1，其由下述序列组成：SEQ ID NO：6，SEQ ID NO：12，SEQ ID NO： 18或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；
(ii)VH CDR2，其由下述序列组成：SEQ ID NO：7，SEQ ID NO：13，SEQ ID NO：19或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，和
(iii)VH CDR3，其由下述序列组成：SEQ ID NO：8，SEQ ID NO：14，SEQ ID NO：20，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；
和/或
(b)选自下列的轻链可变区(VL)CDR中的一个或多个(例如1个，2个或3个)：
(iv)VL CDR1，其由下述序列组成：SEQ ID NO：9，SEQ ID NO：15或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，
(v)VL CDR2，其由下述序列组成：SEQ ID NO：10，SEQ ID NO：16或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，和
(vi)VL CDR3，其由下述序列组成：SEQ ID NO：11，SEQ ID NO：17或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含如上文所定义的VH CDR1、VH CDR2和VH CDR3。在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含如上文所定义的VL CDR1、VL CDR2和VL CDR3。在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含如上文所定义的VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2和VL CDR3。
在某些优选的实施方案中，所述抗体或其抗原结合片段包含：
(a)重链可变区(VH)，所述VH包含：
(i)VH CDR2，其由下述序列组成：SEQ ID NO：7，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，
(ii)VH CDR3，其由下述序列组成：SEQ ID NO：8，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，
以及
(iii)VH CDR1，其由下述序列组成：任意免疫球蛋白的重链可变区中所包含的VH CDR1的序列；
和/或
(b)轻链可变区(VL)，所述VL包含：
(iv)VL CDR1，其由下述序列组成：SEQ ID NO：9，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，
(v)VL CDR3，其由下述序列组成：SEQ ID NO：11，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，
以及
(vi)VL CDR2，其由下述序列组成：任意免疫球蛋白的轻链可变区(例如，κ轻链可变区)中所包含的VL CDR2的序列。
在某些优选的实施方案中，所述VH CDR1为人免疫球蛋白的重链可变区中所包含的VH CDR1的序列。
在某些优选的实施方案中，所述VH CDR1由下述序列组成：(a)SEQ ID NO：6，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；或，(b)人重链胚系基因所编码的氨基酸序列中所包含的VH CDR1的序列。在某些示例性实施方案中，所述人重链胚系基因选自IGHV4-4＊08和IGHV4-61＊01。
在某些示例性实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：7所示的VH CDR2；如SEQ ID NO：8所示的VH CDR3；以及，具有选自下列的氨基酸序列的VH CDR1：SEQ ID NO：6、SEQ ID NO：137或SEQ ID NO：138。
在某些优选的实施方案中，所述VL CDR2为人免疫球蛋白的轻链可变区(例如，κ轻链可变区)中所包含的VL CDR2的序列。
在某些优选的实施方案中，所述VL CDR2由下述序列组成：(a)SEQ ID NO：10，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；或，(b)人轻链胚系基因所编码的氨基酸序列中所包含的VL CDR2的序列。在某些示例性实施方案中，所述人轻链胚系基因选自IGKV1-39＊01和IGKV1-5＊03。
在某些示例性实施方案中，本发明的抗体或其抗原结合片段的VL包含：如SEQ ID  NO：9所示的VL CDR1；如SEQ ID NO：11所示的VL CDR3；以及，具有选自下列的氨基酸序列的VL CDR2：SEQ ID NO：10、SEQ ID NO：139或SEQ ID NO：140。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：7所示的VH CDR2，如SEQ ID NO：8所示的VH CDR3，以及具有选自下列的氨基酸序列的VH CDR1：SEQ ID NO：6，137或138；并且，该抗体或其抗原结合片段的VL包含：如SEQ ID NO：9所示的VL CDR1，如SEQ ID NO：11所示的VL CDR3，以及具有选自下列的氨基酸序列的VL CDR2：SEQ ID NO：10，139或140。
在某些优选的实施方案中，所述抗体或其抗原结合片段包含：
(a)重链可变区(VH)，所述VH包含：
(i)VH CDR1，其由下述序列组成：SEQ ID NO：12，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，
(ii)VH CDR3，其由下述序列组成：SEQ ID NO：14，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，以及
(iii)VH CDR2，其由下述序列组成：任意免疫球蛋白的重链可变区中所包含的VH CDR2的序列；
和/或
(b)轻链可变区(VL)，所述VL包含：
(iv)VL CDR1，其由下述序列组成：SEQ ID NO：15，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，
(v)VL CDR3，其由下述序列组成：SEQ ID NO：17，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，以及
(vi)VL CDR2，其由下述序列组成：任意免疫球蛋白的轻链可变区(例如，κ轻链可变区)中所包含的VL CDR2的序列。
在某些优选的实施方案中，所述VH CDR2为人免疫球蛋白的重链可变区中所包含的VH CDR2的序列。
在某些优选的实施方案中，所述VH CDR2由下述序列组成：(a)SEQ ID NO：13，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸 置换、缺失或添加)的序列；或，(b)人重链胚系基因所编码的氨基酸序列中所包含的VH CDR2的序列。在某些示例性实施方案中，所述人重链胚系基因选自IGHV4-30-4＊07和IGHV4-4＊01。
在某些示例性实施方案中，本发明的抗体或其抗原结合片段的VH包含：(a)如SEQ ID NO：12所示的VH CDR1；如SEQ ID NO：14所示的VH CDR3；以及，具有选自下列的氨基酸序列的VH CDR2：SEQ ID NO：13、SEQ ID NO：141或SEQ ID NO：142。
在某些优选的实施方案中，所述VL CDR2为人免疫球蛋白的轻链可变区(例如，κ轻链可变区)中所包含的VL CDR2的序列。
在某些优选的实施方案中，所述VL CDR2由下述序列组成：(a)SEQ ID NO：16，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；或，(b)人轻链胚系基因所编码的氨基酸序列中所包含的VL CDR2的序列。在某些示例性实施方案中，所述人轻链胚系基因选自IGKV2-28＊01和IGKV3-15＊01。
在某些示例性实施方案中，本发明的抗体或其抗原结合片段的VL包含：如SEQ ID NO：15所示的VL CDR1；如SEQ ID NO：17所示的VL CDR3；以及，具有选自下列的氨基酸序列的VLCDR2：SEQ ID NO：16、SEQ ID NO：143或SEQ ID NO：144。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：12所示的VH CDR1，如SEQ ID NO：14所示的VH CDR3，以及具有选自下列的氨基酸序列的VH CDR2：SEQ ID NO：13、SEQ ID NO：141或SEQ ID NO：142；并且，该抗体或其抗原结合片段的VL包含：如SEQ ID NO：15所示的VL CDR1，如SEQ ID NO：17所示的VL CDR3，以及具有选自下列的氨基酸序列的VL CDR2：SEQ ID NO：16、SEQ ID NO：143或SEQ ID NO：144。
在某些优选的实施方案中，所述抗体或其抗原结合片段包含：
(a)重链可变区(VH)，所述VH包含：
(i)VH CDR1，其由下述序列组成：SEQ ID NO：18，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；
(ii)VH CDR2，其由下述序列组成：SEQ ID NO：19，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，
和
(iii)VH CDR3，其由下述序列组成：SEQ ID NO：20，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；
和/或
(b)轻链可变区(VL)，所述VL包含：
(iv)VL CDR1，其由下述序列组成：SEQ ID NO：15，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，
(v)VL CDR2，其由下述序列组成：SEQ ID NO：16，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，
和
(vi)VL CDR3，其由下述序列组成：SEQ ID NO：17，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：18所示的VH CDR1，如SEQ ID NO：19所示的VH CDR2，以及如SEQ ID NO：20所示的VH CDR3；并且，该抗体或其抗原结合片段的VL包含：如SEQ ID NO：15所示的VL CDR1，如SEQ ID NO：16所示的VL CDR2，以及如SEQ ID NO：17所示的VL CDR3。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段是人源化的。在某些优选的实施方案中，本发明的抗体或其抗原结合片段的人源化程度为至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％或至少98％。在某些优选的实施方案中，本发明的抗体或其抗原结合片段的非CDR区包含不超过19个，不超过15个，不超过14个，不超过13个，不超过12个，不超过11个，不超过10个，不超过9个，不超过8个，不超过7个，不超过6个，不超过5个，不超过4个或不超过3个的非人源(例如，食蟹猴源)氨基酸残基。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段进一步包含框架区(FR)。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：
(a)选自下列的重链可变区(VH)构架区(FR)中的一个或多个(例如1个，2个，3个或4个)：
(i)VH FR1，其由下述序列组成：SEQ ID NO：21，SEQ ID NO：29，SEQ ID NO：37，SEQ ID NO：44，SEQ ID NO：50，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个、3个或4个氨基酸置换、缺失或添加)的序列；
(ii)VH FR2，其由下述序列组成：SEQ ID NO：22，SEQ ID NO：30，SEQ ID NO：38，SEQ ID NO：51，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个、3个或4个氨基酸置换、缺失或添加)的序列；
(iii)VH FR3，其由下述序列组成：SEQ ID NO：23，SEQ ID NO：31，SEQ ID NO：39，SEQ ID NO：45，SEQ ID NO：52，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个、3个或4个氨基酸置换、缺失或添加)的序列；和
(iv)VH FR4，其由下述序列组成：SEQ ID NO：24，SEQ ID NO：32，SEQ ID NO：46，SEQ ID NO：53，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；
和/或
(b)选自下列的轻链可变区(VL)构架区(FR)中的一个或多个(例如1个，2个，3个或4个)：
(v)VL FR1，其由下述序列组成：SEQ ID NO：25，SEQ ID NO：33，SEQ ID NO：47，SEQ ID NO：54，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个、3个或4个氨基酸置换、缺失或添加)的序列；
(vi)VL FR2，其由下述序列组成：SEQ ID NO：26，SEQ ID NO：34，SEQ ID NO：48，SEQ ID NO：55，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个、3个或4个氨基酸置换、缺失或添加)的序列；
(vii)VL FR3，其由下述序列组成：SEQ ID NO：27，SEQ ID NO：35，SEQ ID NO：49，SEQ ID NO：56，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个、3个或4个氨基酸置换、缺失或添加)的序列；和
(viii)VL FR4，其由下述序列组成：SEQ ID NO：28，SEQ ID NO：36，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个、3个或4个氨基酸置换、缺失或添加)的序列。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含如上文所定义的VH FR1、VH FR2、VH FR3和VH FR4。在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含如上文所定义的VL FR1、VL FR2、VL FR3和VL FR4。在某些优选的 实施方案中，本发明的抗体或其抗原结合片段包含如上文所定义的VH FR1、VH FR2、VH FR3、VH FR4、VL FR1、VL FR2、VL FR3和VL FR4。
在某些优选的实施方案中，所述抗体或其抗原结合片段包含：
(a)重链框架区，其与选自下列的重链可变区中所包含的重链框架区相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性：如SEQ ID NOs：1，3，5，57，59，157，158，163和164所示的重链可变区；
和/或
(b)轻链框架区，其与选自下列的轻链可变区中所包含的轻链框架区具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性：如SEQ ID NOs：2，4，58，60，159，160，165和166任一项所示的轻链可变区。
在某些优选的实施方案中，所述抗体或其抗原结合片段包含：
(a)选自下列的重链可变区中含有的4个FR：
如SEQ ID NOs：1，3，5，57，59，157，158，163和164任一项所示的重链可变区；
和/或
(b)选自下列的轻链可变区中含有的4个FR：
如SEQ ID NOs：2，4，58，60，159，160，165和166任一项所示的轻链可变区。
在某些优选的实施方案中，所述抗体或其抗原结合片段的VH包含：
(a)如SEQ ID NO：21所示的VH FR1、如SEQ ID NO：22所示的VH FR2、如SEQ ID NO：23所示的VH FR3、和如SEQ ID NO：24所示的VH FR4；
(b)如SEQ ID NO：29所示的VH FR1、如SEQ ID NO：30所示的VH FR2、如SEQ ID NO：31所示的VH FR3、和如SEQ ID NO：32所示的VH FR4；
(c)如SEQ ID NO：37所示的VH FR1、如SEQ ID NO：38所示的VH FR2、如SEQ ID NO：39所示的VH FR3、和如SEQ ID NO：32所示的VH FR4；
(d)如SEQ ID NO：44所示的VH FR1、如SEQ ID NO：22所示的VH FR2、如SEQ ID NO：45所示的VH FR3、和如SEQ ID NO：46所示的VH FR4；或
(e)如SEQ ID NO：50所示的VH FR1、如SEQ ID NO：51所示的VH FR2、如SEQ ID NO：52所示的VH FR3、和如SEQ ID NO：53所示的VH FR4；
和/或，所述抗体或其抗原结合片段的VL包含：
(a)如SEQ ID NO：25所示的VL FR1、如SEQ ID NO：26所示的VL FR2、如SEQ ID NO：27所示的VL FR3、和如SEQ ID NO：28所示的VL FR4；
(b)如SEQ ID NO：33所示的VL FR1、如SEQ ID NO：34所示的VL FR2、如SEQ ID NO：35所示的VL FR3、和如SEQ ID NO：36所示的VL FR4；
(c)如SEQ ID NO：47所示的VL FR1、如SEQ ID NO：48所示的VL FR2、如SEQ ID NO：49所示的VL FR3、和如SEQ ID NO：28所示的VL FR4；或
(d)如SEQ ID NO：54所示的VL FR1、如SEQ ID NO：55所示的VL FR2、如SEQ ID NO：56所示的VL FR3、和如SEQ ID NO：36所示的VL FR4。
在某些优选的实施方案中，所述抗体或其抗原结合片段包含人免疫球蛋白的框架区(例如，人胚系抗体基因所编码的氨基酸序列中所包含的框架区)，所述框架区任选地包含一个或多个从人源残基至食蟹猴源残基的回复突变。
在某些优选的实施方案中，所述抗体或其抗原结合片段包含：人重链胚系基因所编码的氨基酸序列中所包含的重链框架区，和/或人轻链胚系基因所编码的氨基酸序列中所包含的轻链框架区。
在某些优选的实施方案中，所述抗体或其抗原结合片段包含：IGHV4-4＊08所编码的氨基酸序列中所包含的重链框架区，和IGKV1-39＊01所编码的氨基酸序列中所包含的轻链框架区，所述重链框架区和/或轻链框架区任选地包含一个或多个从人源残基至食蟹猴源残基的回复突变。
在某些优选的实施方案中，所述抗体或其抗原结合片段包含：IGHV4-4＊02所编码的氨基酸序列中所包含的重链框架区，和IGKV4-1＊01所编码的氨基酸序列中所包含的轻链框架区，所述重链框架区和/或轻链框架区任选地包含一个或多个从人源残基至食蟹猴源残基的回复突变。
在某些优选的实施方案中，所述抗体或其抗原结合片段包含：
(a)如SEQ ID NO：21所示的VH FR1、如SEQ ID NO：22所示的VH FR2、如SEQ ID NO：23所示的VH FR3、和如SEQ ID NO：24所示的VH FR4；以及，如SEQ ID NO：25所示的VL FR1、如SEQ ID NO：26所示的VL FR2、如SEQ ID NO：27所示的VL FR3、和如SEQ ID NO：28所示的VL FR4；
(b)如SEQ ID NO：29所示的VH FR1、如SEQ ID NO：30所示的VH FR2、如SEQ ID NO：31所示的VH FR3、和如SEQ ID NO：32所示的VH FR4；以及，如SEQ ID NO： 33所示的VL FR1、如SEQ ID NO：34所示的VL FR2、如SEQ ID NO：35所示的VL FR3、和如SEQ ID NO：36所示的VL FR4；
(c)如SEQ ID NO：37所示的VH FR1、如SEQ ID NO：38所示的VH FR2、如SEQ ID NO：39所示的VH FR3、和如SEQ ID NO：32所示的VH FR4；以及，如SEQ ID NO：33所示的VL FR1、如SEQ ID NO：34所示的VL FR2、如SEQ ID NO：35所示的VL FR3、和如SEQ ID NO：36所示的VL FR4；
(d)如SEQ ID NO：44所示的VH FR1、如SEQ ID NO：22所示的VH FR2、如SEQ ID NO：45所示的VH FR3、如SEQ ID NO：46所示的VH FR4；以及，如SEQ ID NO：47所示的VL FR1、如SEQ ID NO：48所示的VL FR2、如SEQ ID NO：49所示的VL FR3、和如SEQ ID NO：28所示的VL FR4；或
(e)如SEQ ID NO：50所示的VH FR1、如SEQ ID NO：51所示的VH FR2、如SEQ ID NO：52所示的VH FR3、和如SEQ ID NO：53所示的VH FR4；以及，如SEQ ID NO：54所示的VL FR1、如SEQ ID NO：55所示的VL FR2、如SEQ ID NO：56所示的VL FR3、和如SEQ ID NO：36所示的VL FR4。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的重链可变区的氨基酸序列与选自下列的重链可变区的氨基酸序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性：如SEQ ID NOs：1，3，5，57，59，157，158，163和164所示的重链可变区。在某些优选的实施方案中，所述抗体或其抗原结合片段的重链可变区选自如SEQ ID NOs：1，3，5，57，59，157，158，163和164任一项所示的重链可变区。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的轻链可变区的氨基酸序列与选自下列的轻链可变区的氨基酸序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性：如SEQ ID NOs：2，4，58，60，159，160，165和166任一项所示的轻链可变区。在某些优选的实施方案中，所述抗体或其抗原结合片段的轻链可变区选自如SEQ ID NOs：2，4，58，60，159，160，165和166任一项所示的轻链可变区。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含如上文所定义的重 链可变区和如上文所定义的轻链可变区。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的重链可变区选自如SEQ ID NOs：1，57，157和158任一项所示的重链可变区；并且，所述抗体或其抗原结合片段的轻链可变区选自如SEQ ID NOs：2，58，159和160任一项所示的轻链可变区；或
本发明的抗体或其抗原结合片段的重链可变区选自如SEQ ID NOs：3，59，163和164任一项所示的重链可变区；并且，所述抗体或其抗原结合片段的轻链可变区选自如SEQ ID NOs：4，60，165和166任一项所示的轻链可变区。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：
(1)如SEQ ID NO：1所示的VH和如SEQ ID NO：2所示的VL；
(2)如SEQ ID NO：3所示的VH和如SEQ ID NO：4所示的VL；
(3)如SEQ ID NO：5所示的VH和如SEQ ID NO：4所示的VL；
(4)如SEQ ID NO：57所示的VH和如SEQ ID NO：58所示的VL；
(5)如SEQ ID NO：59所示的VH和如SEQ ID NO：60所示的VL；
(6)如SEQ ID NO：157所示的VH和如SEQ ID NO：58所示的VL；
(7)如SEQ ID NO：158所示的VH和如SEQ ID NO：58所示的VL；
(8)如SEQ ID NO：57所示的VH和如SEQ ID NO：159所示的VL；
(9)如SEQ ID NO：57所示的VH和如SEQ ID NO：160所示的VL；
(10)如SEQ ID NO：163所示的VH和如SEQ ID NO：60所示的VL；
(11)如SEQ ID NO：164所示的VH和如SEQ ID NO：60所示的VL；
(12)如SEQ ID NO：59所示的VH和如SEQ ID NO：165所示的VL；或
(13)如SEQ ID NO：59所示的VH和如SEQ ID NO：166所示的VL。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段选自scFv、Fab、Fab’、(Fab’) 2、Fv片段、双抗体(diabody)、双特异性抗体、多特异性抗体、嵌合抗体或人源化抗体。在某些优选的实施方案中，所述抗体为嵌合抗体或人源化抗体。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段为IgG类别(例如，IgG1、IgG2、IgG3或IgG4类别)。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段能够特异性结合HBsAg，中和HBV的毒力，和/或降低HBV DNA和/或HBsAg在受试者体内的血清水平。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段带有标记。在某些优选 的实施方案中，所述抗体或其抗原结合片段带有可检测的标记，例如放射性同位素，荧光物质，发光物质，有色物质或酶(例如辣根过氧化物酶)。
在另一个方面，本发明提供了一种分离的核酸分子，其包含编码本发明的抗体或其抗原结合片段，或其重链可变区和/或轻链可变区的核苷酸序列。
在另一个方面，本发明提供了一种载体(例如克隆载体或表达载体)，其包含根据本发明的分离的核酸分子。
在另一个方面，本发明提供了一种宿主细胞，其包含根据本发明的分离的核酸分子或根据本发明的载体。
在另一个方面，提供了制备根据本发明的抗体或其抗原结合片段的方法，其包括，在允许所述抗体或其抗原结合片段表达的条件下，培养根据本发明的宿主细胞，和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段。
在另一个方面，本发明提供了一种试剂盒，其包括本发明的抗体或其抗原结合片段。在一个优选的实施方案中，本发明的抗体或其抗原结合片段还包括可检测的标记。在一个优选的实施方案中，所述试剂盒还包括第二抗体，其特异性识别本发明的抗体或其抗原结合片段。优选地，所述第二抗体还包括可检测的标记。此类可检测的标记是本领域技术人员熟知的，包括但不限于，放射性同位素，荧光物质，发光物质，有色物质和酶(例如辣根过氧化物酶)等。
在另一个方面，本发明提供了检测HBsAg蛋白在样品中的存在或其水平的方法，其包括使用本发明的抗体或其抗原结合片段。在一个优选的实施方案中，本发明的抗体或其抗原结合片段还包括可检测的标记。在另一个优选的实施方案中，所述方法还包括，使用携带可检测的标记的第二抗体来检测本发明的抗体或其抗原结合片段。所述方法可以用于诊断目的，或者非诊断目的(例如，所述样品是细胞样品，而非来自患者的样品)。
在另一个方面，本发明提供了诊断受试者是否感染了HBV的方法，其包括：使用本发明的抗体或其抗原结合片段检测HBsAg蛋白在来自所述受试者的样品中的存在。在一个优选的实施方案中，本发明的抗体或其抗原结合片段还包括可检测的标记。在另一个优选的实施方案中，所述方法还包括，使用携带可检测的标记的第二抗体来检测本发明的抗体或其抗原结合片段。
在另一个方面，提供了本发明的抗体或其抗原结合片段在制备试剂盒中的用途，所述试剂盒用于检测HBsAg蛋白在样品中的存在或其水平，或用于诊断受试者是否感 染了HBV。
本发明的抗体或其抗原结合片段可用于预防或治疗受试者(例如人)的HBV感染或与HBV感染相关的疾病(例如乙肝)，用于体外或在受试者(例如人)体内中和HBV的毒力，以及用于在受试者(例如人)体内降低HBV DNA和/或HBsAg的血清水平。
因此，在另一个方面，本发明提供了一种药物组合物，其含有根据本发明的抗体或其抗原结合片段，以及药学上可接受的载体和/或赋形剂。在一个优选的实施方案中，本发明的药物组合物还可包含另外的药学活性剂。在一个优选的实施方案中，所述另外的药学活性剂是用于预防或治疗HBV感染或与HBV感染相关的疾病(例如乙肝)的药物，例如干扰素类药物，如干扰素或聚乙二醇干扰素。
在另一个方面，提供了根据本发明的抗体或其抗原结合片段或根据本发明的药物组合物在制备药物中的用途，所述药物用于预防或治疗受试者(例如人)的HBV感染或与HBV感染相关的疾病(例如乙肝)，用于体外或在受试者(例如人)体内中和HBV的毒力，和/或用于在受试者(例如人)体内降低HBV DNA和/或HBsAg的血清水平。
在另一个方面，本发明提供了一种方法，其用于预防或治疗受试者(例如人)的HBV感染或与HBV感染相关的疾病(例如乙肝)，用于在受试者(例如人)体内中和HBV的毒力，和/或用于在受试者(例如人)体内降低HBV DNA和/或HBsAg的血清水平，所述方法包括，给有此需要的受试者施用有效量的根据本发明的抗体或其抗原结合片段或根据本发明的药物组合物。
本发明所提供的药物和药物组合物可以单独使用或联合使用，也可以与另外的药学活性剂(例如其它抗病毒试剂，例如干扰素类药物，如干扰素或聚乙二醇干扰素)联合使用。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1显示了实施例2中测定3株食蟹猴-人嵌合单克隆抗体M1-23、M3-23、M3-13与抗原HBsAg的结合活性的ELISA检测结果，其中，横坐标表示抗体浓度(Log 10 ng/ml)，纵坐标表示OD值。结果显示，3株食蟹猴-人嵌合单克隆抗体均具有良好的抗原结合活性。
图2显示了在实施例2中6D11鼠源抗体交叉阻断3株食蟹猴-人嵌合单克隆抗体M1-23、M3-23、M3-13与抗原HBsAg的结合的竞争ELISA法检测结果。结果显示，M1-23、M3-23、M3-13与抗原HBsAg的结合可以被6D11显著抑制。
图3A-3B显示了在实施例2中食蟹猴-人嵌合单克隆抗体M1-23、M3-23在HBV转基因小鼠体内疗效评估结果。图3A：小鼠血清中HBsAg水平的变化情况，其中，横坐标表示注射嵌合抗体后的天数，纵坐标表示小鼠血清中HBsAg的相对减少水平(Log 10IU/ml)；图3B：小鼠血清中HBV DNA水平的变化情况，其中，横坐标表示注射嵌合抗体后的天数，纵坐标表示小鼠血清中HBV DNA的相对减少水平(Log 10IU/ml)。结果显示，M1-23、M3-23均可明显清除动物体内HBsAg和HBV DNA。
图4显示了在实施例4中人源化抗体M1D、M3D与抗原HBsAg的结合活性的ELISA检测结果，其中，横坐标表示抗体浓度(Log 10ng/ml)，纵坐标表示OD值。结果显示，2株人源化抗体均具有良好的抗原结合活性，其对HBsAg的亲和力优于参比抗体162。
图5显示了在实施例4中6D11鼠源抗体交叉阻断人源化抗体M1D、M3D与抗原HBsAg的结合的竞争ELISA法检测结果。结果显示，人源化抗体M1D、M3D与抗原HBsAg的结合可以被6D11显著抑制。
图6A-6B显示了在实施例4中人源化抗体M1D对HBV病毒的中和活性的测定结果，其中，图6A为以HBIG为标准品做的标准曲线，图6B为样品的中和活性测定结果，纵坐标表示每mg的抗体具有多少mIU标准单位的HBIG的中和活性。结果显示，M1D对HBV的中和活性与参比抗体162相当。
图7显示了实施例5中人源化抗体M1D、M3D以及嵌合抗体M3-13在HBV转基因小鼠体内疗效评估结果。图7A：小鼠血清中HBsAg水平的变化情况，其中，横坐标表示注射人源化抗体后的天数，纵坐标表示小鼠血清中HBsAg的相对减少水平(Log 10IU/ml)；图7B：小鼠血清中HBV DNA水平的变化情况，其中，横坐标表示注射嵌合抗体后的天数，纵坐标表示小鼠血清中HBV DNA的相对减少水平(Log10IU/ml)。结果显示，3株抗体均可明显清除动物体内HBsAg和HBV DNA，其效果优于参比抗体162。
图8A-8E显示了实施例6中人源化抗体M1D进行CDR区替换后的抗体与抗原HBsAg结合活性的ELISA检测结果，其中，横坐标表示抗体浓度(Log10ng/ml)，纵坐 标表示OD值。结果显示，对抗体M1D的HCDR1和LCDR2区替换后不影响与抗原HBsAg的结合活性，说明抗体M1D的HCDR1和LCDR2区不是与抗原HBsAg结合的关键CDR区。
图9A-9E显示了实施例6中人源化抗体M3D进行CDR区替换后的抗体与抗原HBsAg结合活性的ELISA检测结果，其中，横坐标表示抗体浓度(Log10ng/ml)，纵坐标表示OD值。结果显示，重链CDR2(HCDR2)和轻链CDR2(LCDR2)替换后，不影响与抗原HBsAg的结合活性，说明抗体M3D的HCDR2和LCDR2区不是与抗原HBsAg结合的关键CDR区。
图10显示了实施例8中抗体M1D、M3D、162与多种单点突变的HBsAg aa113-135多肽片段结合情况的western blot检测结果。结果显示，抗体162与在aa120、aa122、aa123位点突变为丙氨酸后的HBsAg失去结合活性，其核心表位为CKTC(aa121-124)；M1D与在aa118-124位点突变为丙氨酸后的HBsAg失去结合活性，其结合的核心表位TGPCKTCT(aa118-124)，比参比抗体162更长，且位置比162靠前；M3D与在aa121-125位点突变为丙氨酸后的HBsAg失去结合活性结合，其核心表位为CKTCT(aa121-125)，核心表位位置比162靠后。
图11A-11B显示实施例9中了人源化抗体M1D、M3D与不同亚型对应的HBsAg-aa113-135的结合活性的ELISA检测结果，其中，横坐标表示抗体浓度(Log10ng/ml)，纵坐标表示OD值。结果显示M1D、M3D均可以结合大部分HBV亚型。
图12A-12B显示了实施例11中单次静脉注射20mg/kg的人源化抗体M1D、M3D后，每一只食蟹猴血清(第1组)中人源化抗体M1D的血药浓度随时间变化的曲线图。其中，横坐标时间(h)，纵坐标表示血药浓度(ng/mL)。
图13A-13B显示了实施例12中人源化抗体M1D、M3D在三种不同缓冲液中的Tm值。横坐标表示温度(℃)，纵坐标表示CP(cal/℃)。结果显示，人源化抗体M1D的Tm onset为61.7℃以上，而M3D的Tm onset值均大于61.6℃，说明M1D和M3D具有很好的热稳定性。
序列信息
本发明涉及的部分序列的信息提供于下面的表1中。
表1：序列的描述
Figure PCTCN2018082157-appb-000001
Figure PCTCN2018082157-appb-000002
Figure PCTCN2018082157-appb-000003
Figure PCTCN2018082157-appb-000004
Figure PCTCN2018082157-appb-000005
Figure PCTCN2018082157-appb-000006
Figure PCTCN2018082157-appb-000007
Figure PCTCN2018082157-appb-000008
Figure PCTCN2018082157-appb-000009
Figure PCTCN2018082157-appb-000010
Figure PCTCN2018082157-appb-000011
发明详述
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、生物化学、核酸化学、免疫学实验室等操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“抗体”是指，通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(V H)和重链恒定区(C H)组成。重链恒定区由3个结构域(C H1、C H2和C H3)组成。各轻链由轻链可变区(V L)和轻链恒定区(C L)组成。轻链恒定区由一个结构域C L组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。V H和V L区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各V H和V L由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(V H和V L)分别形成抗体结合部位。氨 基酸至各区域或结构域的分配遵循Kabat，Sequences of Proteins of Immunological Interest(National Institutes of Health，Bethesda，Md.(1987and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196：901-917；Chothia等人(1989)Nature 342：878-883的定义。
如本文中所使用的，术语“互补决定区”或“CDR”是指抗体可变区中负责抗原结合的氨基酸残基，其通常可包括，轻链可变区中的残基24-34{LCDR1}、50-56{LCDR2}、89-97{LCDR3}以及重链可变区中的残基31-35{HCDR1}、50-65{HCDR2}、95-102{HCDR3}(参见例如，Kabat等，Sequences of Proteins of lmmunological lnterest，第五版，Public Health Service，美国国立卫生研究院，贝塞斯达、马里兰州(1991))，或者轻链可变区中的残基26-32{L1}、50-52{L2}、91-96{L3}以及重链可变区中的残基26-32{H1}、53-55{H2}、96-101{H3}(参见，Chothia和Lesk J.Mol.Biol.196：901-917(1987))。
如本文中所使用的，术语“构架区”或“FR”残基是指，抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。
术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语抗体的“抗原结合片段”是指包含全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology，Ch.7(Paul，W.，ed.，第2版，Raven Press，N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。在一些情况下，抗原结合片段包括Fab、Fab′、F(ab′) 2、Fd、Fv、dAb和互补决定区(CDR)片段、单链抗体(例如，scFv)、嵌合抗体、双抗体(diabody)和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。
如本文中所使用的，术语“Fd片段”意指由V H和C H1结构域组成的抗体片段；术语“dAb片段”意指由VH结构域组成的抗体片段(Ward等人，Nature 341：544 546(1989))；术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段；术语“F(ab′)2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段。
如本文中所使用的，术语“Fv片段”意指由抗体的单臂的V L和V H结构域组成的抗体片段。Fv片段通常被认为是，能形成完整的抗原结合位点的最小抗体片段。一般认 为，六个CDR赋予抗体的抗原结合特异性。然而，即便是一个可变区(例如Fd片段，其仅仅含有三个对抗原特异的CDR)也能够识别并结合抗原，尽管其亲和力低于完整的结合位点。
在一些情况下，抗体的抗原结合片段是单链抗体(例如，scFv)，其中V L和V H结构域通过使其能够产生为单个多肽链的连接体配对形成单价分子(参见，例如，Bird等人，Science 242：423-426(1988)；Huston等人，Proc.Natl.Acad.Sci.USA 85：5879-5883(1988)；和Pluckthun，The Pharmacology of Monoclonal Antibodies，第113卷，Roseburg和Moore编，Springer-Verlag，纽约，第269-315页(1994))。此类scFv分子可具有一般结构：NH 2-V L-接头-V H-COOH或NH 2-V H-接头-V L-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS) 4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90：6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8：725-731，Choi等人(2001)，Eur.J.Immunol.31：94-106，Hu等人(1996)，Cancer Res.56：3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293：41-56和Roovers等人(2001)，Cancer Immunol.描述。
如本文中所使用的，术语“单链抗体-Fc”或“scFv-Fc”意指，将scFv连接至抗体的Fc片段而形成的工程抗体。如本文中所使用的，术语“Fc片段”意指，由抗体的第一重链的第二、第三恒定区与第二重链的第二、第三恒定区经二硫键结合而形成的抗体片段。抗体的Fc片段具有多种不同的功能，但不参与抗原的结合。
在一些情况下，抗体的抗原结合片段是双抗体，即，双价抗体，其中V H和V L结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如，Holliger P.等人，Proc.Natl.Acad.Sci.USA 90：6444-6448(1993)，和Poljak R.J.等人，Structure 2：1121-1123(1994))。
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本发明提供的单克隆抗体)获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。
在本文中，除非上下文明确指出，否则当提及术语“抗体”时，其不仅包括完整抗体，而且包括抗体的抗原结合片段。
如本文中所使用的，术语“单抗”和“单克隆抗体”是指，来自一群高度同源的抗体分子中的一个抗体或抗体的一个片段，也即，除可能自发出现的自然突变外，一群完全相同的抗体分子。单抗对抗原上的单一表位具有高特异性。多克隆抗体是相对于单克隆抗体而言的，其通常包含至少2种或更多种的不同抗体，这些不同的抗体通常识别抗原上的不同表位。单克隆抗体通常可采用Kohler等首次报道的杂交瘤技术获得(Nature，256：495，1975)，但也可采用重组DNA技术获得(如参见U.S.P 4,816,567)。
例如，可以如下来制备单克隆抗体。首先用免疫原(必要时候添加佐剂)免疫注射非人灵长类动物(例如，食蟹猴)或其它合适的宿主动物。免疫原或佐剂的注射方式通常为皮下多点注射或腹腔注射。可将免疫原预先偶联到某些已知蛋白，如血清白蛋白或大豆胰酶抑制剂上，以增强抗原在宿主内的免疫原性。佐剂可以是弗氏佐剂或MPL-TDM等。动物在接受免疫后，体内将产生分泌特异性结合免疫原的抗体的淋巴细胞。另外，淋巴细胞也可以利用体外免疫获得。收集目的淋巴细胞，并用合适的融合剂，如PEG，使其与骨髓瘤细胞融合以获得杂交瘤细胞(Goding，Monoclonal Antibodies：Principles and Practice，pp.59-103，Academic Press，1996)。上述制备的杂交瘤细胞可以接种到合适的培养液中生长，培养液中优选含有一种或多种能够抑制未融合的、母体骨髓瘤细胞生长的物质。例如，对于缺乏次黄嘌呤鸟嘌呤磷酸转移酶(HGPRT或HPRT)的母体骨髓瘤细胞，在培养液中添加次黄嘌呤、氨基喋呤和胸腺嘧啶(HAT培养基)等物质将可以抑制HGPRT-缺陷细胞的生长。优选的骨髓瘤细胞应该具有融合率高，抗体分泌能力稳定，对HAT培养液敏感等特征。生长杂交瘤细胞的培养液用于检测针对特异抗原的单抗的产生。测定杂交瘤细胞产生的单抗的结合特异性的方法包括例如，免疫沉淀或体外结合试验，如放射免疫试验(RIA)、酶联免疫吸附试验(ELISA)。例如，可利用Munson等在Anal.Biochem.107：220(1980)描述的Scatchard分析法来测定单抗的亲和力。当确定了杂交瘤产生的抗体的特异性、亲和力和反应性之后，目的细胞株可以通过(Goding，Monoclonal Antibodies：Principles and Practice，pp.59-103，Academic Press，1996)所描述的标准的有限稀释法进行亚克隆化。合适的培养液可以是DMEM或RPMI-1640等。另外，杂交瘤细胞还可以腹水瘤的形式在动物体内生长。利用传统的免疫球蛋白纯化方法，如蛋白A琼脂糖凝胶、羟基磷灰石层析、凝胶电泳、透析或亲和层析等，可以将亚克隆细胞分泌的单抗从细胞培养液、腹水或血清中分离出来。
还可以通过基因工程重组技术获得单克隆抗体。利用特异性结合单抗重链和轻链基因的核酸引物进行PCR扩增，可以从杂交瘤细胞中分离得到编码单抗重链和轻链基因的DNA分子。将所得的DNA分子插入表达载体内，然后转染宿主细胞(如E.coli细胞、COS细胞、CHO细胞、或其它不产生免疫球蛋白的骨髓瘤细胞)，并在合适的条件下进行培养，可以获得重组表达的目标抗体。
如本文中所使用的，术语“抗原表位”和“表位”是指，抗原上被免疫球蛋白或抗体特异性结合的部位。“表位”在本领域内也称为“抗原决定簇”。表位或抗原决定簇通常由分子的化学活性表面基团例如氨基酸或碳水化合物或糖侧链组成并且通常具有特定的三维结构特征以及特定的电荷特征。例如，表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸，其可以是“线性的”或“构象的”。参见，例如，Epitope Mapping Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)。在线性表位中，蛋白质与相互作用分子(例如抗体)之间的所有相互作用的点沿着蛋白质的一级氨基酸序列线性存在。在构象表位中，相互作用的点跨越彼此分开的蛋白质氨基酸残基而存在。可使用本领域技术人员已知的常规技术，就与相同表位的结合竞争性筛选抗体。例如，可进行竞争和交叉竞争研究，以获得彼此竞争或交叉竞争与抗原结合的抗体。基于它们的交叉竞争来获得结合相同表位的抗体的高通量方法描述于国际专利申请WO 03/48731中。
如本文中所使用的，术语“胚系抗体基因(germline antibody gene)”或“胚系抗体基因片段(germline antibody gene segment)”是指，存在于生物体的基因组中的编码免疫球蛋白的序列，其没有经历过能够导致表达特异性免疫球蛋白的遗传学重排及突变的成熟过程。在本发明中，表述“重链胚系基因”是指，编码免疫球蛋白重链的胚系抗体基因或基因片段，其包括V基因(variable)、D基因(diversity)、J基因(joining)和C基因(constant)；类似地，表述“轻链胚系基因”是指，编码免疫球蛋白轻链的胚系抗体基因或基因片段，其包括V基因(variable)、J基因(joining)和C基因(constant)。在本发明中，由所述胚系抗体基因或胚系抗体基因片段所编码的氨基酸序列也称为“胚系序列(germline sequence)”。胚系抗体基因或胚系抗体基因片段及其相应的胚系序列是本领域技术人员熟知的，并且可从专业数据库(例如，IMGT、UNSWIg、NCBI或VBASE2)获得或查询。
如本文中使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。在某些实施方式中，特异性结合某抗原的抗体(或对 某抗原具有特异性的抗体)是指，抗体以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的亲和力(K D)结合该抗原。
如本文中所使用的，术语“K D”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体(例如，本发明的抗体)以小于大约10 -5M，例如小于大约10 -6M、10 -7M、10 -8M、10 -9M或10 -10M或更小的解离平衡常数(K D)结合抗原(例如，HBsAg)，例如，如使用表面等离子体共振术(SPR)在BIACORE仪中测定的。
如本文中使用的，术语“免疫原性(immunogenicity)”是指，能够刺激机体形成特异抗体或致敏淋巴细胞的能力。其既指，抗原能刺激特定的免疫细胞，使免疫细胞活化、增殖、分化，最终产生免疫效应物质如抗体和致敏淋巴细胞的特性，也指抗原刺激机体后，机体免疫系统能形成抗体或致敏T淋巴细胞的特异性免疫应答。当给受试者施用异源抗体时，异源抗体在受试者体内的免疫原性是不想要的，因为此类免疫原性将导致受试者的免疫系统/免疫细胞对异源抗体的排斥作用，从而导致异源抗体在受试者体内的功效下降或者在受试者体内引起不想要的副作用。因此，在施用给人受试者之前，通常需要对异源抗体(例如食蟹猴源抗体)进行改造，以尽可能减小其免疫原性。
如本文中所使用的，术语“嵌合抗体”是指这样的抗体，其轻链或/和重链的一部分源自一个抗体(其可以源自某一特定物种或属于某一特定抗体类或亚类)，且轻链或/和重链的另一部分源自另一个抗体(其可以源自相同或不同的物种或属于相同或不同的抗体类或亚类)，但无论如何，其仍保留对目标抗原的结合活性(U.S.P 4,816,567to Cabilly et al.；Morrison et al.，Proc.Natl.Acad.Sci.USA，81：6851 6855(1984))。例如，术语“嵌合抗体”可包括这样的抗体(例如人猴嵌合抗体)，其中抗体的重链和轻链可变区来自第一抗体(例如食蟹猴源抗体)，而抗体的重链和轻链可变区来自第二抗体(例如人抗体)。
如本文中所使用的，术语“人源化抗体”是指，经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。通常而言，人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。人源化抗体通常保留了供体抗体的预期性质，包括但不限于，抗原特异性、亲和性、反应性、中和病毒能力和/或清除病毒能力等。供体抗体可以是有预期性质(例如，抗原特异性、亲和性、反应性、中 和病毒能力和/或清除病毒能力)的小鼠、大鼠、兔或非人灵长类动物(例如，食蟹猴)抗体。
人源化抗体既能够保留非人源供体抗体(例如食蟹猴源抗体)的预期性质，又能够有效降低非人源供体抗体(例如食蟹猴源抗体)在人受试者中的免疫原性，因此，是特别有利的。然而，由于供体抗体的CDR与受体抗体的FR之间的匹配问题，人源化抗体的预期性质(例如，抗原特异性、亲和性、反应性、中和病毒能力和/或清除病毒能力)通常低于非人源供体抗体(例如食蟹猴源抗体)。
因此，尽管本领域的研究人员已对抗体的人源化展开了深入的研究，并取得了一些进展(参见例如，Jones et al.，Nature，321：522 525(1986)；Reichmann et al.，Nature，332：323 329(1988)；Presta，Curr.Op.Struct.Biol.，2：593 596(1992)；and Clark，Immunol.Today 21：397 402(2000))，但是，如何对某一供体抗体进行充分的人源化，以使得所产生的人源化抗体既具有尽可能高的人源化程度，又能够尽可能地保留供体抗体的预期性质，现有技术并没有提供详尽的指导。技术人员需要针对具体供体抗体进行摸索、探究和改造，付出大量的创造性劳动才有可能获得，既具有高人源化程度(例如至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的人源化程度)、又保留具体供体抗体的预期性质的人源化抗体。
在本发明中，为了使人源化抗体尽可能保留供体抗体的性质(包括例如，特异性、亲和性、反应性、中和病毒能力和/或清除病毒能力)，本发明的人源化抗体中构架区(FR)可以既包含人源受体抗体的氨基酸残基，也包含相应的非人源供体抗体的氨基酸残基。
此外，为了进一步提高人源化的程度，以尽可能地减少非人氨基酸残基引起的免疫原性，在本发明中，可以对人源化抗体中来源于供体抗体的CDR区的部分氨基酸残基进行替换，例如将其替换为相应的人源免疫球蛋白CDR区的氨基酸残基，或其他氨基酸残基。
此外，为了进一步完善或优化人源化抗体的性能，在本发明中还可以对人源化抗体的重链和轻链可变区中的部分氨基酸残基进行替换，例如将其替换为既非来源于受体抗体，也非来源于供体抗体的氨基酸残基。
具体而言，在本申请中，发明人首先开发了具有优良性质的3株食蟹猴-人嵌合单克隆抗体，分别命名为M1-23，M3-23，M3-13(其重链和轻链可变区分别如SEQ ID NO：1- 5所示)：三株抗体不仅能够特异性识别/结合HBsAg，能够中和HBV的毒力，而且能够在受试者体内降低HBV DNA和/或HBsAg的血清水平，能够有效清除体内的HBV和被HBV感染的细胞。因此，M1-23，M3-23及M3-13抗体具有用于预防和治疗HBV感染以及与HBV感染相关的疾病(例如乙肝)的潜力。
在此基础上，发明人又付出了大量的创造性劳动，对M1-23，M3-23这两株嵌合抗体进行了深入的研究和改造，从而开发了M1-23和M3-23嵌合抗体的人源化抗体：本发明的人源化抗体不仅具有极高的人源化程度(人源化程度可高达98％)，而且具有与亲本抗体基本上相同的(或甚至更优良的)预期性质(包括但不限于，HBsAg结合活性，中和HBV的活性，清除体内的HBV DNA或HBsAg的活性，或清除体内的HBV和被HBV感染的细胞的活性等)。
因此，本发明的抗体(特别是人源化抗体)是极为有利的，其不仅保留了亲本抗体的功能和性质，从而具有用于预防和治疗HBV感染以及与HBV感染相关的疾病(例如乙肝)的潜力；而且具有极高的人源化程度(人源化程度可高达98％)，从而可安全地施用给人受试者，而不引发免疫原性反应。本发明的抗体(特别是人源化抗体)具有重大的临床价值。
在本申请中，本发明抗体的预期性质包括，特异性结合HBsAg的活性，中和HBV的活性，清除体内的HBV DNA或HBsAg的活性，和/或，清除体内的HBV和被HBV感染的细胞的活性。根据本发明的人源化抗体保留了亲本抗体(食蟹猴-人嵌合抗体)的上述预期性质中的一项或多项，优选保留了亲本抗体(食蟹猴-人嵌合抗体)的所有上述预期性质。
在本申请中，通过对本发明的亲本抗体(食蟹猴-人嵌合抗体)进行改造，例如对其FR中的部分氨基酸残基进行置换(例如保守置换)，以获得本发明的人源化抗体。此类置换例如可以(1)降低抗体对蛋白水解的敏感性；(2)降低抗体对氧化作用的易感性；(3)改变(例如增强)抗体对抗原的结合亲和力；(4)改变(例如增强)抗体中和HBV的活性；(5)改变(例如增强)抗体清除HBV的活性；(6)进一步提高抗体的人源化程度，降低抗体的免疫原性；或(7)改变抗体的其它生物化学特点或功能特性；而仍然保留抗体的预期性质。此类置换可以存在于CDR区和/或FR区中，并且可以是单个氨基酸置换或多个氨基酸置换。
如本文中所使用的，术语“人源化程度”是用于表示人源化抗体中非人源氨基酸残基的数量的指标。人源化抗体的人源化程度例如可通过下述来进行计算：人源化程度＝(FR区氨基酸个数-FR区保留的非人源氨基酸个数)/FR区氨基酸个数×100％。
如本文中所使用的，“中和抗体”是指，能够显著降低或完全抑制目标病毒的毒力(例如，感染细胞的能力)的抗体或其抗原结合片段。通常而言，中和抗体能够识别并结合目标病毒，并且阻止目标病毒进入/感染受试者的细胞。本发明的抗体即是中和抗体。
然而，应当理解的是，在本申请中，抗体的中和病毒的能力并不直接等同于抗体的清除病毒的能力。如本文中所使用的，“中和病毒”是指，通过抑制目标病毒进入/感染受试者的细胞的过程，从而中和目标病毒的毒力(即，显著降低或完全抑制目标病毒的毒力)。如本文中所使用的，“清除病毒”是指，机体内的目标病毒(无论其是否感染了细胞)被从机体中消除，从而机体朝向未被病毒感染之前的状态转变(例如，病毒的血清学检测结果转变为阴性)。因此，在一般情况下，中和抗体并不一定具备清除病毒的能力。然而，在本申请中，发明人出人意料地发现，本发明的抗体不仅具有中和HBV的能力，而且具有清除病毒的能力(即，能够清除体内的HBV DNA和/或HBsAg，清除体内的HBV和被HBV感染的细胞)，从而具有重大的临床价值。
如本文中所使用的，术语“分离的”是指，经人工手段从天然状态下获得。如果自然界中出现某一种“被分离”的物质或成分，那么可能是其所处的天然环境发生了改变或从天然环境下离分出该物质，或二者情况均有发生。比如，对于某一活体动物体内天然存在的某种未被分离的多聚核苷酸或多肽而言，从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为“分离的”。术语“分离的”不排除人工或合成的物质的存在，也不排除不影响物质活性的其它不纯物质的存在。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2 果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar，Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4：11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48：444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
如本文中使用的，术语“保守置换”和“保守氨基酸置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的预期性质的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选 用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32：1180-1187(1993)；Kobayashi等人Protein Eng.12(10)：879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94：412-417(1997)，其通过引用并入本文)。
本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如，Immunology-A Synthesis(2nd Edition，E.S.Golub and D.R.Gren，Eds.，Sinauer Associates，Sunderland，Mass.(1991))，其以引用的方式并入本文中。在本发明中，术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。此外，如本文中所使用的，术语“单克隆抗体”和“单抗”具有相同的含义且可互换使用；术语“多克隆抗体”和“多抗”具有相同的含义且可互换使用。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington′s Pharmaceutical Sciences.Edited by Gennaro AR，19th ed.Pennsylvania：Mack Publishing Company，1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂，稀释剂，维持渗透压的试剂，延迟吸收的试剂，防腐剂。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。
如本文中所使用的，术语“佐剂”是指非特异性免疫增强剂，当其与抗原一起或预先递送入机体时，其可增强机体对抗原的免疫应答或改变免疫应答类型。佐剂有很多种，包括但不限于铝佐剂(例如氢氧化铝)、弗氏佐剂(例如完全弗氏佐剂和不完全弗氏佐剂)、短小棒状杆菌、脂多糖、细胞因子等。弗氏佐剂是目前动物试验中最常用的佐剂。氢氧化铝佐剂则在临床实验中使用较多。
如本文中所使用的，术语“预防”是指，为了阻止或延迟疾病或病症或症状(例如，HBV感染或与HBV感染相关的疾病)在受试者体内的发生而实施的方法。如本文中所使用的，术语“治疗”是指，为了获得有益或所需临床结果而实施的方法。为了本发明的目的，有益或所需的临床结果包括(但不限于)减轻症状、缩小疾病的范围、稳定(即， 不再恶化)疾病的状态，延迟或减缓疾病的发展、改善或减轻疾病的状态、和缓解症状(无论部分或全部)，无论是可检测或是不可检测的。此外，“治疗”还可以指，与期望的存活期相比(如果未接受治疗)，延长存活期。在本申请中，本发明的抗体具有中和HBV的能力，从而可用于预防/阻止未患病受试者或其细胞感染HBV。此外，本发明的抗体具有清除HBV的能力(即，能够清除体内的HBV DNA和/或HBsAg，清除体内的HBV和被HBV感染的细胞)，从而可用于治疗患病受试者的HBV感染或与HBV感染相关的疾病。
如本文中使用的，术语“受试者”是指哺乳动物，例如灵长类哺乳动物，例如人。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如HBV感染或与HBV感染相关的疾病)有效量是指，足以预防，阻止，或延迟疾病(例如HBV感染或与HBV感染相关的疾病)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
本发明的抗体
在本申请中，发明人首先开发了具有优良性质的3株食蟹猴-人嵌合单克隆抗体，分别命名为M1-23，M3-23，M3-13(其重链和轻链可变区分别如SEQ ID NO：1-5所示)：三株抗体不仅能够特异性识别/结合HBsAg，能够中和HBV的毒力，而且能够在受试者体内降低HBV DNA和/或HBsAg的血清水平，能够有效清除体内的HBV和被HBV感染的细胞。因此，M1-23，M3-23及M3-13抗体具有用于预防和治疗HBV感染以及与HBV感染相关的疾病(例如乙肝)的潜力。
在此基础上，发明人对M1-23，M3-23这两株抗体进行了深入的研究和改造，从而开发了M1-23和M3-23抗体的人源化抗体：本发明的人源化抗体不仅具有极高的人源化程度(人源化程度可高达98％)，而且具有与亲本抗体基本上相同的(或甚至更优良的)预期性质(包括但不限于，HBsAg结合活性，中和HBV的活性，清除体内的HBV DNA或HBsAg的活性，或清除体内的HBV和被HBV感染的细胞的活性等)。
因此，本发明的抗体(特别是人源化抗体)是极为有利的，其不仅保留了亲本抗体的功能和性质，从而具有用于预防和治疗HBV感染以及与HBV感染相关的疾病(例如乙肝) 的潜力；而且具有极高的人源化程度(人源化程度可高达98％)，从而可安全地施用给人受试者，而不引发免疫原性反应。本发明的抗体(特别是人源化抗体)具有重大的临床价值。
因此，在一个方面，本发明提供了一种能够特异性结合HBsAg的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
(a)选自下列的重链可变区(VH)互补决定区(CDR)中的一个或多个(例如1个，2个或3个)：
(i)VH CDR1，其由下述序列组成：SEQ ID NO：6，SEQ ID NO：12，SEQ ID NO：18或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；
(ii)VH CDR2，其由下述序列组成：SEQ ID NO：7，SEQ ID NO：13，SEQ ID NO：19或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，和
(iii)VH CDR3，其由下述序列组成：SEQ ID NO：8，SEQ ID NO：14，SEQ ID NO：20，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；
和/或
(b)选自下列的轻链可变区(VL)CDR中的一个或多个(例如1个，2个或3个)：
(iv)VL CDR1，其由下述序列组成：SEQ ID NO：9，SEQ ID NO：15或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，
(v)VL CDR2，其由下述序列组成：SEQ ID NO：10，SEQ ID NO：16或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，和
(vi)VL CDR3，其由下述序列组成：SEQ ID NO：11，SEQ ID NO：17或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含如上文所定义的VH CDR1、VH CDR2和VH CDR3。在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含如上文所定义的VL CDR1、VL CDR2和VL CDR3。在某些优选的实施方案 中，本发明的抗体或其抗原结合片段包含如上文所定义的VH CDR1、VH CDR2、VH CDR3、VL CDR1、VL CDR2和VL CDR3。
在某些优选的实施方案中，所述抗体或其抗原结合片段包含：
(a)重链可变区(VH)，所述VH包含：
(i)VH CDR2，其由下述序列组成：SEQ ID NO：7，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，
(ii)VH CDR3，其由下述序列组成：SEQ ID NO：8，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，
以及
(iii)VH CDR1，其由下述序列组成：任意免疫球蛋白的重链可变区中所包含的VH CDR1的序列；
和/或
(b)轻链可变区(VL)，所述VL包含：
(iv)VL CDR1，其由下述序列组成：SEQ ID NO：9，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，
(v)VL CDR3，其由下述序列组成：SEQ ID NO：11，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，
以及
(vi)VL CDR2，其由下述序列组成：任意免疫球蛋白的轻链可变区(例如，κ轻链可变区)中所包含的VL CDR2的序列。
在某些优选的实施方案中，所述VH CDR1为人免疫球蛋白的重链可变区中所包含的VH CDR1的序列。
在某些优选的实施方案中，所述VH CDR1由下述序列组成：(a)SEQ ID NO：6，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；或，(b)人重链胚系基因所编码的氨基酸序列中所包含的VH CDR1的序列。在某些示例性实施方案中，所述人重链胚系基因选自IGHV4-4＊08和IGHV4-61＊01。
在某些示例性实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：7所示的VH CDR2；如SEQ ID NO：8所示的VH CDR3；以及，具有选自下列的氨基酸序列的VH CDR1：SEQ ID NO：6、SEQ ID NO：137或SEQ ID NO：138。
在某些优选的实施方案中，所述VL CDR2为人免疫球蛋白的轻链可变区(例如，κ轻链可变区)中所包含的VL CDR2的序列。
在某些优选的实施方案中，所述VL CDR2由下述序列组成：(a)SEQ ID NO：10，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；或，(b)人轻链胚系基因所编码的氨基酸序列中所包含的VL CDR2的序列。在某些示例性实施方案中，所述人轻链胚系基因选自IGKV1-39＊01和IGKV1-5＊03。
在某些示例性实施方案中，本发明的抗体或其抗原结合片段的VL包含：如SEQ ID NO：9所示的VL CDR1；如SEQ ID NO：11所示的VL CDR3；以及，具有选自下列的氨基酸序列的VL CDR2：SEQ ID NO：10、SEQ ID NO：139或SEQ ID NO：140。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：7所示的VH CDR2；如SEQ ID NO：8所示的VH CDR3；以及VH CDR1，其由下述序列组成：任意免疫球蛋白的重链可变区中所包含的VH CDR1的序列(例如，SEQ ID NO：6，137或138)；
并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO：9所示的VL CDR1，如SEQ ID NO：11所示的VL CDR3，以及如SEQ ID NO：10所示的VL CDR2。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：7所示的VH CDR2；如SEQ ID NO：8所示的VH CDR3；以及如SEQ ID NO：6所示的VH CDR1；
并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO：9所示的VL CDR1，如SEQ ID NO：11所示的VL CDR3，以及VL CDR2，其由下述序列组成：任意免疫球蛋白的轻链可变区(例如，κ轻链可变区)中所包含的VL CDR2的序列(例如，SEQ ID NO：10，139或140)。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：7所示的VH CDR2，如SEQ ID NO：8所示的VH CDR3，以及具有选自下列的氨基酸序列的VH CDR1：SEQ ID NO：6，137或138；并且，该抗体或其抗原结合片段的VL包含：如SEQ ID NO：9所示的VL CDR1，如SEQ ID NO：11所示的VL CDR3，以及具有选自下列的氨基酸序列的VL CDR2：SEQ ID NO：10，139或140。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：6所示的VH CDR1、如SEQ ID NO：7所示的VH CDR2、和如SEQ ID NO：8所示 的VH CDR3；并且，该抗体或其抗原结合片段的VL包含：如SEQ ID NO：9所示的VL CDR1、如SEQ ID NO：10所示的VL CDR2、和如SEQ ID NO：11所示的VL CDR3。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：137所示的VH CDR1、如SEQ ID NO：7所示的VH CDR2、和如SEQ ID NO：8所示的VH CDR3；并且，该抗体或其抗原结合片段的VL包含：如SEQ ID NO：9所示的VL CDR1、如SEQ ID NO：10所示的VL CDR2、和如SEQ ID NO：11所示的VL CDR3。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：138所示的VH CDR1、如SEQ ID NO：7所示的VH CDR2、和如SEQ ID NO：8所示的VH CDR3；并且，该抗体或其抗原结合片段的VL包含：如SEQ ID NO：9所示的VL CDR1、如SEQ ID NO：10所示的VL CDR2、和如SEQ ID NO：11所示的VL CDR3。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：6所示的VH CDR1、如SEQ ID NO：7所示的VH CDR2、和如SEQ ID NO：8所示的VH CDR3；并且，该抗体或其抗原结合片段的VL包含：如SEQ ID NO：9所示的VL CDR1、如SEQ ID NO：139所示的VL CDR2、和如SEQ ID NO：11所示的VL CDR3。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：6所示的VH CDR1、如SEQ ID NO：7所示的VH CDR2、和如SEQ ID NO：8所示的VH CDR3；并且，该抗体或其抗原结合片段的VL包含：如SEQ ID NO：9所示的VL CDR1、如SEQ ID NO：140所示的VL CDR2、和如SEQ ID NO：11所示的VL CDR3。
在某些优选的实施方案中，所述抗体或其抗原结合片段包含：
(a)重链可变区(VH)，所述VH包含：
(i)VH CDR1，其由下述序列组成：SEQ ID NO：12，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，
(ii)VH CDR3，其由下述序列组成：SEQ ID NO：14，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，以及
(iii)VH CDR2，其由下述序列组成：任意免疫球蛋白的重链可变区中所包含的VH CDR2的序列；
和/或
(b)轻链可变区(VL)，所述VL包含：
(iv)VL CDR1，其由下述序列组成：SEQ ID NO：15，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，
(v)VL CDR3，其由下述序列组成：SEQ ID NO：17，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，
以及
(vi)VL CDR2，其由下述序列组成：任意免疫球蛋白的轻链可变区(例如，κ轻链可变区)中所包含的VL CDR2的序列。
在某些优选的实施方案中，所述VH CDR2为人免疫球蛋白的重链可变区中所包含的VH CDR2的序列。
在某些优选的实施方案中，所述VH CDR2由下述序列组成：(a)SEQ ID NO：13，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；或，(b)人重链胚系基因所编码的氨基酸序列中所包含的VH CDR2的序列。在某些示例性实施方案中，所述人重链胚系基因选自IGHV4-30-4＊07和IGHV4-4＊01。
在某些示例性实施方案中，本发明的抗体或其抗原结合片段的VH包含：(a)如SEQ ID NO：12所示的VH CDR1；如SEQ ID NO：14所示的VH CDR3；以及，具有选自下列的氨基酸序列的VH CDR2：SEQ ID NO：13、SEQ ID NO：141或SEQ ID NO：142。
在某些优选的实施方案中，所述VL CDR2为人免疫球蛋白的轻链可变区(例如，κ轻链可变区)中所包含的VL CDR2的序列。
在某些优选的实施方案中，所述VL CDR2由下述序列组成：(a)SEQ ID NO：16，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；或，(b)人轻链胚系基因所编码的氨基酸序列中所包含的VL CDR2的序列。在某些示例性实施方案中，所述人轻链胚系基因选自IGKV2-28＊01和IGKV3-15＊01。
在某些示例性实施方案中，本发明的抗体或其抗原结合片段的VL包含：如SEQ ID NO：15所示的VL CDR1；如SEQ ID NO：17所示的VL CDR3；以及，具有选自下列的氨基酸序列的VL CDR2：SEQ ID NO：16、SEQ ID NO：143或SEQ ID NO：144。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：12所示的VH CDR1，如SEQ ID NO：14所示的VH CDR3，以及VH CDR2，其由下述序列组成：任意免疫球蛋白的重链可变区中所包含的VH CDR2的序列(例如， SEQ ID NO：13、SEQ ID NO：141或SEQ ID NO：142)；
并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO：15所示的VL CDR1，如SEQ ID NO：17所示的VL CDR3，以及如SEQ ID NO：16所示的VL CDR2。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：12所示的VH CDR1；如SEQ ID NO：14所示的VH CDR3；以及如SEQ ID NO：13所示的VH CDR2；
并且，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO：15所示的VL CDR1，如SEQ ID NO：17所示的VL CDR3，以及VL CDR2，其由下述序列组成：任意免疫球蛋白的轻链可变区(例如，κ轻链可变区)中所包含的VL CDR2的序列(例如，SEQ ID NO：16、SEQ ID NO：143或SEQ ID NO：144)。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：12所示的VH CDR1，如SEQ ID NO：14所示的VH CDR3，以及VH CDR2，所述VH CDR2具有选自下列的序列：SEQ ID NO：13，141或142；并且，该抗体或其抗原结合片段的VL包含：如SEQ ID NO：15所示的VL CDR1，如SEQ ID NO：17所示的VL CDR3，以及VL CDR2，所述VL CDR2具有选自下列的序列：SEQ ID NO：16，143或144。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：12所示的VH CDR1、如SEQ ID NO：13所示的VH CDR2、和如SEQ ID NO：14所示的VH CDR3；并且，该抗体或其抗原结合片段的VL包含：如SEQ ID NO：15所示的VL CDR1、如SEQ ID NO：16所示的VL CDR2、和如SEQ ID NO：17所示的VL CDR3。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：12所示的VH CDR1、如SEQ ID NO：141所示的VH CDR2、和如SEQ ID NO：14所示的VH CDR3；并且，该抗体或其抗原结合片段的VL包含：如SEQ ID NO：15所示的VL CDR1、如SEQ ID NO：16所示的VL CDR2、和如SEQ ID NO：17所示的VL CDR3。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：12所示的VH CDR1、如SEQ ID NO：142所示的VH CDR2、和如SEQ ID NO：14所示的VH CDR3；并且，该抗体或其抗原结合片段的VL包含：如SEQ ID NO：15所示的VL CDR1、如SEQ ID NO：16所示的VL CDR2、和如SEQ ID NO：17所示的VL  CDR3。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：12所示的VH CDR1、如SEQ ID NO：13所示的VH CDR2、和如SEQ ID NO：14所示的VH CDR3；并且，该抗体或其抗原结合片段的VL包含：如SEQ ID NO：15所示的VL CDR1、如SEQ ID NO：143所示的VL CDR2、和如SEQ ID NO：17所示的VL CDR3。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：12所示的VH CDR1、如SEQ ID NO：13所示的VH CDR2、和如SEQ ID NO：14所示的VH CDR3；并且，该抗体或其抗原结合片段的VL包含：如SEQ ID NO：15所示的VL CDR1、如SEQ ID NO：144所示的VL CDR2、和如SEQ ID NO：17所示的VL CDR3。
在某些优选的实施方案中，所述抗体或其抗原结合片段包含：
(a)重链可变区(VH)，所述VH包含：
(i)VH CDR1，其由下述序列组成：SEQ ID NO：18，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；
(ii)VH CDR2，其由下述序列组成：SEQ ID NO：19，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，
和
(iii)VH CDR3，其由下述序列组成：SEQ ID NO：20，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；
和/或
(b)轻链可变区(VL)，所述VL包含：
(iv)VL CDR1，其由下述序列组成：SEQ ID NO：15，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，
(v)VL CDR2，其由下述序列组成：SEQ ID NO：16，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，
和
(vi)VL CDR3，其由下述序列组成：SEQ ID NO：17，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的VH包含：如SEQ ID NO：18所示的VH CDR1，如SEQ ID NO：19所示的VH CDR2，以及如SEQ ID NO：20所示的VH CDR3；并且，该抗体或其抗原结合片段的VL包含：如SEQ ID NO：15所示的VL CDR1，如SEQ ID NO：16所示的VL CDR2，以及如SEQ ID NO：17所示的VL CDR3。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段进一步包含框架区。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：
(a)选自下列的重链可变区(VH)构架区(FR)中的一个或多个(例如1个，2个，3个或4个)：
(i)VH FR1，其由下述序列组成：SEQ ID NO：21，SEQ ID NO：29，SEQ ID NO：37，SEQ ID NO：44，SEQ ID NO：50，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个、3个或4个氨基酸置换、缺失或添加)的序列；
(ii)VH FR2，其由下述序列组成：SEQ ID NO：22，SEQ ID NO：30，SEQ ID NO：38，SEQ ID NO：51，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个、3个或4个氨基酸置换、缺失或添加)的序列；
(iii)VH FR3，其由下述序列组成：SEQ ID NO：23，SEQ ID NO：31，SEQ ID NO：39，SEQ ID NO：45，SEQ ID NO：52，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个、3个或4个氨基酸置换、缺失或添加)的序列；和
(iv)VH FR4，其由下述序列组成：SEQ ID NO：24，SEQ ID NO：32，SEQ ID NO：46，SEQ ID NO：53，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个、3个或4个氨基酸置换、缺失或添加)的序列；
和/或
(b)选自下列的轻链可变区(VL)构架区(FR)中的一个或多个(例如1个，2个，3个或4个)：
(v)VL FR1，其由下述序列组成：SEQ ID NO：25，SEQ ID NO：33，SEQ ID NO：47，SEQ ID NO：54，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个、3个或4个氨基酸置换、缺失或添加)的序列；
(vi)VL FR2，其由下述序列组成：SEQ ID NO：26，SEQ ID NO：34，SEQ ID NO：48，SEQ ID NO：55，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、 2个、3个或4个氨基酸置换、缺失或添加)的序列；
(vii)VL FR3，其由下述序列组成：SEQ ID NO：27，SEQ ID NO：35，SEQ ID NO：49，SEQ ID NO：56，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个、3个或4个氨基酸置换、缺失或添加)的序列；和
(viii)VL FR4，其由下述序列组成：SEQ ID NO：28，SEQ ID NO：36，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个、3个或4个氨基酸置换、缺失或添加)的序列。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含如上文所定义的VH FR1、VH FR2、VH FR3和VH FR4。在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含如上文所定义的VL FR1、VL FR2、VL FR3和VL FR4。在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含如上文所定义的VH FR1、VH FR2、VH FR3、VH FR4、VL FR1、VL FR2、VL FR3和VL FR4。
在某些优选的实施方案中，所述抗体或其抗原结合片段的VH包含：
(a)如SEQ ID NO：21所示的VH FR1、如SEQ ID NO：22所示的VH FR2、如SEQ ID NO：23所示的VH FR3、和如SEQ ID NO：24所示的VH FR4；
(b)如SEQ ID NO：29所示的VH FR1、如SEQ ID NO：30所示的VH FR2、如SEQ ID NO：31所示的VH FR3、和如SEQ ID NO：32所示的VH FR4；或
(c)如SEQ ID NO：37所示的VH FR1、如SEQ ID NO：38所示的VH FR2、如SEQ ID NO：39所示的VH FR3、和如SEQ ID NO：32所示的VH FR4。
在某些优选的实施方案中，所述抗体或其抗原结合片段的VL包含：
(a)如SEQ ID NO：25所示的VL FR1、如SEQ ID NO：26所示的VL FR2、如SEQ ID NO：27所示的VL FR3、和如SEQ ID NO：28所示的VL FR4；或
(b)如SEQ ID NO：33所示的VL FR1、如SEQ ID NO：34所示的VL FR2、如SEQ ID NO：35所示的VL FR3、和如SEQ ID NO：36所示的VL FR4。
在某些优选的实施方案中，所述抗体或其抗原结合片段包含：
(a)如SEQ ID NO：21所示的VH FR1、如SEQ ID NO：22所示的VH FR2、如SEQ ID NO：23所示的VH FR3、和如SEQ ID NO：24所示的VH FR4；以及，如SEQ ID NO：25所示的VL FR1、如SEQ ID NO：26所示的VL FR2、如SEQ ID NO：27所示的VL FR3、和如SEQ ID NO：28所示的VL FR4；
(b)如SEQ ID NO：29所示的VH FR1、如SEQ ID NO：30所示的VH FR2、如SEQ  ID NO：31所示的VH FR3、和如SEQ ID NO：32所示的VH FR4；以及，如SEQ ID NO：33所示的VL FR1、如SEQ ID NO：34所示的VL FR2、如SEQ ID NO：35所示的VL FR3、和如SEQ ID NO：36所示的VL FR4；或
(c)如SEQ ID NO：37所示的VH FR1、如SEQ ID NO：38所示的VH FR2、如SEQ ID NO：39所示的VH FR3、和如SEQ ID NO：32所示的VH FR4；以及，如SEQ ID NO：33所示的VL FR1、如SEQ ID NO：34所示的VL FR2、如SEQ ID NO：35所示的VL FR3、和如SEQ ID NO：36所示的VL FR4。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段是人源化的。在某些优选的实施方案中，本发明的抗体或其抗原结合片段的人源化程度为至少80％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％，或100％。在某些优选的实施方案中，本发明的抗体或其抗原结合片段的非CDR区包含不超过19个，不超过15个，不超过14个，不超过13个，不超过12个，不超过11个，不超过10个，不超过9个，不超过8个，不超过7个，不超过6个，不超过5个，不超过4个，不超过3个，不超过2个，或不超过1个的非人源(例如，食蟹猴源)氨基酸残基，或者其不包含非人源(例如，食蟹猴源)氨基酸残基。在某些优选的实施方案中，本发明的抗体或其抗原结合片段的FR区包含不超过19个，不超过15个，不超过14个，不超过13个，不超过12个，不超过11个，不超过10个，不超过9个，不超过8个，不超过7个，不超过6个，不超过5个，不超过4个，不超过3个，不超过2个，或不超过1个的非人源(例如，食蟹猴源)氨基酸残基，或者其不包含非人源(例如，食蟹猴源)氨基酸残基。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：人免疫球蛋白的框架区，例如人胚系抗体基因所编码的氨基酸序列中所包含的框架区。在某些优选的实施方案中，所述抗体或其抗原结合片段包含：人重链胚系基因所编码的氨基酸序列中所包含的重链框架区，和/或人轻链胚系基因所编码的氨基酸序列中所包含的轻链框架区。
在此类实施方案中，本发明的抗体或其抗原结合片段的重链框架区和/或轻链框架区可以包含一个或多个非人源(例如，食蟹猴源)氨基酸残基。在某些优选的实施方案中，所述重链框架区和/或轻链框架区包含一或多个氨基酸残基，其被回复突变至相应的食蟹 猴源残基或相应食蟹猴源残基的保守氨基酸氨基酸置换(此类突变称为回复突变(back mutation))。
在某些优选的实施方案中，所述抗体或其抗原结合片段的VH包含：人重链胚系基因IGHV4-4＊08所编码的氨基酸序列中所包含的重链框架区，所述重链框架区任选地包含一个或多个从人源残基至食蟹猴源残基的回复突变。在某些优选的实施方案中，所述IGHV4-4＊08所编码的氨基酸序列如SEQ ID NO：40所示。
在某些优选的实施方案中，所述抗体或其抗原结合片段的VH包含：
(i)VH FR1，其为SEQ ID NO：21，或者与SEQ ID NO：21的差异在于选自下列的一个或几个氨基酸置换(例如，1个，2个或3个氨基酸置换)：
(01)位于H17的T；
(02)位于H20的L；和
(03)位于H23的T；
(ii)VH FR2，其为SEQ ID NO：22；
(iii)VH FR3，其为SEQ ID NO：23，或者与SEQ ID NO：23的差异在于选自下列的一个或几个氨基酸置换(例如，1个，2个或3个氨基酸置换)：
(01)位于H64的K；
(02)位于H69的I；和
(03)位于H78的F；
(iv)VH FR4，其为SEQ ID NO：24，或者与SEQ ID NO：24的差异在于选自下列的一个或几个氨基酸置换(例如，1个或2个氨基酸置换)：
(01)位于H107的T；和
(02)位于H108的T；
其中，上述提及的所有氨基酸位置均是根据Kabat编号系统的位置。
在某些示例性实施方案中，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO：44所示的VH FR1、如SEQ ID NO：22所示的VH FR2、如SEQ ID NO：45所示的VH FR3、和如SEQ ID NO：46所示的VH FR4。
在某些优选的实施方案中，所述抗体或其抗原结合片段的VH包含：人重链胚系基因IGHV4-4＊02所编码的氨基酸序列中所包含的重链框架区，所述重链框架区任选地包含一个或多个从人源残基至食蟹猴源残基的回复突变。在某些优选的实施方案中，所述 IGHV4-4＊02所编码的氨基酸序列如SEQ ID NO：42所示。
在某些优选的实施方案中，所述抗体或其抗原结合片段的VH包含：
(i)VH FR1，其为SEQ ID NO：29，或者与SEQ ID NO：29的差异在于选自下列的一个或几个氨基酸置换(例如，1个或2个)：
(01)位于H2的V；和
(02)位于H16的G；
(ii)VH FR2，其为SEQ ID NO：30，或者与SEQ ID NO：30的差异在于下列的氨基酸置换：位于H37的V；
(iii)VH FR3，其为SEQ ID NO：31，或者与SEQ ID NO：31的差异在于选自下列的一个或几个氨基酸置换(例如，1个，2个，3个或4个氨基酸置换)：
(01)位于H65的S；
(02)位于H71的V；
(03)位于H73的K；和
(04)位于H81的K；
(iv)VH FR4，其为SEQ ID NO：32，或者与SEQ ID NO：32的差异在于下列的一个氨基酸置换：位于H107的T；
其中，上述中提及的所有氨基酸位置是根据Kabat编号系统的位置。
在某些示例性实施方案中，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO：50所示的VH FR1、如SEQ ID NO：51所示的VH FR2、如SEQ ID NO：52所示的VH FR3、和如SEQ ID NO：53所示的VH FR4。
在某些优选的实施方案中，所述抗体或其抗原结合片段的VL包含：人轻链胚系基因IGKV1-39＊01所编码的氨基酸序列中所包含的轻链框架区，所述轻链框架区任选地包含一个或多个从人源残基至食蟹猴源残基的回复突变。在某些优选的实施方案中，所述IGKV1-39＊01所编码的氨基酸序列如SEQ ID NO：41所示。
在某些优选的实施方案中，所述抗体或其抗原结合片段的VL包含：
(i)VL FR1，其为SEQ ID NO：25，或者与SEQ ID NO：25的差异在于选自下列的一个或几个氨基酸置换(例如，1个，或2个氨基酸置换)：
(01)位于L1的D；和
(02)位于L3的Q；
(ii)VL FR2，其为SEQ ID NO：26，或者与SEQ ID NO：26的差异在于下列的氨基酸置换：位于L45的K；
(iii)VL FR3，其为SEQ ID NO：27，或者与SEQ ID NO：27的差异在于选自下列的一个或几个氨基酸置换(例如，1个，2个，3个或4个氨基酸置换)：
(01)位于L55的Q；
(02)位于L63的S；
(03)位于L70的D；和
(04)位于L84的A；
(iv)VL FR4，其为SEQ ID NO：28；
其中，上述提及的所有氨基酸位置是根据Kabat编号系统的位置。
在某些示例性实施方案中，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO：47所示的VL FR1、如SEQ ID NO：48所示的VL FR2、如SEQ ID NO：49所示的VL FR3、和如SEQ ID NO：28所示的VL FR4。
在某些优选的实施方案中，所述抗体或其抗原结合片段的VL包含：人轻链胚系基因IGKV4-1＊01所编码的氨基酸序列中所包含的轻链框架区，所述轻链框架区任选地包含一个或多个从人源残基至食蟹猴源残基的回复突变。在某些优选的实施方案中，所述IGKV4-1＊01所编码的氨基酸序列如SEQ ID NO：43所示。
在某些优选的实施方案中，所述抗体或其抗原结合片段的VL包含：
(i)VL FR1，其为SEQ ID NO：33，或者与SEQ ID NO：33的差异在于选自下列的一个或几个氨基酸置换(例如，1个，2个或3个氨基酸置换)：
(01)位于L3的V；
(02)位于L5的T；和
(03)位于L19的A；
(ii)VL FR2，其为SEQ ID NO：34，或者与SEQ ID NO：34的差异在于下列的氨基酸置换：位于L43的V；
(iii)VL FR3，其为SEQ ID NO：35，或者与SEQ ID NO：35的差异在于选自下列的一个或几个氨基酸置换(例如，1个，2个或3个氨基酸置换)：
(01)位于L60的D；
(02)位于L76的S；和
(03)位于L77的S；
(iv)VL FR4，其为SEQ ID NO：36；
其中，上述提及的氨基酸位置是根据Kabat编号系统的位置。
在某些示例性实施方案中，所述抗体或其抗原结合片段的VL包含：如SEQ ID NO：54所示的VL FR1、如SEQ ID NO：55所示的VL FR2、如SEQ ID NO：56所示的VL FR3、和如SEQ ID NO：36所示的VL FR4。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段包含：
(a)IGHV4-4＊08所编码的氨基酸序列中所包含的重链框架区，以及，IGKV1-39＊01所编码的氨基酸序列中所包含的轻链框架区；或
(b)IGHV4-4＊02所编码的氨基酸序列中所包含的重链框架区，以及，IGKV4-1＊01所编码的氨基酸序列中所包含的轻链框架区。
在某些优选的实施方案中，所述抗体或其抗原结合片段包含：
(a)如SEQ ID NO：44所示的VH FR1、如SEQ ID NO：22所示的VH FR2、如SEQ ID NO：45所示的VH FR3、如SEQ ID NO：46所示的VH FR4；以及，如SEQ ID NO：47所示的VL FR1、如SEQ ID NO：48所示的VL FR2、如SEQ ID NO：49所示的VL FR3、和如SEQ ID NO：28所示的VL FR4；或
(b)如SEQ ID NO：50所示的VH FR1、如SEQ ID NO：51所示的VH FR2、如SEQ ID NO：52所示的VH FR3、和如SEQ ID NO：53所示的VH FR4；以及，如SEQ ID NO：54所示的VL FR1、如SEQ ID NO：55所示的VL FR2、如SEQ ID NO：56所示的VL FR3、和如SEQ ID NO：36所示的VL FR4。
在某些优选的实施方式中，本发明的抗体或其抗原结合片段的VH包含：
(a)如SEQ ID NO：21所示的VH FR1、如SEQ ID NO：22所示的VH FR2、如SEQ ID NO：23所示的VH FR3、和如SEQ ID NO：24所示的VH FR4；
(b)如SEQ ID NO：29所示的VH FR1、如SEQ ID NO：30所示的VH FR2、如SEQ ID NO：31所示的VH FR3、和如SEQ ID NO：32所示的VH FR4；
(c)如SEQ ID NO：37所示的VH FR1、如SEQ ID NO：38所示的VH FR2、如SEQ ID NO：39所示的VH FR3、和如SEQ ID NO：32所示的VH FR4；
(d)如SEQ ID NO：44所示的VH FR1、如SEQ ID NO：22所示的VH FR2、如SEQ ID NO：45所示的VH FR3、和如SEQ ID NO：46所示的VH FR4；或者
(e)如SEQ ID NO：50所示的VH FR1、如SEQ ID NO：51所示的VH FR2、如SEQ ID NO：52所示的VH FR3、和如SEQ ID NO：53所示的VH FR4。
在某些优选的实施方式中，本发明的抗体或其抗原结合片段的VL包含：
(a)如SEQ ID NO：25所示的VL FR1、如SEQ ID NO：26所示的VL FR2、如SEQ ID NO：27所示的VL FR3、和如SEQ ID NO：28所示的VL FR4；
(b)如SEQ ID NO：33所示的VL FR1、如SEQ ID NO：34所示的VL FR2、如SEQ ID NO：35所示的VL FR3、和如SEQ ID NO：36所示的VL FR4；
(c)如SEQ ID NO：47所示的VL FR1、如SEQ ID NO：48所示的VL FR2、如SEQ ID NO：49所示的VL FR3、和如SEQ ID NO：28所示的VL FR4；或者
(d)如SEQ ID NO：54所示的VL FR1、如SEQ ID NO：55所示的VL FR2、如SEQ ID NO：56所示的VL FR3、和如SEQ ID NO：36所示的VL FR4。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的重链可变区包含如上文所定义的VH FR1、VH CDR1、VH FR2、VH CDR2、VH FR3、VH CDR3和VH FR4。在某些优选的实施方案中，本发明的抗体或其抗原结合片段的轻链可变区包含如上文所定义的VL FR1、VL CDR1、VL FR2、VL CDR2、VL FR3、VL CDR3和VL FR4。在某些优选的实施方案中，本发明的抗体或其抗原结合片段的重链可变区包含如上文所定义的VH FR1、VH CDR1、VH FR2、VH CDR2、VH FR3、VH CDR3和VH FR4；并且，轻链可变区包含如上文所定义的VL FR1、VL CDR1、VL FR2、VL CDR2、VL FR3、VL CDR3和VL FR4。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的重链可变区的氨基酸序列与选自下列的重链可变区的氨基酸序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性：如SEQ ID NOs：1，3，5，57，59，157，158，163和164任一项所示的重链可变区。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的重链可变区选自如SEQ ID NOs：1，3，5，57，59，157，158，163和164任一项所示的重链可变区。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的轻链可变区的氨基酸序列与选自下列的轻链可变区的氨基酸序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性：如SEQ ID NOs：2，4，58，60，159，160，165和166任一项所示的轻链可变区。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的轻链可变区选自如SEQ ID NOs：2，4，58，60，159，160，165和166任一项所示的轻链可变区。
在某些优选的实施方案中，本发明的抗体包含如上文所定义的重链可变区和如上文所定义的轻链可变区。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的重链可变区选自如SEQ ID NOs：1，57，157和158任一项所示的重链可变区；并且，所述抗体或其抗原结合片段的轻链可变区选自如SEQ ID NOs：2，58，159和160任一项所示的轻链可变区。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段的重链可变区选自如SEQ ID NOs：3，59，163和164任一项所示的重链可变区；并且，所述抗体或其抗原结合片段的轻链可变区选自如SEQ ID NOs：4，60，165和166任一项所示的轻链可变区。
在某些优选的实施方案中，本发明的抗体包含：
(1)如SEQ ID NO：1所示的VH和如SEQ ID NO：2所示的VL；
(2)如SEQ ID NO：3所示的VH和如SEQ ID NO：4所示的VL；
(3)如SEQ ID NO：5所示的VH和如SEQ ID NO：4所示的VL；
(4)如SEQ ID NO：57所示的VH和如SEQ ID NO：58所示的VL；
(5)如SEQ ID NO：59所示的VH和如SEQ ID NO：60所示的VL；
(6)如SEQ ID NO：157所示的VH和如SEQ ID NO：58所示的VL；
(7)如SEQ ID NO：158所示的VH和如SEQ ID NO：58所示的VL；
(8)如SEQ ID NO：57所示的VH和如SEQ ID NO：159所示的VL；
(9)如SEQ ID NO：57所示的VH和如SEQ ID NO：160所示的VL；
(10)如SEQ ID NO：163所示的VH和如SEQ ID NO：60所示的VL；
(11)如SEQ ID NO：164所示的VH和如SEQ ID NO：60所示的VL；
(12)如SEQ ID NO：59所示的VH和如SEQ ID NO：165所示的VL；或
(13)如SEQ ID NO：59所示的VH和如SEQ ID NO：166所示的VL。
本发明的抗体可以通过基因工程重组技术来获得。例如，通过化学合成或PCR扩增获得编码本发明抗体的重链和轻链基因的DNA分子。将所得DNA分子插入表达载体内，然后转染宿主细胞，如E.coli细胞、猿猴COS、CHO细胞、或其它不产生免疫球蛋白的骨髓瘤细胞。然后，在特定条件下培养转染后的宿主细胞，并表达本发明的抗体。
本发明的抗体对HBsAg蛋白具有高特异性和高亲和力。例如，本发明的抗体结合HBsAg的KD值可小于1x 10 -5M；优选地，KD值小于1x 10 -6M；更优选地，KD值小于1x 10 -7M；最优选地，KD值小于1x 10 -8M。
本发明的抗体可以是包含两条重链和两条轻链的、具有传统的“Y”型结构的抗体。另外，本发明的抗体也可以是具有传统的“Y”型结构的抗体的Fab片段、Fab′、F(ab) 2、Fv、或其它类型的片段，其保持了对HBsAg蛋白的亲和力，并且其结合HBsAg蛋白的亲和力可以高于或低于具有传统的“Y”型结构的抗体。
本发明的抗原结合片段可以通过水解完整的抗体分子获得(参见Morimoto et al.，J.Biochem.Biophys.Methods 24：107-117(1992)and Brennan et al.，Science 229：81(1985))。另外，这些抗原结合片段也可以直接由重组宿主细胞产生(reviewed in Hudson，Curr.Opin.Immunol.11：548-557(1999)；Little et al.，Immunol.Today，21：364-370(2000))。比如，Fab′片段可以直接从E.coli细胞中获得；可以将Fab′片段化学藕联形成F(ab′) 2片段(Carter et al.，Bio/Technology，10：163-167(1992))。另外，Fv、Fab或F(ab′) 2片段也可以直接从重组宿主细胞培养液中直接分离得到。本领域的普通技术人员完全知晓制备这些抗原结合片段的其它技术。
因此，在某些优选的实施方案中，本发明的抗体或其抗原结合片段选自scFv、Fab、Fab’、(Fab’) 2、Fv片段、双抗体(diabody)、双特异性抗体、多特异性抗体、嵌合抗体或人源化抗体。特别优选地，本发明的抗体或其抗原结合片段为嵌合抗体或人源化抗体。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段能够特异性结合HBsAg，中和HBV的毒力，和/或降低HBV DNA和/或HBsAg在受试者体内的血清水平。
在某些优选的实施方案中，本发明的抗体或其抗原结合片段是IgG类别。例如，本发明的抗体或其抗原结合片段可以为IgG1或IgG2或IgG3或IgG4类别。
融合抗体
在另一个方面，可制备融合抗体或免疫粘合素，例如，可以将本发明的抗体或其抗原结合片段连接于另一个多肽。在某些优选的实施方案中，融合抗体包含抗本发明抗体 的重链可变区和轻链可变区。在某些优选的实施方案中，融合抗体包含本发明抗体的VH结构域和VL结构域；其中，所述VH结构域连接于第一个多肽，而所述VL结构域连接于第二个多肽。
衍生的抗体
本发明的抗体或其抗原结合片段可进行衍生化，例如被连接至另一个分子(例如另一个多肽或蛋白)。通常，抗体或其抗原结合片段的衍生化(例如，标记)不会不利影响其对HBsAg的结合。因此，本发明的抗体或其抗原结合片段还意欲包括此类衍生化的形式。例如，可以将本发明的抗体或其抗原结合片段功能性连接(通过化学偶合、基因融合、非共价连接或其它方式)于一个或多个其它分子基团，例如另一个抗体(例如，形成双特异性抗体)，检测试剂，药用试剂，和/或能够介导抗体或抗原结合片段与另一个分子结合的蛋白或多肽(例如，抗生物素蛋白或多组氨酸标签)。
一种类型的衍生化抗体(例如，双特异性抗体)是通过交叉连接2个或多个抗体(属于同一类型或不同类型)而产生的。适当的交联剂包括例如异基双功能剂，其含有由适当的间隔区隔开的2个不同的反应基团(例如m-丁烯酰胺苯甲酸-N-羟基琥珀酰亚胺酯)；以及，同基双功能剂(双琥珀酰亚胺辛二酸酯)。此类交联剂可从Pierce Chemical Company，Rockford，II购买得到。
另一种类型的衍生化抗体是标记的抗体。例如，可以将本发明的抗体或其抗原结合片段连接至有用的检测试剂。此类检测试剂包括例如，荧光化合物，例如荧光素、异硫氯酸荧光素、罗丹明、5-二甲氨基-1-蔡磺酰氯、藻红蛋白、镧磷光剂等等。此外，抗体也可以用酶进行标记，例如辣根过氧化物酶、β-半乳糖苷酶、萤光素酶、碱性磷酸酶、葡萄糖氧化酶等等。当抗体用酶进行标记时，可通过加入能够被酶利用并产生可识别的信号或反应产物的试剂来检测标记的抗体。例如，当使用辣根过氧化物酶标记抗体时，可以加入过氧化氢和二氨基联苯，以产生可检测的有色反应产物，从而检测标记的抗体的存在或量。此外，抗体也可以用生物素进行标记。在这种情况下，可以通过间接测定抗生物素蛋白的结合来检测标记的抗体的存在或量。此外，抗体也可以用可被第二个报告分子识别的标签进行标记(例如亮氨酸拉链互补序列、金属结合结构域、附加表位等等)。在某些具体实施方式中，标签通过不同长度的间隔臂连接至抗体，以降低潜在的位阻。
此外，本发明的抗体或其抗原结合片段还可以用化学基团进行衍生，例如聚乙二醇(PEG)，甲基或乙基，或者糖基。这些基团可用于改善抗体的生物学特性，例如增加血清 半衰期。
核酸分子、载体和宿主细胞
在另一个方面，本发明提供了一种分离的核酸分子，其包含编码本发明的抗体或其抗原结合片段，或其重链可变区和/或轻链可变区的核苷酸序列。在某些优选的实施方案中，本发明的分离的核酸分子编码根据本发明的抗体或其抗原结合片段，或其重链可变区和/或轻链可变区。
在另一个方面，本发明提供了一种载体(例如克隆载体或表达载体)，其包含根据本发明的分离的核酸分子。在某些优选的实施方案中，本发明的载体是例如质粒，粘粒，噬菌体等。在某些优选的实施方案中，所述载体能够在受试者(例如哺乳动物，例如人)体内表达本发明的抗体或其抗原结合片段。
在另一个方面，本发明提供了一种宿主细胞，其包含根据本发明的分离的核酸分子或根据本发明的载体。此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。本发明的细胞还可以是细胞系，例如293T细胞。
在另一个方面，提供了制备根据本发明的抗体或其抗原结合片段的方法，其包括，在允许所述抗体或其抗原结合片段表达的条件下，培养根据本发明的宿主细胞，和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段。
诊断方法和试剂盒
本发明的抗体或其抗原结合片段能够特异性结合HBsAg，从而可用于检测HBsAg蛋白在样品中的存在或其水平，可用于诊断受试者是否感染了HBV。
因此，在另一个方面，本发明提供了一种试剂盒，其包括本发明的抗体或其抗原结合片段。在一个优选的实施方案中，本发明的抗体或其抗原结合片段还包括可检测的标记。在一个优选的实施方案中，所述试剂盒还包括第二抗体，其特异性识别本发明的抗体或其抗原结合片段。优选地，所述第二抗体还包括可检测的标记。
可按照上文详细描述的方法来对本发明的抗体或其抗原结合片段或者第二抗体进行标记。例如，可以将本发明的抗体或其抗原结合片段连接至可检测的标记。此类可检测的标记是本领域技术人员熟知的，包括但不限于，放射性同位素，荧光物质，发光物质，有色物质和酶(例如辣根过氧化物酶)等。此外，此类可检测的标记还包括例如，放射 性同位素，例如125碘；荧光物质例如荧光素、异硫氯酸荧光素、罗丹明、5-二甲氨基-1-蔡磺酰氯、藻红蛋白、镧磷光剂等等；能够产生可识别的信号或反应产物的酶，例如辣根过氧化物酶、β-半乳糖苷酶、萤光素酶、碱性磷酸酶、葡萄糖氧化酶等等；能够被第二个报告分子识别的标签，例如生物素、抗生物素蛋白、亮氨酸拉链互补序列、金属结合结构域、附加表位等等。在某些具体实施方式中，可通过不同长度的接头将检测试剂(例如标签)连接至抗体，以降低潜在的位阻。
在另一个方面，本发明提供了检测HBsAg蛋白在样品中的存在或其水平的方法，其包括使用本发明的抗体或其抗原结合片段。在一个优选的实施方案中，本发明的抗体或其抗原结合片段还包括可检测的标记。在另一个优选的实施方案中，所述方法还包括，使用携带可检测的标记的第二抗体来检测本发明的抗体或其抗原结合片段。所述方法可以用于诊断目的，或者非诊断目的(例如，所述样品是细胞样品，而非来自患者的样品)。
在另一个方面，本发明提供了诊断受试者是否感染了HBV的方法，其包括：使用本发明的抗体或其抗原结合片段检测HBsAg蛋白在来自所述受试者的样品中的存在。在一个优选的实施方案中，本发明的抗体或其抗原结合片段还包括可检测的标记。在另一个优选的实施方案中，所述方法还包括，使用携带可检测的标记的第二抗体来检测本发明的抗体或其抗原结合片段。
在另一个方面，提供了本发明的抗体或其抗原结合片段在制备试剂盒中的用途，所述试剂盒用于检测HBsAg蛋白在样品中的存在或其水平，或用于诊断受试者是否感染了HBV。
治疗方法和药物组合物
本发明的抗体或其抗原结合片段可用于预防或治疗受试者(例如人)的HBV感染或与HBV感染相关的疾病(例如乙肝)，用于体外或在受试者(例如人)体内中和HBV的毒力，以及用于在受试者(例如人)体内降低HBV DNA和/或HBsAg的血清水平。
因此，在另一个方面，本发明提供了一种药物组合物，其含有根据本发明的抗体或其抗原结合片段，以及药学上可接受的载体和/或赋形剂。在一个优选的实施方案中，本发明的药物组合物还可包含另外的药学活性剂。在一个优选的实施方案中，所述另外的药学活性剂是用于预防或治疗HBV感染或与HBV感染相关的疾病(例如乙肝)的药物，例如其它抗病毒试剂，例如干扰素类药物，如干扰素或聚乙二醇干扰素。
在另一个方面，提供了根据本发明的抗体或其抗原结合片段或根据本发明的药物组 合物在制备药物中的用途，所述药物用于预防或治疗受试者(例如人)的HBV感染或与HBV感染相关的疾病(例如乙肝)，用于体外或在受试者(例如人)体内中和HBV的毒力，和/或用于在受试者(例如人)体内降低HBV DNA和/或HBsAg的血清水平。
在另一个方面，本发明提供了一种用于预防或治疗受试者的HBV感染或与HBV感染相关的疾病(例如乙肝)，用于在受试者(例如人)体内中和HBV的毒力，和/或用于在受试者(例如人)体内降低HBV DNA和/或HBsAg的血清水平的方法，所述方法包括，给有此需要的受试者施用有效量的本发明的抗体或其抗原结合片段，或者本发明的药物组合物。
本发明的抗体或其抗原结合片段或者本发明的药物组合物的施用方式可以是传统的施用途径，包括但不限于，口服、口腔、舌下、眼球、局部、肠胃外、直肠、叶鞘内、内胞浆网槽内、腹股沟、膀胱内、局部(如，粉剂、药膏或滴剂)，或鼻腔途径。本发明的抗体或其抗原结合片段可通过本领域已知的多种方法给药。但是，对于许多治疗用途而言，优选的给药途径/方式是胃肠外给药(例如静脉注射，皮下注射，腹膜内注射，肌内注射)。技术人员应理解，给药途径和/或方式将根据预期目的而发生变化。在一个优选的实施方案中，本发明的抗体或其抗原结合片段通过静脉输注或注射给予。
本发明的抗体或其抗原结合片段或者本发明的药物组合物可以配制成多种剂型，例如液体、半固体和固体剂型，例如溶液剂(例如注射剂)、分散剂或悬浮剂、片剂、粉剂、颗粒剂、乳剂、丸剂、糖浆剂、粉剂、脂质体、胶囊剂和栓剂。优选剂型取决于预期的给药方式和治疗用途。
例如，一种优选的剂型是注射剂。此类注射剂可以是无菌注射溶液。例如，可通过下述方法来制备无菌注射溶液：在适当的溶剂中掺入必需剂量的本发明的抗体或其抗原结合片段，以及任选地，同时掺入其他期望的成分(包括但不限于，pH调节剂，表面活性剂，佐剂，离子强度增强剂，等渗剂、防腐剂、稀释剂，或其任何组合)，随后过滤除菌。此外，可以将无菌注射溶液制备为无菌冻干粉剂(例如，通过真空干燥或冷冻干燥)以便于储存和使用。此类无菌冻干粉剂可在使用前分散于合适的载体中，例如无菌无热原水。
另一种优选的剂型是分散剂。可通过下述方法来制备分散剂：将本发明的抗体或其抗原结合片段掺入无菌载体中，其含有一种基础分散介质以及任选的其他期望的成分(包括但不限于，pH调节剂，表面活性剂，佐剂，离子强度增强剂，等渗剂、防腐剂、稀释剂，或其任何组合)。此外，还可以在分散剂中掺入可延迟吸收的试剂，例如单硬脂酸盐 和明胶，以获得期望的药代动力学特征。
另一种优选的剂型是口服固相剂型，包括胶囊、片剂、粉剂、颗粒剂等。此类固相剂型通常含有下述中的至少一种：(a)惰性药物赋形剂(或载体)，如柠檬酸钠、磷酸钙；(b)填充剂，如淀粉、乳糖、蔗糖、甘露糖和硅酸；(c)粘合剂，如羧甲基纤维素、藻酸盐、明胶、聚乙烯吡咯烷酮、蔗糖和阿拉伯树胶；(d)湿润剂，如甘油；(e)碎裂剂，如琼脂，碳酸钙，马铃薯粉或木薯粉；(f)缓凝剂，如石蜡；(g)促吸收剂，如四氨基混合物；(h)保湿剂，如十六烷基醇和单硬脂酸甘油酯；(i)吸附剂，如高岭土和斑脱土；(j)润滑剂，如滑石，硬脂酸钙，硬脂酸镁，固体聚乙二醇，硫酸十二烷醇钠，或其任何组合。在片剂和胶囊剂型的情况下，还可以含有缓冲剂。
此外，还可以在口服固相剂型中添加释放速率改造剂(即，能改变药物释放速率的试剂)，以获得改良释放或脉冲释放剂型。此类释放速率改造剂包括但不限于，羧丙基甲基纤维素，甲基纤维素，碳甲基纤维素钠，纤维素乙烷，醋酸纤维素，聚乙烯氧化物，黄原胶糖，异丙烯酸氨共聚物，氢化调味油，巴西棕榈蜡，石蜡，邻苯二酸醋酸纤维素，邻苯二甲酸羧丙基甲基纤维素，甲基丙烯酸共聚物，或其任何组合。改良释放和脉冲释放剂型可含有一种或一组释放速率改造剂。
另一种优选的剂型是口服的液体剂型，包括例如乳剂、溶液剂、悬浮剂、糖浆剂等。除了活性成分之外，此类口服的液体剂型还可含有本领域常用的一些惰性溶液，例如水或其它溶剂，如乙烷基醇，异丙基醇，丙烯乙二醇，1，3-丁烯乙二醇，油(例如棉籽油，落花生油，玉米油，橄榄油，调味油和芝麻油)，甘油，聚乙二醇和脂肪酸山梨醇酯，以及其任何组合。除了这些惰性溶液之外，此类口服的液体剂型还可包括保湿剂、乳化剂、悬浮剂、糖化剂、调味剂和香味剂等。
此外，本发明的抗体或其抗原结合片段可以以单位剂量形式存在于药物组合物中，以便于施用。本发明的药物组合物应当是无菌的并在生产和储存条件下稳定。
本发明所提供的药物和药物组合物可以单独使用或联合使用，也可以与另外的药学活性剂(例如其它抗病毒试剂，例如干扰素类药物，如干扰素或聚乙二醇干扰素)联合使用。在某些优选的实施方案中，将本发明的抗体或其抗原结合片段与其它抗病毒试剂联合使用，以预防和或治疗乙型肝炎病毒感染相关疾病。本发明的抗体或其抗原结合片段可以和此类抗病毒试剂同时、分开或连续给药。此类抗病毒试剂包括但不限于，干扰素类药物、利巴韦林、金刚烷、羧基脲、IL-2、L-12和五羧链胞酸等。
本发明的药物组合物可包括“治疗有效量”或“预防有效量”的本发明抗体或抗原结合 片段。“预防有效量”是指，足以预防，阻止，或延迟疾病(例如HBV感染或与HBV感染相关的疾病)的发生的量。“治疗有效量”是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。本发明抗体或抗原结合片段的治疗有效量可根据如下因素发生变化：待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
可调整给药方案以获得最佳目的反应(例如治疗或预防反应)。例如，可以单次给药，可以在一段时间内多次给药，或者可以随治疗情况的紧急程度按比例减少或增加剂量。
本发明抗体或抗原结合片段的治疗或预防有效量的典型非极限范围是0.025到50mg/kg，更优选0.1到50mg/kg，更优选0.1-25mg/kg，O.1-10mg/kg。应注意的是，剂量可随需要治疗的症状的类型和严重性不同而发生变化。此外，本领域技术人员理解，对于任一特定患者，特定的给药方案应根据患者需要和医生的专业评价而随时间调整；此处给出的剂量范围只用于举例说明目的，而不限定本发明药物组合物的使用或范围。
发明的有益效果
与现有技术相比，本发明的技术方案具有以下有益效果：
(1)本发明的抗体不仅能够特异性识别/结合HBsAg，能够中和HBV的毒力，而且能够在受试者体内降低HBV DNA和/或HBsAg的血清水平，能够有效清除体内的HBV和被HBV感染的细胞。因此，本发明的抗体具有用于预防和治疗HBV感染以及与HBV感染相关的疾病(例如乙肝)的潜力。
(2)本发明的抗体(特别是人源化抗体)不仅保留了亲本猴源抗体的功能和性质，从而具有用于预防和治疗HBV感染以及与HBV感染相关的疾病(例如乙肝)的潜力；而且具有极高的人源化程度(人源化程度可高达98％)，从而可安全地施用给人受试者，而不引发免疫原性反应。因此，本发明的抗体(特别是人源化抗体)具有重大的临床价值。
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons，Inc.，1995中所述的方法进行；限制性内切酶的使用依照产品制造商推荐的条件。本 领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
实施例1：特异性结合HBsAg的人-食蟹猴嵌合单克隆抗体的制备
1.1食蟹猴免疫
使用实验室前期研发的重组HBV疫苗(该重组疫苗详细描述于中国专利申请201710085194.3)对大于6个月，体重为4±1kg的食蟹猴进行肌肉注射免疫，注射剂量为20μg/monkey/time。免疫时间点分别在第0、2、6、10、14、18、22、26周。在免疫第4针(10周)食蟹猴血清滴度达到平台期后，我们分别在第13、17、25、29周(即每次免疫后的第三周，产生大量记忆B细胞)，采集食蟹猴外周血10ml/monkey/time。
1.2食蟹猴外周血单核细胞分离
食蟹猴外周血单核细胞的分离参照SepMate TM使用说明书进行。SepMate TM试管用于通过密度梯度离心从人外周全血和脐带血样本中分离单个核细胞(MNCs)。分离前应确保样本、含2％胎牛血清(FBS)的PBS缓冲液(PBS+2％FBS)、密度梯度离心液以及离心机均处于室温(15-25℃)。具体步骤如下：
1)使用移液器将密度梯度离心液通过SepMate TM插件中央小孔小心加入SepMate TM试管中。密度梯度离心液上端应淹没过插件。注意：密度梯度离心液在使用移液器后可能会产生小气泡，这不会对使用效果产生影响。
2)使用等体积的PBS+2％FBS稀释样本。轻柔混匀。
3)保持SepMate TM试管垂直，用移液器沿试管壁加入稀释后的样本。样本将会在插件上面和密度梯度离心液混合。注意：样本可以沿管壁直接倒入。注意不要讲稀释的样本直接倒在中央小孔上。
4)在室温下1200×g离心10分钟，无需关闭离心机刹车。注意：对于存放超过24小时的样本，推荐离心时间为20分钟。
5)倒出上层液体至一个新的试管，该层含富集的MNCs。请勿将SepMate TM试管在倒置状态下超过2秒。注意：离心后可能会有部分红细胞(RBCs)出现在SepMate TM插件的表层，但不会对使用效果产生影响。
6)使用PBS+2％FBS清洗MNCs。重复清洗一次。注意：清洗细胞时建议在室温下1200×g离心8分钟，无需关闭离心机刹车。
1.3记忆B细胞分离
因市面上无专门的食蟹记忆B细胞分选试剂，我们采用EasySep TM HUMAN PAN-B CELL ENRICHMENT KIT进行。
1)按照上述1.2的方法分离出的PBMC细胞以300g，离心10分钟，然后用10mL无血清的1640培养基洗涤细胞一次。
2)用10mL过滤后的MACS buffer重复洗涤一次。
3)将细胞用1mL MACS buffer重悬，然后转移至5mL聚苯乙烯圆底试管中。
4)按50μL/mL细胞加入EasySep TM Human Pan-B Cell Enrichment Cocktail。
5)室温孵育10min，按75μL/mL细胞加入EasySep TM D Magnetic Particles。
6)补加MACS buffer至2.5mL，轻轻上下混匀。
7)将试管放入磁铁分离器上5分钟，之后将需要的阳性细胞转移至新的试管中，而不需要的阴性细胞保留在原始试管中。
1.4体外刺激培养
1.4.1 HEK293-CD154辐照
辐照剂量为40Gy，具体操作步骤参见《RS 2000型生物学X-ray辐照仪使用说明书》。
1.4.2细胞铺板
RPMI 1640培养基重悬好的细胞按5个/孔与配制好的50ng/mL的IL21和辐射过后的HEK293-CD154按10 4个/孔混匀后，共同孵育铺入96孔板中，每孔250μL。静置放于37℃的CO 2培养箱中，刺激培养14天。
1.4.3细胞培养阳性孔检测
取商业化的HBsAg板(购于北京万泰)，每孔加入100μL待测上清，并在37℃温育1h。随后，用PBST洗涤ELISA板5次，然后添加以1∶5000稀释的GAH-HRP 100μL，并在37℃温育30min。随后，用PBST洗涤ELISA板5次，并添加底物TMB溶液。显色15min后，用H 2SO 4终止显色反应，并于OD450/620测定读值。以RPMI 1640培养基为阴性对照。
1.5B细胞抗体可变区基因的钓取
1.5.1细胞处理
将含有阳性B细胞培养孔的培养板先在300×g条件下离心5min，将细胞上清吸出。加入150μL的Lysis Buffer(购自北京GenMagBio)裂解液，用枪头反复吹吸几次后移入无RNA酶的1.5mL Eppendorf管中，放置于-80℃冰箱中，可以保存一年。
1.5.2 RNA提取
RNA提取方法参照病毒DNA/RNA共提取试剂盒说明书(GenMagBio)。
1)在1.5mL无核酸酶Eppendorf管中加入10μL Proteinase K、4μL Acryl Carrier、300μL Lysis Buffer、20μL磁珠(使用前颠倒或使用移液器吹吸混匀磁珠)，加入200μL样品，震荡混匀各离心管，室温上下颠倒混匀10min，使磁珠和核酸充分结合。
2)将离心管置于磁力架上1min，使管内磁珠被吸附，用移液器移走管内液体，取下离心管。
3)加入500μL的Wash Buffer I，使磁珠重新悬浮，利用磁力架吸附磁铁，1min后移走管内液体，取下离心管。
4)加入500μL的Wash Buffer II，使磁珠重新悬浮，利用磁力架吸附磁珠，1min后移走管内液体，同时保持离心管置于磁力架上，使磁珠继续被吸附。
5)加入550μL的Wash BufferIII，尽量不要冲起磁珠，1min后移走管内液体，取下离心管。
6)加入50μL的Elution Buffer，使磁珠重新悬浮，55℃水浴5min，期间轻轻晃动两次使核酸充分洗脱。
7)将离心管置于磁力架1min，使磁珠被吸附，将液体转移至新的1.5mL无核酸酶的离心管中。
1.5.3B细胞抗体可变区RT-PCR
反转录使用的引物(参见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.)如表2所示。
表2：食蟹猴抗体可变区基因反转录引物
Figure PCTCN2018082157-appb-000012
具体步骤如下：
(1)首先按下列配方配置I液：
Buffers Volume
DEPC水 6.8μL
引物/条 0.6μL
RNAsin 0.1μL
Mrna 5μL
(2)75℃温浴5min。
(3)迅速放入冰浴中5min。
(4)之后按下列配方配置II液：
Buffers Volume
5×AMV buffer 4μL
10mmol dNTP 1μL
AMV反转录酶 0.2μL
(5)混匀，加5.2μL到各管中。
(6)混匀，42℃水浴反转录40min。
(7)分装保存，短期内使用置于-20℃，短期内不使用置于-80℃。
1.5.4抗体可变区基因扩增
B细胞抗体H链、κ轻链及λ轻链可变区基因采用巢式PCR扩增，扩增引物参考(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).并将巢式第二轮引物上与载体的同源序列替换成本实验室已有的真核表达载体(参见实施例1.5.5.1)上的一段作为同源序列。按照表3配制PCR反应体系，每个体系为25μL。巢式第一轮反应以反转录的cDNA为模板，反应条件为：95℃，5min；95℃，30s；55℃，60s；72℃，90s；72℃，7min；扩增循环数为35个；巢式第二轮反应以第一轮扩增产物为模板，反应条件为：95℃，5min；95℃，30s；58℃，60s；72℃，90s；72℃，7min；扩增循环数为35个。随后通过琼脂糖凝胶电泳分析扩增产物，并用DNA纯化回收试剂盒(TianGen，DP214-03)回收扩增产物。
表3：巢式PCR反应体系
Figure PCTCN2018082157-appb-000013
Figure PCTCN2018082157-appb-000014
1.5.5抗体基因的表达与纯化
1.5.5.1用于真核表达的重组载体的构建
本发明中，需要进行大量的抗体重组，因此需要构建一套可以高效重组抗体的轻重链载体。本发明对实验室现有的真核表达载体pTT5进行了特殊改造，构建一套供双质粒共转染用轻重链重组载体。轻重链信号肽采用MGWSCIILFLVATATGVHS。信号肽下游分别接入编码人抗体轻重链恒定区的序列，构建成一套方便抗体重组的pTT5-CH，pTT5-Cκ和pTT5-Cλ真核表达载体。利用实验室自制的Gibson装配液将上述构建好的真核表达载体与1.5.4中回收到的抗体可变区基因PCR产物连接(引物带有与载体同源序列)，得到重组载体VH+pTT5-CH，VH+pTT5-Cκ和VH+pTT5-Cλ。将重组载体转化入到大肠杆菌DH5α菌株，涂布LB平板，并在37℃培养箱培养过夜。从平板上挑取单克隆菌落，并进行测序，测序结果使用IgBlast对序列进行比对，以确认其基因正确性，排除假基因。
1.5.5.2抗体基因的小量及大量表达
将构建好的重组载体VH+pTT5-CH和VH+pTT5-Cκ和VH+pTT5-Cλ共转染HEK293-细胞，小量表达双质粒共转染至每孔500μL的24孔板中，如小量表达的细胞上清具有抗原活性，将转染体系放大到100mL(根据抗体用量决定)的CHO-S悬浮细胞(细胞密度为约2×10 6cells/ml)。转染后的细胞在32℃，5％CO2培养箱摇瓶培养，表达7天后收上清。
1.5.5.3抗体纯化
收集细胞表达上清，根据制造商的说明书，用Protein A层析柱进行纯化。具体步骤如下：收获的细胞培养上清，8000rpm，离心10min，保留上清，利用干粉Na 2HPO 4调节其PH值为8.4，然后用0.22μm孔径滤膜过滤。取偶联上Protein A的Sepharose 4B介质10mL装柱，连接至AKTA Explorer100系统，将A泵接入0.2M磷酸氢二钠溶液，B泵接入0.2M柠檬酸溶液。选取检测波长UV 280nm，流速5mL/min，调节A/B泵进样比例，先取100％B(pH 2.3)冲洗柱子出去杂蛋白，取10％B(pH 8.0)平衡pH，待检测波长稳定后归零，上样，待穿透峰过后取10％B继续平衡至检测波长降至零点并稳定后，用70％B(pH 4.0)洗脱，收取洗脱峰。将洗脱峰样品透析至PBS缓冲液中，并 测定浓度及SDS-PAGE分析，以确定IgG抗体的纯度。
实施例2：特异性结合HBsAg的食蟹猴-人嵌合单克隆抗体的性质分析
按照实施例1的方法，共制备获得了3株特异性结合HBsAg的食蟹猴-人嵌合单克隆抗体，分别命名为M1-23，M3-23，M3-13。三株抗体的VH和VL氨基酸序列如下表所示(SEQ ID NO：1-5)。另外，还利用IMGT确定了三株抗体的CDR序列，其重链可变区和轻链可变区的CDR的氨基酸序列如表5(SEQ ID NO：6-20)所示。
表4：M1-23/M3-23/M3-13轻重链可变区氨基酸序列
Figure PCTCN2018082157-appb-000015
表5：M1-23/M3-23/M3-13轻重链CDR的序列
Figure PCTCN2018082157-appb-000016
Figure PCTCN2018082157-appb-000017
发明人对纯化后的3株单克隆抗体进行了一系列的性质分析。首先通过ELISA法分别检测了M1-23，M3-23及M3-13与HBsAg的结合能力，按首孔浓度为10μg/mL，进行3倍浓度梯度稀释，共稀释12个浓度梯度。随后，将稀释好的抗体加入商业化的HBsAg板中(购于北京万泰)，并在37℃孵育1h，用PBST洗板5次，甩干。随后，加入GAH-HRP酶标二抗，孵育30min，用PBST洗板5次，甩干。并添加底物TMB溶液。显色15min后，用H 2SO 4终止显色反应，并于OD450/630测定读值。结果如图1所示，M1-23，M3-23及M3-13均具有良好的抗原结合活性。
通过竞争ELISA法考察识别HBsAg SA类型表位(HBsAg aa113-135)的鼠源单抗6D11(详细描述于中国专利申请201610879693.5)阻断M1-23，M3-23及M3-13与HBsAg的结合的情况。竞争ELISA法的具体步骤参见实施例4.2。如图2所示，结果显示鼠源单抗6D11可显著阻断M1-23，M3-23，M3-13分别与HBsAg的结合，这表明M1-23，M3-23，M3-13与鼠源单抗6D11识别的均为相同HBsAg SA线性表位。
通过HBV转基因小鼠来评价食蟹猴-人嵌合抗体在动物体内的病毒清除能力。将食蟹猴-人嵌合抗体按10mg/kg的剂量单剂尾静脉注射的方式施用给HBV转基因小鼠，每组6只HBV转基因小鼠。随后，通过眼眶后静脉丛取血采集小鼠的血液，并检测小鼠血清中的HBsAg水平和HBV DNA水平的变化。小鼠血清中的HBsAg水平和HBV DNA水平的检测方法参见实施例5.1及5.2。结果表明，M1-23，M3-23及M3-13(M1-23、M3-23结果示于图3A-3B；M3-13对HBsAg的清除结果示于图7A-7B)具有用于治疗HBV感染或与HBV感染相关的疾病(例如乙肝)的潜力。
实施例3：抗体M1-23与M3-23的人源化
为了减少异源抗体施用给人受试者时引起的免疫原性，发明人对M1-23，M3-23这两株抗体进行了人源化。由于食蟹猴与人类的免疫球蛋白之间氨基酸序列具有高度一致性，因此对与M1-23，M3-23这两株抗体而言，为使其适用于人类治疗仅需额外地将可变区FR少数氨基酸替换为人可变区FR序列中相应位置的氨基酸。然而，虽然抗体主要通过CDR来接触和识别抗原，但是抗体FR区中的部分残基可能也参与抗原-抗体的相互作用，影响CDR的空间构型，例如在将鼠源抗体的FR区替换为人抗体的FR区后，鼠源抗体的CDR的空间构型往往发生改变，导致人源化抗体识别/结合抗原的亲和力显著下降，甚至导致人源化抗体丧失与抗原结合的能力(葛彦，人源化抗体研制策略分析及应用研究[J].国外医学，免疫学分册，2004，27(5)：271)。因此，在对M1-23，M3-23进行人源化的过程中，可变区FR序列中点突变位置及替换后的氨基酸的选择是十分重要的。
通过使用IMGT信息系统(http：//-www.imgt.org)，并结合实验经验，在大量的分析和实验的基础上，发明人出人意料地发现，选择人胚系基因序列IGHV4-4＊08(SEQ ID NO：40)与IGKV1-39＊01(SEQ ID NO：41)作为M1-23人源化的模板，IGHV4-4＊02(SEQ ID NO：42)与IGKV4-1＊01(SEQ ID NO：43)作为M3-23人源化的模板是特别有利的，所涉及的胚系基因序列列于下表6。
表6：人胚系基因氨基酸序列
Figure PCTCN2018082157-appb-000018
Figure PCTCN2018082157-appb-000019
根据胚系基因序列，同时考虑到改变靠近CDR的FR区氨基酸可能会影响人源化抗体识别/结合抗原的亲和力，我们在保留CDR区附近的氨基酸基础上，设计了如下单点突变对两株抗体的FR区进行改造。分别将人源化后的M1-23、M3-23命名为M1D、M3D。
表7：抗体M1-23的FR区氨基酸残基的单点突变
Figure PCTCN2018082157-appb-000020
※：上表中提及的氨基酸位置是根据Kabat编号系统的位置。
表8：抗体M3-23的FR区氨基酸残基的单点突变
Figure PCTCN2018082157-appb-000021
Figure PCTCN2018082157-appb-000022
※：其中上表中提及的氨基酸位置是根据Kabat编号系统的位置。
表9：M1-23，M3-23FR区人源化改造引物
Figure PCTCN2018082157-appb-000023
Figure PCTCN2018082157-appb-000024
根据表7，表8中列出的单点突变，设计了相关引物(见表9)，并通过PCR的方法，分别以M1-23-H，M1-23-L，M3-23-H和M3-23-L为模板，将对应位置的原始氨基酸全部进行突变。以M1-23重链可变区(M1-23-H)的人源化为例，设计寡核苷酸引物PTT5-1-23H-F，1-23H-F1，1-23H-F2，1-23H-R1，1-23H-F3及PTT5-1-23H-R(其序列示于上表)。PCR的具体方案如下：以M1-23-H的编码基因为模板，分别用引物对1-23H-F1/1-23H-R1和1-23H-F3/PTT5-1-23H-R扩增出片段1和片段2；以片段1为模板，用引物对1-23H-F2/1-23H-R1扩增出片段3；以片段3为模板，用引物对PTT5-1-23H-F/1-23H-R1扩增出片段4；通过重叠延伸PCR将片段4和片段2连接到一起，再以PTT5-1-23H-F/PTT5-1-23H-R为上下游引物进行扩增，获得人源化后的基因片段M1D-H。
按照上述方法，获得M1-23-L的人源化基因片段M1D-L，M3-23-H的人源化基因片段M3D-H，M3-23-L的人源化基因片段M3D-L。
用实验室自制的Cibson装配液将获得的人源化基因片段M1D-H、M3D-H分别与真 核表达载体pTT5-CH连接，M1D-L、M3D-L分别与真核表达载体pTT5-Cκ连接，得到重组载体。将重组载体转化入到大肠杆菌DH5α菌株，涂布LB平板，并在37℃培养箱培养过夜。从平板上挑取单克隆菌落，并进行测序，挑选测序正确的质粒，按照实施例1中方法，进行抗体的表达与纯化。
人源化抗体M1D、M3D氨基酸序列示于表11。另外，还通过下式计算了M1D、M3D的人源化程度。如下表10所示，这两株抗体经过上述人源化后，人源化程度由90％左右提高到了98％左右。
表10：人源化抗体的序列分析
Figure PCTCN2018082157-appb-000025
人源化程度＝(FR区氨基酸个数-FR区保留的猴源氨基酸个数)/FR区氨基酸个数×100％
表11：抗体M1D，M3D轻重链可变区氨基酸序列
Figure PCTCN2018082157-appb-000026
实施例4：人源化抗体M1D，M3D的体外结合活性评估
4.1抗体的抗原结合活性的测定
通过ELISA方法(酶联免疫吸附法)检测了人源化抗体M1D，M3D与抗原HBsAg的结合活性。按首孔浓度为10μg/mL，进行3倍浓度梯度稀释，共稀释12个浓度梯度，以人源化抗体162(其详细描述于中国专利申请CN201610879693.5中)作为参比抗体。 随后，将稀释好的抗体加入商业化的HBsAg板中(购于北京万泰)，并在37℃孵育1h，用PBST洗板5次，甩干。随后，加入GAH-HRP酶标二抗，孵育30min，用PBST洗板5次，甩干。并添加底物TMB溶液。显色15min后，用H 2SO 4终止显色反应，并于OD450/630测定读值。结果如图4所示，人源化抗体M1D，M3D均具有很好的抗原结合活性，且其对抗原HBsAg的亲和力都优于参比抗体162。上述结果表明，经过人源化改造的这两株抗体不仅具有极高的人源化程度(97％以上)，而且基本上保留了亲本抗体的抗原结合活性，甚至表现出比亲本抗体更好的抗原结合活性。
4.2抗体的初步表位鉴定
发明人还对抗体的表位进行了初步鉴定，以判断所筛选得到的人猴嵌合抗体及人源化抗体是否识别HBsAg上的SA表位。使用识别SA类型表位的高浓度的6D11交叉阻断待测抗体与HBsAg的结合情况，若有明显阻断(竞争)效果，则说明待测抗体与6D11识别同一表位，具体步骤如下：
(1)测定抗体浓度，按照10μg/孔的用量加入商业化的HBsAg ELISA板中，然后用20％NBS补足体积至50μL，37℃孵育30min。
(2)6D11-HRP用ED-11以1∶20000稀释后，按照50μL/孔加入ELISA板，37℃孵育30min。
(3)用PBST洗板5次，甩干。
(4)并添加底物TMB溶液，显色15min后，用H 2SO 4终止显色反应。
(5)于OD450/630测定读值。
结果如图5所示，其中162在本实验中作阳性对照。M1D，M3D与HBsAg的结合均可被鼠源抗体6D11竞争，且竞争效果跟阳性对照162以及亲本抗体M1-23，M3-23相当。上述结果说明人源化抗体M1D，M3D同样识别SA表位。
4.3抗体的中和活性的测定
发明人利用实验室自制的细胞模型HepaG2-NTCP，来评估抗体中和HBV感染(100MOI HBV感染滴度)的能力。将一定量的HBV与梯度稀释的抗体共孵育于HepaG2细胞中，HBV能够侵入分化后的HepaG2细胞并在细胞中进行复制，随着不同抗体的加入及加入抗体量的变化，HBV的侵入和复制将会被不同程度的减弱或抑制，从而细胞上清中将呈现不同水平的HBV抗原(HBeAg)。因此，通过测定HBV抗原(HBeAg)的水平，即可 判断不同人源化抗体中和HBV的能力。
对人源化抗体进行两倍浓度梯度稀释，并以162抗体作为阳性对照，从10μg/mL开始，共稀释18个梯度。将病毒感染体系分别与抗体混合，预孵育1h。然后加入预先铺好HepaG2的细胞板中，37℃培养24h，每隔两天对细胞进行换液。感染后第7天，取细胞培养上清，检测其中的HBeAg水平。检测方法如下：
(1)反应板的制备：将anti-HBeAg单克隆抗体用20mM PB缓冲液(Na 2HPO 4/NaH 2PO 4缓冲液，pH 7.4)稀释到2μg/mL。向96孔化学反光板中加入稀释的anti-HBeAg单克隆抗体(100μL/孔)，37℃孵育2h后4℃过夜。所使用的原料均购自北京万泰生物药业股份有限公司。用PBST洗涤96孔化学反光板一次，甩干。向96孔化学反光板中加入封闭液，每孔200μL，37℃封闭2h。随后，弃去封闭液，将平板放入干燥间干燥，于2-8℃保存备用。
(2)孵育：向上述制备好的反应板中加入100μL/孔的待检样品，并在37℃温育60min。
(3)洗涤：用PBST洗涤96孔化学反光板5次，甩干。
(4)孵育：加入辣根过氧化物酶标记的anti-HBeAg抗体(100μl/孔)，并在37℃温育30min。原料购自北京万泰生物药业股份有限公司。
(5)洗涤：用PBST洗涤96孔化学反光板5次，甩干。
(6)显色：加入鲁米诺化学发光试剂(100μl/孔)。
(7)读板：利用化学发光酶标仪读取数值。
实验结果如图6A-6B所示，M1D对HBV的中和活性与参比抗体162相当。
实施例5：人源化抗体M1D，M3D的动物治疗效果的测定
将上述5株抗体按10mg/kg的剂量单剂尾静脉注射的方式施用给HBV转基因小鼠，每组6只HBV转基因小鼠。随后，通过眼眶后静脉丛取血采集小鼠的血液，并检测小鼠血清中的HBsAg水平和HBV DNA水平的变化。
5.1HBsAg的定量检测
(1)反应板的制备：将小鼠单克隆抗体HBs-45E9用20mM PB缓冲液(Na 2HPO 4/NaH 2PO 4缓冲液，pH 7.4)稀释到2μg/mL，在化学发光板中每孔添加100μL的包被液，并在2-8℃下包被16-24h，随后再37℃包被2h，用PBST洗涤液洗涤平板一次，甩干。洗涤后，在各孔中加入200μL的封闭液，并在37℃封闭2h。随后， 弃去封闭液，将平板放入干燥间干燥，于2-8℃保存备用。
(2)样品稀释：将采集的小鼠血清用含20％NBS(新生牛血清)的PBS溶液，稀释成1∶30和1∶150两个梯度，用于后续的定量检测。
(3)样品变性处理：取15μL经上述稀释的血清样品与7.5μL变性缓冲液(15％SDS，溶于20mM PB7.4)充分混匀，并在37℃反应1h。随后，加入90μL的中和缓冲液(4％CHAPS，溶于20mM PB7.4)，充分混匀。
(4)样品反应：在反应板中加入100μL经上述变性处理的血清样品，37℃反应1h。随后用PBST洗涤反应板5次，甩干。
(5)酶标记物反应：向化学发光板中按100μL/孔加入HBs-A6A7-HRP反应液，并在37℃反应1h。随后用PBST洗涤平板5次，甩干。
(6)发光反应和测量：向化学发光板中加入发光液(100μL/孔)，并进行光强检测。
(7)小鼠血清样品中的HBsAg浓度的计算：使用标准品进行平行实验，根据标准品的测定结果绘制标准曲线。然后，将小鼠血清样品的光强测量值代入标准曲线，计算出待检血清样品中的HBsAg浓度。
5.2HBV DNA的定量检测
HBV DNA的定量检测按照HBV DNA实时荧光定量检测试剂盒说明书进行(试剂盒购于上海科华生物工程股份有限公司)。
在本实施例中，测定了人源化抗体M1D、M3D、食蟹猴-人嵌合抗体M3-13以及参比抗体162在动物体内的病毒清除能力。实验结果如图7A-7B所示，结果显示人源化抗体M1D、M3D以及猴-人嵌合抗体M3-13均具有良好的病毒清除能力，其清除动物体内的HBsAg及HBV DNA的能力优于参比抗体162。
上述结果表明，本发明的人源化抗体不仅具有极高的人源化程度(最高可达98％)，能够减少免疫排斥反应的可能性，而且展示出与猴-人嵌合抗体相当且优于参比抗体162的病毒清除能力。这样的技术效果是显著的和出人意料的。
实施例6：人源化抗体M1D，M3D的非关键CDR区确定
在抗体可变区结构域中，与抗原直接结合的部位主要是CDR区，但很多研究表明，在抗体的6个CDR中并不都是抗原-抗体结合的关键区域，非关键CDR区可以通过CDR替换的方法来确定。本发明依据最适匹配原则选择CDR替换序列，通过IMGT信息系统，将人源化抗体M1D，M3D的VH和VL氨基酸序列分别输入查找框中，然后将 系统搜索出的胚系基因对应的抗体轻重链氨基酸序列，按照FR区同源性最高，CDR区序列差异性最大的原则选择人源化抗体CDR替换序列，除重链CDR3以外的每个CDR区最终选择两条相对应的替换序列。替换序列见下表：
表12：人源化抗体M1D，M3D CDR替换序列
Figure PCTCN2018082157-appb-000027
根据上表所述CDR替换序列，设计相关引物，并通过PCR的方法，替换M1D，M3D抗体中相对应的CDR区，引物设计见下表。以M1D重链可变区CDR1(M1D- HCDR1)的第一种替换(M1D-H-CDR1-A)为例，设计寡核苷酸引物M1D-H-CDR1-A R及M1D-H-CDR1-A F(其序列示于下表)，并将该替换CDR后的抗体命名为M1D HCDR1-1，其他替换后抗体的命名方式以此类推。PCR的具体方案如下：以M1D-H的编码基因为模板，用引物对pTT5-M1D-H-F/M1D-H-CDR1-A R和M1D-H-CDR1-A F/pTT5-M1D-H-R分别扩增出上、下游片段；通过重叠延伸PCR将片段连接到一起，再以pTT5-M1D-H-F/pTT5-M1D-H-R为上下游引物进行扩增，获得在M1D-HCDR1发生CDR替换的基因片段。
表13：CDR替换引物序列
Figure PCTCN2018082157-appb-000028
Figure PCTCN2018082157-appb-000029
Figure PCTCN2018082157-appb-000030
按照上述方法，将获得的重链CDR替换的基因片段分别与真核表达载体pTT5-CH连接，并与相对应的没有进行CDR替换的轻链表达载体小量转染HEK293-细胞，并检测小量表达的细胞上清是否具有抗原活性；同样，将获得的轻链CDR替换的基因片段分别与真核表达载体pTT5-Cκ连接，并与相对应的没有进行CDR替换的重链表达载体进 行小量转染并检测转染上清的抗原结合活性。
如果人源化抗体的某个CDR区被替换后，对应的细胞转染上清没有抗原结合活性或者结合活性显著下降，则说明被替换的CDR区是抗体抗原结合的关键CDR区。如果替换后，对应的细胞转染上清抗原结合活性基本不变，则说明被替换的CDR区不是抗体抗原结合的关键区域。
M1D替换结果如图8A-8E所示，M1D重链CDR1(M1D-H-CDR1)和轻链CDR2(M1D-L-CDR2)替换后，与HBsAg抗原结合活性基本没变；而重链CDR2和轻链CDR1替换后，抗原结合活性显著下降；而轻链CDR3替换后，完全失去抗原结合活性。上述结果表明，抗体M1D的HCDR1和LCDR2不是与抗原HBsAg结合的关键CDR区，对其替换并不会影响抗体与抗原的结合活性。
M3D替换结果如图9A-9E所示，M3D重链CDR2(HCDR2)和轻链CDR2(LCDR2)替换后，抗原结合活性基本没变；而轻链CDR1替换后，一种替换(M3D-LCDR1-1)抗原结合活性明显下降，另一种替换(M3D-LCDR1-2)，基本没有抗原结合活性；重链CDR1(HCDR1)和轻链CDR3(LCDR3)替换后，基本没有抗原结合活性。上述结果表明，抗体M3D的HCDR2和LCDR2不是与抗原结合的关键CDR区，对其替换并不会影响抗体与抗原的结合活性。
实施例7：人源化抗体M1D，M3D与抗原结合关键氨基酸确定
为了确定抗体抗原相互作用的关键氨基酸位点，我们对M1D的4个CDR区HCDR2、HCDR3、LCDR1、LCDR3和M3D的4个CDR区HCDR1、HCDR3、LCDR1、LCDR3进行丙氨酸扫描。关键氨基酸往往具有一定的保守性，比如维持抗体构象的氨基酸残基和被包埋的氨基酸残基，这些氨基酸突变成丙氨酸以后会导致抗体失去活性。突变后的抗体通过Elisa的方法检测同HBsAg的结合活性并计算突变后抗体的rEC50(WT/Mutation，原始抗体EC50相对值为1，完全失去结合活性的用0标示)，将突变后rEC50＜0.2即活性下降5倍的用蓝色标示出来，包括D、E、R、H、L、W、Y等氨基酸残基。结果如表14所示，其中，酸性氨基酸D、E，碱性氨基酸R、H在抗体抗原结合时可以形成盐桥，从而在抗体抗原的结合反应中起重要作用，把这些氨基酸突变成丙氨酸后会破坏离子键的形成从而使抗体结合活性大大降低；脂肪族氨基酸L，属于疏水性氨基酸W、Y属于芳香族氨基酸，在维持抗体构象中起重要作用。其中，M1D的H56Y、H97D、H100cD、H100dL、94L、L96L突变成丙氨酸后活性显著下降， 为不可变点；M3D的H33Y、H34D、H35W、H94R、H95D、H101E、L27dY、L32Y、L91H突变成丙氨酸后活性显著下降，为不可变点。
表14：M1D、M3D关键氨基酸位点确定
Figure PCTCN2018082157-appb-000031
Figure PCTCN2018082157-appb-000032
实施例8：M1D、M3D识别的核心表位鉴定
M1D、M3D识别SA线性表位，为了进一步确定两株抗体识别的核心表位，我们将HBsAg的aa113-135(SEQ ID NO：168)进行单点突变共合成下列表格中的14条多肽。多肽用CB稀释，100ng/mL包板，抗体用20％NBS溶液释到1000ng/mL，之后按照3倍稀释12个梯度，并用Elisa的方法检测抗体与不同单点突变多肽的结合活性，用PRISM软件绘制结合曲线。若突变后结合活性丧失，说明该突变位点为SA线性表位上的关键氨基酸。
表15：SEQ13-B点突变肽序列
Figure PCTCN2018082157-appb-000033
M1D、M3D及参比抗体162与上述单点突变多肽结合的EC50值汇总到下列表16(完全失去结合活性的用＞500表示)，由结果可知，162结合的核心表位为CKTC(aa121-124)，而M1D结合的核心表位TGPCKTCT(aa118-124)，比参比抗体162更长，且在aa113-135位置靠前；M3D结合的核心表位为CKTCT(aa121-125)，核心表位位置比162靠后。
表16：M1D、M3D与HBsAg(aa113-135)单点突变多肽结合的EC50值
Figure PCTCN2018082157-appb-000034
为了进一步验证抗体在抗原上结合的关键位置，我们将上述实验在HBsAg抗原蛋白进行验证。设计相关引物(表17)，并通过PCR的方法扩增14条带点突变HBsAg基因片段，扩增出的片段分别用Gibson连接入经过EcoRI和BamHI双酶切的PTT5空载中，测序正确即载体构建完成。将构建好的质粒小量转染HEK293T细胞，转染两天后，收集细胞，做western blot进行验证，具体步骤如下：
(1)收集细胞，用5mL PBS洗涤细胞两次，细胞计数后1000rpm离心5分钟；
(2)按照每1×10^6个细胞加入30μL DDM裂解细胞(也可用BCA法定量细胞蛋白量)，4℃裂解2h，期间用振荡器振荡几次；
(3)13300rpm离心10分钟，吸取上清制样，并取30μL进行还原SDS-PAGE凝胶电泳；
(4)用电转仪转膜300mA恒流电转90min，商业化封闭液37℃封闭1h；
(5)1mg/mL抗体M1D、M3D和162分别用ED-11稀释1000倍并在37℃条件下孵育1h；
(6)PBST每间隔5分钟洗涤1次，共3次；
(7)加入酶标二抗GAH-HRP(1∶5000)，37℃孵育30min；
(8)PBST每间隔5分钟洗涤1次，共3次；
(9)显色拍照；(10)以β-actin作内参，将步骤(4)中PVDF膜β-actin对应位置裁下，直接37℃孵育HRP偶联的mouse anti-β-actin抗体(1∶5000)30min，其他步骤同上。结果如图10所示，可以看出，在HBsAg抗原蛋白验证的三个抗体的核心表位与在线性多肽上完全一致。
表17：C-HBsAg单点突变引物
Figure PCTCN2018082157-appb-000035
Figure PCTCN2018082157-appb-000036
实施例9：M1D、M3D广谱活性鉴定
为了进一步确认M1D、M3D的广谱活性，我们将不同亚型A-G(其中F型包括F1、F2)对应的HBsAg片段(aa113-135)包板(序列见表18)，多肽用CB稀释，100ng/mL包板，抗体用20％NBS溶液释到1000ng/mL，之后按照3倍稀释12个梯度，并用Elisa的方法检测M1D、M3D与不同亚型的HBsAg(aa113-135)的结合活性，用PRISM软件绘制结合曲线。结果如图11A-11B所示，M1D、M3D均可以与大部分HBV亚型结合。
表18：不同亚型的HBsAg-aa113-135氨基酸序列
Figure PCTCN2018082157-appb-000037
实施例10：M1D、M3D的亲和力测定
以5μg/mL的HBsAg溶解于醋酸钠(pH 4.5)在Biacore 3000设备上运行包被芯片程序，将HBsAg包被至CM5芯片上，HBsAg的包被量为2400RU。将分析物从100nM，2倍梯度稀释，制备7个浓度的样品。在Biacore 3000设备上运行亲和力测定程序，设置流速为50μl/min，结合时间为90s，解离时间为600s，样品室的温度设置为10℃，再生液为50mM NaOH，再生流速为50μL/min，再生时间为60s。表19展示了M1D分子和M3D分子与抗原HBsAg的亲和力，分别为1.06nM和1.12nM。
表19：M1D、M3D与抗原的亲和力
Figure PCTCN2018082157-appb-000038
实施例11：给食蟹猴单次静脉注射抗体M1D、M3D后的药代动力学及初步毒性的评估
将6只未曾经历过大分子实验(＞2000Dalton)的雄性食蟹猴分成2组，3只/组。其中，通过静脉推注给第1组和第2组的食蟹猴施用剂量为20mg/kg的抗体M1D和M3D溶液。评估给食蟹猴单次静脉注射抗体M1D和M3D后的药代动力学特征及初步毒性反应。具体的实验设计如表20。
表20：评估M1D、M3D分子药代动力学特征及初步毒性反应的实验设计
Figure PCTCN2018082157-appb-000039
在给药前(0小时)，每天两次(上午9：30和下午4：00)笼边观察实验动物的健康及外观的状态。在实验开展前对实验动物进行体检，以确认动物的健康状态。在给药当天，在每个采血点前后分别观察实验动物的状态，包括实验动物的一般状态、行为、活动量、 排泄、呼吸及其它异常症状。结果显示，所有动物在给药期间及给药后均未表现出任何异常反应。此外，还在给药后，跟踪实验动物的体重和体温。结果示于表21-24中。
表21：M1D给药后的实验动物的体重
Figure PCTCN2018082157-appb-000040
表22：M1D给药后的实验动物的体温
Figure PCTCN2018082157-appb-000041
表23：M3D给药后的实验动物的体重
Figure PCTCN2018082157-appb-000042
表24：M3D给药后的实验动物的体温
Figure PCTCN2018082157-appb-000043
Figure PCTCN2018082157-appb-000044
上述实验结果表明，抗体M1D和M3D在施用给实验动物后，不会引起实验动物的不良副作用，从而初步证实了两株抗体良好的安全性。
此外，还分别于给药前(0小时)，给药后0.25h(15分钟)、0.5h(30分钟)、1h、2h、4h、10h、24h(Day 1)、48h(Day 2)、72h(Day 3)、96h(Day 4)、144h(Day 6)、192h(Day 8)、240h(Day 10)、336h(Day 14)、408h(Day 17)、504h(Day 21)及672h(Day 28)，从实验动物的头静脉采集约1.0mL全血。对收集的血清和全血进行下述分析。
采用化学发光免疫分析法(CLIA)，检测食蟹猴血清中抗体M1D和M3D的浓度。血清中人源化抗体M1D和M3D的定量下限(LLOQ)为0.063ng/mL，定量上限(ULOQ)为4.0ng/mL。检测结果示于图12A-12B中。
图12A显示了单次静脉注射20mg/kg的人源化抗体M1D后，每一只食蟹猴血清(第1组)中人源化抗体M1D的血药浓度随时间变化的曲线图。
图12B显示了单次静脉注射20mg/kg的人源化抗体M3D后，每一只食蟹猴血清(第2组)中人源化抗体M3D的血药浓度随时间变化的曲线图。
使用药动学软件WinNonlinTM Version 6.2.1(Pharsight，Mountain View，CA)，以静脉注射非房室模型(IV bolus input)对人源化抗体M1D、M3D的实验数据进行处理。
使用对数线性梯形法计算下列参数：起始血清药物浓度(C0)、最后一个可检测点的时间(T last)，消除半衰期(T 1/2)，表观分布容积(Vdss)，总体清除率(CL)，从零时间点到最后一个可检测到浓度的时间点的平均滞留时间(MRT 0-last)，从零时间点到无穷大的平均滞留时间(MRT 0-inf)，从零时间点到最后一个可检测到浓度的时间点的血清浓度-时间曲线下面积(AUC 0-last)，及从零时间点到无穷大的血清浓度-时间曲线下面积(AUC 0-inf)。
结果显示：单次静脉注射20mg/kg的人源化抗体M1D后，雄性食蟹猴对人源化抗体M1D的CL为0.00962±0.00335mL/min/kg。人源化抗体M1D的平均消除半衰期(t 1/2)为81.3±78.7小时。食蟹猴血清中人源化抗体M1D的Vdss和AUC 0-inf值分别为0.05±0.0209L/kg和38053±14915μg.h/mL。
单次静脉注射20mg/kg的人源化抗体M3D后，雄性食蟹猴对人源化抗体M3D的CL为0.0049±0.0025mL/min/kg。人源化抗体M3D的平均消除半衰期(t 1/2)为134±94.6小时。食蟹猴血清中人源化抗体M3D的Vdss和AUC 0-inf值分别为0.0497±0.0143L/kg和79419±33500μg.h/mL。
上述实验结果还概述于表25和表26中。
表25：第1组雄性食蟹猴的血清中人源化抗体M1D的主要药动学参数
PK参数 C1001 C1002 C1003 Mean IV   SD CV(％)
Rsq_adj 0.800 0.830 0.581 -- ± -- --
No.points used for T 1/2 17.0 17.0 14.0 ND ± -- --
C0(ng/mL) 593886 520570 359594 491350 ± 119848 24.4
T 1/2(h) 172 32.8 39.0 81.3 ± 78.7 96.8
Vdss(L/kg) 0.0583 0.0406 0.0472 0.0487 ± 0.00894 18.3
Cl(mL/min/kg) 0.00670 0.0128 0.00969 0.00971 ± 0.00303 31.2
T last(h) 672 672 672 672 ± -- --
AUC 0-last(ng.h/mL) 49777730 26142490 33521840 36480687 ± 12092238 33.1
AUC 0-inf(ng.h/mL) 49781190 26142960 34385180 36769777 ± 11998175 32.6
MRT 0-last(h) 145 53.1 64.0 87.4 ± 50.3 57.5
MRT 0-inf(h) 145 53.1 81.2 93.2 ± 47.2 50.6
AUCExtra(％) 0.00695 0.00181 2.51 0.840 ± 1.45 172
AUMCExtra(％) 0.0369 0.0245 23.2 7.74 ± 13.3 173
表26：第2组雄性食蟹猴的血清中人源化抗体M3D的主要药动学参数
PK参数 C2001 C2002 C2003 Mean PO   SD CV(％)
Rsq_adj 0.949 0.889 0.881 -- ± -- --
No.points used for T 1/2 16.0 16.0 11.0 ND ± -- --
C0(ng/mL) 740468 562963 674855 659429 ± 89752 13.6
T 1/2(h) 119 47.2 235 134 ± 94.6 70.7
Vdss(L/kg) 0.0428 0.0401 0.0660 0.0497 ± 0.0143 28.7
Cl(mL/min/kg) 0.00381 0.00782 0.00308 0.00490 ± 0.00255 52.0
T last(h) 672 672 672 672 ± -- --
AUC 0-last(ng.h/mL) 86427070 42620190 89311120 72786127 ± 26164236 35.9
AUC 0-inf(ng.h/mL) 87449170 42633960 108174500 79419210 ± 33500012 42.2
MRT 0-last(h) 179 85.3 219 161 ± 68.8 42.7
MRT 0-inf(h) 187 85.5 357 210 ± 137 65.4
AUCExtra(％) 1.17 0.0323 17.4 6.21 ± 9.74 157
AUMCExtra(％) 5.27 0.280 49.3 18.3 ± 27.0 148
另外，还对食蟹猴的血清进行血常规化学分析。食蟹猴血清中的各项指标(包括胆红素，丙氨酸转氨酶，天冬氨酸转氨酶，总蛋白，白蛋白，碱性磷酸酶，γ-谷氨酰转移酶，葡萄糖，尿素，肌酸酐，钙离子，磷，总胆固醇，甘油三酯，钠离子，钾离子，氯离子和球蛋白等的水平)均未见明显异常。这些实验结果表明，在指定的剂量条件下人源化抗体M1D或M3D的单次静脉注射是安全的。因此，本发明的人源化抗体M1D或M3D可施用给受试者(例如人)，以预防和/或治疗HBV感染或与HBV感染相关的疾病(例如乙肝)。
实施例12：M1D及M3D的成药性评估
1.人源化抗体M1D及M3D的等电点测定
利用毛细管等电聚焦电泳法(cIEF)对人源化抗体M1D和M3D的等电点进行测定。实验结果如表27所示。结果显示，人源化抗体M1D的pl值为8.8，M3D的pl为9.0。
表27：M1D、M3D的等电点
Figure PCTCN2018082157-appb-000045
2.人源化抗体M1D、M3D的稳定性测定
通常使用Tm值来描述抗体分子的稳定性。Tm值越高，抗体分子的热稳定性越好。将人源化抗体M1D和M3D分别溶解于三种缓冲液中，并稀释至1mg/ml，并通过差式扫描量热法(DSC)进行测试。开始扫描温度设置为10℃，结束扫描温度设置为110℃，扫描速度为200℃/hr，冷却速度设置为Exp，最终仪器保持温度设置为25℃，数据采集频率设置为10sec，进样前毛细管温度设置为30℃。实验结果示于图13A-13B、表28-29中。结果显示，人源化抗体M1D的Tm onset为61.7℃以上，而M3D的Tmonset值均大于61.6℃，说明M1D和M3D具有很好的热稳定性。
表28：M1D的Tm值
Figure PCTCN2018082157-appb-000046
表29：M3D的Tm值
Buffer TM onset(℃) TM1(℃) TM2(℃)
Acetate 5.0 61.6 68.8 82.2
Histidine 6.0 62.8 71.1 82.3
PBS7.2 65.7 78.5 /
此外，将人源化抗体M1D和M3D，以60mg/ml的浓度溶解于pH6.0的组氨酸缓冲液，以2mg/ml的浓度溶解于pH7.2的磷酸缓冲液，然后分别储存于40℃的条件下。分别于第0周(T0)、第1周(T1W)和第2周(T2W)，对样品的理化性质(包括外观、pH值、蛋白浓度等)进行监测。结果描述于表30和表31中。
表30：M1D稳定性测试
Figure PCTCN2018082157-appb-000047
Figure PCTCN2018082157-appb-000048
表31：M3D稳定性测试
Figure PCTCN2018082157-appb-000049
外观的监测
将样品瓶擦拭干净，并使用澄明度检测仪(黑背景和白背景)来观察样品的颜色和澄清度。上述表30-31的结果显示，两种缓冲液及40℃储存条件下的人源化抗体M1D和M3D样品均未出现明显变化。
pH值和蛋白浓度的监测
检测样品在A280处的吸光值，利用比尔-朗伯定律(Beer-Lambert law)，计算样品中的蛋白浓度。另外，使用pH计测定样品的pH值，重复测定2次，取平均值为最终结果。实验结果示于上述表30-31中。结果显示，人源化抗体M1D和M3D在不同缓冲液，40℃下储存2周后，pH值和蛋白浓度未发生显著改变。
SEC-HPLC检测
采用Agilent 1260infinity，TSK G3000SWXL凝胶柱(5μm，7.8mm×300mm)，通过SEC-HPLC对经历不同储存条件的人源化抗体M1D和M3D样品进行分析，其中，流动相组成为50mM PB+300mM NaCl，pH7.0±0.2；流速为1.0mL/min；检测波长为280nm；样品浓度为10mg/mL，进样量为10μL。实验结果示于上述表30-31中。结果显示，在不同储存条件(不同缓冲液，不同储存时间)下，含有人源化抗体M1D和M3D的样品的主峰均大于95％。这说明，人源化抗体M1D和M3D是稳定的。
cIEF检测
利用毛细管电泳对样品进行测试分析，结果显示M1D分子在组氨酸缓冲液中40℃储存2周后cIEF主峰下降6.0％，而在PBS缓冲液中储存2周后，主峰下降24.9％。对于M3D而言，在组氨酸缓冲液中40℃储存2周后cIEF主峰下降13.7％，而在PBS缓冲液中储存2周后，主峰下降30.6％。
3.人源化抗体M1D及M3D变体的可溶性分析
将人源化抗体M1D和M3D分别以60mg/mL的浓度溶解在含有5％蔗糖和0.02％PS80的25mM组氨酸溶液(pH 6.0)中，并进行离心超滤浓缩(离心温度为5℃，转速为4850rpm)，直至样品溶液的液面随着离心时间的增加而不再有明显下降。用移液器小心回收样品，观察此时样品的性状。结果显示，溶液样品呈黄色。
然后，移取1000μL的溶液样品至1.5mL EP管中，以10000rpm离心20分钟。离心结果显示，样品无分层，液体澄清，且无沉淀出现。用移液器分别小心吸取离心后样品的上层和下层，并通过UV280测定蛋白浓度。结果显示，M1D的溶解度为171mg/ml；M3D的溶解度170mg/ml。进一步，对溶解在含有5％蔗糖和0.02％PS80的25mM组氨酸溶液(pH 6.0)中的人源化抗体M1D和M3D变体的粘度进行测定。在该缓冲体系中，人源化抗体M1D在171mg/mL浓度下的粘度为13.2cp(1cP＝1mPa.s)，而M3D在170mg/mL浓度下的粘度为19.8cp，在100mg/ml下粘度为11.5cp。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (26)

  1. 能够特异性结合HBsAg的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
    (a)选自下列的重链可变区(VH)互补决定区(CDR)中的一个或多个(例如1个，2个或3个)：
    (i)VH CDR1，其由下述序列组成：SEQ ID NO：6，SEQ ID NO：12，SEQ ID NO：18或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；
    (ii)VH CDR2，其由下述序列组成：SEQ ID NO：7，SEQ ID NO：13，SEQ ID NO：19或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，和
    (iii)VH CDR3，其由下述序列组成：SEQ ID NO：8，SEQ ID NO：14，SEQ ID NO：20，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；
    和/或
    (b)选自下列的轻链可变区(VL)CDR中的一个或多个(例如1个，2个或3个)：
    (iv)VL CDR1，其由下述序列组成：SEQ ID NO：9，SEQ ID NO：15或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，
    (v)VL CDR2，其由下述序列组成：SEQ ID NO：10，SEQ ID NO：16或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，和
    (vi)VL CDR3，其由下述序列组成：SEQ ID NO：11，SEQ ID NO：17或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列。
  2. 权利要求1所述的抗体或其抗原结合片段，其包含：
    (a)重链可变区(VH)，所述VH包含：
    (i)VH CDR2，其由下述序列组成：SEQ ID NO：7，或者与其相比具有一个或几个氨 基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，
    (ii)VH CDR3，其由下述序列组成：SEQ ID NO：8，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，以及
    (iii)VH CDR1，其由下述序列组成：任意免疫球蛋白的重链可变区中所包含的VH CDR1的序列；
    和/或
    (b)轻链可变区(VL)，所述VL包含：
    (iv)VL CDR1，其由下述序列组成：SEQ ID NO：9，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，(v)VL CDR3，其由下述序列组成：SEQ ID NO：11，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，以及
    (vi)VL CDR2，其由下述序列组成：任意免疫球蛋白的轻链可变区(例如，κ轻链可变区)中所包含的VL CDR2的序列；
    优选地，所述VH CDR1为人免疫球蛋白的重链可变区中所包含的VH CDR1的序列；
    优选地，所述VH CDR1由下述序列组成：(a)SEQ ID NO：6，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；或，(b)人重链胚系基因所编码的氨基酸序列中所包含的VH CDR1的序列；
    优选地，所述人重链胚系基因选自IGHV4-4＊08和IGHV4-61＊01；
    优选地，所述VH包含：如SEQ ID NO：7所示的VH CDR2；如SEQ ID NO：8所示的VH CDR3；以及，具有选自下列的氨基酸序列的VH CDR1：SEQ ID NO：6、SEQ ID NO：137或SEQ ID NO：138；
    优选地，所述VL CDR2为人免疫球蛋白的轻链可变区(例如，κ轻链可变区)中所包含的VL CDR2的序列；
    优选地，所述VL CDR2由下述序列组成：(a)SEQ ID NO：10，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；或，(b)人轻链胚系基因所编码的氨基酸序列中所包含的VL CDR2的序列；
    优选地，所述人轻链胚系基因选自IGKV1-39＊01和IGKV1-5＊03；
    优选地，所述VL包含：如SEQ ID NO：9所示的VL CDR1；如SEQ ID NO：11所 示的VL CDR3；以及，具有选自下列的氨基酸序列的VL CDR2：SEQ ID NO：10、SEQ ID NO：139或SEQ ID NO：140；
    优选地，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO：7所示的VH CDR2，如SEQ ID NO：8所示的VH CDR3，以及具有选自下列的氨基酸序列的VH CDR1：SEQ ID NO：6，137或138；并且，该抗体或其抗原结合片段的VL包含：如SEQ ID NO：9所示的VL CDR1，如SEQ ID NO：11所示的VL CDR3，以及具有选自下列的氨基酸序列的VL CDR2：SEQ ID NO：10，139或140。
  3. 权利要求2所述的抗体或其抗原结合片段，其包含：
    (i)如SEQ ID NO：7所示的VH CDR2；如SEQ ID NO：8所示的VH CDR3；以及VH CDR1，其由下述序列组成：任意免疫球蛋白的重链可变区中所包含的VH CDR1的序列(例如，SEQ ID NO：6，137或138)；和，如SEQ ID NO：9所示的VL CDR1，如SEQ ID NO：11所示的VL CDR3，以及如SEQ ID NO：10所示的VL CDR2；或
    (ii)如SEQ ID NO：7所示的VH CDR2；如SEQ ID NO：8所示的VH CDR3；以及如SEQ ID NO：6所示的VH CDR1；和，如SEQ ID NO：9所示的VL CDR1，如SEQ ID NO：11所示的VL CDR3，以及VL CDR2，其由下述序列组成：任意免疫球蛋白的轻链可变区(例如，κ轻链可变区)中所包含的VL CDR2的序列(例如，SEQ ID NO：10，139或140)；
    优选地，所述VH CDR1为人免疫球蛋白的重链可变区中所包含的VH CDR1的序列；
    优选地，所述VL CDR2为人免疫球蛋白的轻链可变区(例如，κ轻链可变区)中所包含的VL CDR2的序列；
    优选地，所述抗体或其抗原结合片段包含：
    (1)如SEQ ID NO：6所示的VH CDR1、如SEQ ID NO：7所示的VH CDR2、和如SEQ ID NO：8所示的VH CDR3；以及，如SEQ ID NO：9所示的VL CDR1、如SEQ ID NO：10所示的VL CDR2、和如SEQ ID NO：11所示的VL CDR3；
    (2)如SEQ ID NO：137所示的VH CDR1、如SEQ ID NO：7所示的VH CDR2、和如SEQ ID NO：8所示的VH CDR3；以及，如SEQ ID NO：9所示的VL CDR1、如SEQ ID NO：10所示的VL CDR2、和如SEQ ID NO：11所示的VL CDR3；
    (3)如SEQ ID NO：138所示的VH CDR1、如SEQ ID NO：7所示的VH CDR2、和如SEQ ID NO：8所示的VH CDR3；和，如SEQ ID NO：9所示的VL CDR1、如SEQ  ID NO：10所示的VL CDR2、和如SEQ ID NO：11所示的VL CDR3；
    (4)如SEQ ID NO：6所示的VH CDR1、如SEQ ID NO：7所示的VH CDR2、和如SEQ ID NO：8所示的VH CDR3；和，如SEQ ID NO：9所示的VL CDR1、如SEQ ID NO：139所示的VL CDR2、和如SEQ ID NO：11所示的VL CDR3；或
    (5)如SEQ ID NO：6所示的VH CDR1、如SEQ ID NO：7所示的VH CDR2、和如SEQ ID NO：8所示的VH CDR3；和，如SEQ ID NO：9所示的VL CDR1、如SEQ ID NO：140所示的VL CDR2、和如SEQ ID NO：11所示的VL CDR3。
  4. 权利要求1所述的抗体或其抗原结合片段，其包含：
    (a)重链可变区(VH)，所述VH包含：
    (i)VH CDR1，其由下述序列组成：SEQ ID NO：12，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，
    (ii)VH CDR3，其由下述序列组成：SEQ ID NO：14，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，以及
    (iii)VH CDR2，其由下述序列组成：任意免疫球蛋白的重链可变区中所包含的VH CDR2的序列；
    和/或
    (b)轻链可变区(VL)，所述VL包含：
    (iv)VL CDR1，其由下述序列组成：SEQ ID NO：15，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，
    (v)VL CDR3，其由下述序列组成：SEQ ID NO：17，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，以及
    (vi)VL CDR2，其由下述序列组成：任意免疫球蛋白的轻链可变区(例如，κ轻链可变区)中所包含的VL CDR2的序列；
    优选地，所述VH CDR2为人免疫球蛋白的重链可变区中所包含的VH CDR2的序列；
    优选地，所述VH CDR2由下述序列组成：(a)SEQ ID NO：13，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；或，(b)人重链胚系基因所编码的氨基酸序列中所包含的VH CDR2的序列；
    优选地，所述人重链胚系基因选自IGHV4-30-4＊07和IGHV4-4＊01；
    优选地，所述VH包含：(a)如SEQ ID NO：12所示的VH CDR1；如SEQ ID NO：14所示的VH CDR3；以及，具有选自下列的氨基酸序列的VH CDR2：SEQ ID NO：13、SEQ ID NO：141或SEQ ID NO：142；
    优选地，所述VL CDR2为人免疫球蛋白的轻链可变区中所包含的VL CDR2的序列；
    优选地，所述VL CDR2由下述序列组成：(a)SEQ ID NO：16，或者与其相比具有一个或几个氨基酸置换、缺失或添加的序列；或，(b)人轻链胚系基因所编码的氨基酸序列中所包含的VL CDR2的序列；
    优选地，所述人轻链胚系基因选自IGKV2-28＊01和IGKV3-15＊01；
    优选地，所述VL包含：如SEQ ID NO：15所示的VL CDR1；如SEQ ID NO：17所示的VL CDR3；以及，具有选自下列的氨基酸序列的VL CDR2：SEQ ID NO：16、SEQ ID NO：143或SEQ ID NO：144；
    优选地，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO：12所示的VH CDR1，如SEQ ID NO：14所示的VH CDR3，以及具有选自下列的氨基酸序列的VH CDR2：SEQ ID NO：13、SEQ ID NO：141或SEQ ID NO：142；并且，该抗体或其抗原结合片段的VL包含：如SEQ ID NO：15所示的VL CDR1，如SEQ ID NO：17所示的VL CDR3，以及具有选自下列的氨基酸序列的VL CDR2：SEQ ID NO：16、SEQ ID NO：143或SEQ ID NO：144。
  5. 权利要求4所述的抗体或其抗原结合片段，其包含：
    (i)如SEQ ID NO：12所示的VH CDR1，如SEQ ID NO：14所示的VH CDR3，以及VH CDR2，其由下述序列组成：任意免疫球蛋白的重链可变区中所包含的VH CDR2的序列(例如，SEQ ID NO：13、SEQ ID NO：141或SEQ ID NO：142)；和，如SEQ ID NO：15所示的VL CDR1，如SEQ ID NO：17所示的VL CDR3，以及如SEQ ID NO：16所示的VL CDR2；或
    (ii)如SEQ ID NO：12所示的VH CDR1；如SEQ ID NO：14所示的VH CDR3；以及如SEQ ID NO：13所示的VH CDR2；和，如SEQ ID NO：15所示的VL CDR1，如SEQ ID NO：17所示的VL CDR3，以及VL CDR2，其由下述序列组成：任意免疫球蛋白的轻链可变区中所包含的VL CDR2的序列(例如，SEQ ID NO：16、SEQ ID NO：143或SEQ ID NO：144)；
    优选地，所述VH CDR2为人免疫球蛋白的重链可变区中所包含的VH CDR2的序列；
    优选地，所述VL CDR2为人免疫球蛋白的轻链可变区(例如，κ轻链可变区)中所包含的VL CDR2的序列；
    优选地，所述抗体或其抗原结合片段包含：
    (1)如SEQ ID NO：12所示的VH CDR1、如SEQ ID NO：13所示的VH CDR2、和如SEQ ID NO：14所示的VH CDR3；以及，如SEQ ID NO：15所示的VL CDR1、如SEQ ID NO：16所示的VL CDR2、和如SEQ ID NO：17所示的VL CDR3；
    (2)如SEQ ID NO：12所示的VH CDR1、如SEQ ID NO：141所示的VH CDR2、和如SEQ ID NO：14所示的VH CDR3；以及，如SEQ ID NO：15所示的VL CDR1、如SEQ ID NO：16所示的VL CDR2、和如SEQ ID NO：17所示的VL CDR3；
    (3)如SEQ ID NO：12所示的VH CDR1、如SEQ ID NO：142所示的VH CDR2、和如SEQ ID NO：14所示的VH CDR3；以及，如SEQ ID NO：15所示的VL CDR1、如SEQ ID NO：16所示的VL CDR2、和如SEQ ID NO：17所示的VL CDR3；
    (4)如SEQ ID NO：12所示的VH CDR1、如SEQ ID NO：13所示的VH CDR2、和如SEQ ID NO：14所示的VH CDR3；以及，如SEQ ID NO：15所示的VL CDR1、如SEQ ID NO：143所示的VL CDR2、和如SEQ ID NO：17所示的VL CDR3；或
    (5)如SEQ ID NO：12所示的VH CDR1、如SEQ ID NO：13所示的VH CDR2、和如SEQ ID NO：14所示的VH CDR3；以及，如SEQ ID NO：15所示的VL CDR1、如SEQ ID NO：144所示的VL CDR2、和如SEQ ID NO：17所示的VL CDR3。
  6. 权利要求1所述的抗体或其抗原结合片段，其包含：
    (a)重链可变区(VH)，所述VH包含：
    (i)VH CDR1，其由下述序列组成：SEQ ID NO：18，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；
    (ii)VH CDR2，其由下述序列组成：SEQ ID NO：19，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，和
    (iii)VH CDR3，其由下述序列组成：SEQ ID NO：20，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；
    和/或
    (b)轻链可变区(VL)，所述VL包含：
    (iv)VL CDR1，其由下述序列组成：SEQ ID NO：15，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，(v)VL CDR2，其由下述序列组成：SEQ ID NO：16，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列，和
    (vi)VL CDR3，其由下述序列组成：SEQ ID NO：17，或者与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个或3个氨基酸置换、缺失或添加)的序列；
    优选地，所述抗体或其抗原结合片段的VH包含：如SEQ ID NO：18所示的VH CDR1，如SEQ ID NO：19所示的VH CDR2，以及如SEQ ID NO：20所示的VH CDR3；并且，该抗体或其抗原结合片段的VL包含：如SEQ ID NO：15所示的VL CDR1，如SEQ ID NO：16所示的VL CDR2，以及如SEQ ID NO：17所示的VL CDR3。
  7. 权利要求1-6任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段是人源化的；
    优选地，所述抗体或其抗原结合片段的人源化程度为至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％或至少98％；
    优选地，所述抗体或其抗原结合片段的非CDR区包含不超过19个，不超过15个，不超过14个，不超过13个，不超过12个，不超过11个，不超过10个，不超过9个，不超过8个，不超过7个，不超过6个，不超过5个，不超过4个或不超过3个的非人源(例如，食蟹猴源)氨基酸残基。
  8. 权利要求1-7任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段进一步包含框架区(FR)；
    优选地，所述抗体或其抗原结合片段包含：
    (a)选自下列的重链可变区(VH)构架区(FR)中的一个或多个(例如1个，2个，3个或4个)：
    (i)VH FR1，其由下述序列组成：SEQ ID NO：21，SEQ ID NO：29，SEQ ID NO：37，SEQ ID NO：44，SEQ ID NO：50，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个、3个或4个氨基酸置换、缺失或添加)的序列；
    (ii)VH FR2，其由下述序列组成：SEQ ID NO：22，SEQ ID NO：30，SEQ ID NO：38，SEQ ID NO：51，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个、3个或4个氨基酸置换、缺失或添加)的序列；
    (iii)VH FR3，其由下述序列组成：SEQ ID NO：23，SEQ ID NO：31，SEQ ID NO：39，SEQ ID NO：45，SEQ ID NO：52，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个、3个或4个氨基酸置换、缺失或添加)的序列；和
    (iv)VH FR4，其由下述序列组成：SEQ ID NO：24，SEQ ID NO：32，SEQ ID NO：46，SEQ ID NO：53，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个、3个或4个氨基酸置换、缺失或添加)的序列；
    和/或
    (b)选自下列的轻链可变区(VL)构架区(FR)中的一个或多个(例如1个，2个，3个或4个)：
    (v)VL FR1，其由下述序列组成：SEQ ID NO：25，SEQ ID NO：33，SEQ ID NO：47，SEQ ID NO：54，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个、3个或4个氨基酸置换、缺失或添加)的序列；
    (vi)VL FR2，其由下述序列组成：SEQ ID NO：26，SEQ ID NO：34，SEQ ID NO：48，SEQ ID NO：55，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个、3个或4个氨基酸置换、缺失或添加)的序列；
    (vii)VL FR3，其由下述序列组成：SEQ ID NO：27，SEQ ID NO：35，SEQ ID NO：49，SEQ ID NO：56，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个、3个或4个氨基酸置换、缺失或添加)的序列；和
    (viii)VL FR4，其由下述序列组成：SEQ ID NO：28，SEQ ID NO：36，或与其相比具有一个或几个氨基酸置换、缺失或添加(例如，1个、2个、3个或4个氨基酸置换、缺失或添加)的序列；
    优选地，所述抗体或其抗原结合片段包含：
    (a)重链框架区，其与选自下列的重链可变区中所包含的重链框架区相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性：如SEQ ID NOs：1，3，5，57，59，157，158，163和164所示的重链可变区；
    和/或
    (b)轻链框架区，其与选自下列的轻链可变区中所包含的轻链框架区相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性：如SEQ ID NOs：2，4，58，60，159，160，165和166任一项所示的轻链可变区；
    优选地，所述抗体或其抗原结合片段包含：
    (a)选自下列的重链可变区中含有的4个FR：
    如SEQ ID NOs：1，3，5，57，59，157，158，163和164任一项所示的重链可变区；
    和/或
    (b)选自下列的轻链可变区中含有的4个FR：
    如SEQ ID NOs：2，4，58，60，159，160，165和166任一项所示的轻链可变区。
  9. 权利要求1-8任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段进一步包含人免疫球蛋白的框架区(例如，人胚系抗体基因所编码的氨基酸序列中所包含的框架区)，所述框架区任选地包含一个或多个(例如，1个、2个、3个、4个、5个、6个、7个、8个、9个或10个)从人源残基至食蟹猴源残基的回复突变；
    优选地，所述抗体或其抗原结合片段包含：人重链胚系基因所编码的氨基酸序列中所包含的重链框架区，和/或人轻链胚系基因所编码的氨基酸序列中所包含的轻链框架区；
    优选地，所述抗体或其抗原结合片段包含：IGHV4-4＊08所编码的氨基酸序列中所包含的重链框架区，和IGKV1-39＊01所编码的氨基酸序列中所包含的轻链框架区，所述重链框架区和/或轻链框架区任选地包含一个或多个(例如，1个、2个、3个、4个、5个、6个、7个、8个、9个或10个)从人源残基至食蟹猴源残基的回复突变；
    优选地，所述抗体或其抗原结合片段包含：IGHV4-4＊02所编码的氨基酸序列中所包含的重链框架区，和IGKV4-1＊01所编码的氨基酸序列中所包含的轻链框架区，所述重链框架区和/或轻链框架区任选地包含一个或多个(例如，1个、2个、3个、4个、5个、6个、7个、8个、9个或10个)从人源残基至食蟹猴源残基的回复突变。
  10. 权利要求1-9任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段的重链可变区的氨基酸序列与选自下列的重链可变区的氨基酸序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性：如SEQ ID NOs：1，3， 5，57，59，157，158，163和164所示的重链可变区；
    优选地，所述抗体或其抗原结合片段的重链可变区选自如SEQ ID NOs：1，3，5，57，59，157，158，163和164任一项所示的重链可变区。
  11. 权利要求1-10任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段的轻链可变区的氨基酸序列与选自下列的轻链可变区的氨基酸序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性：如SEQ ID NOs：2，4，58，60，159，160，165和166任一项所示的轻链可变区；
    优选地，所述抗体或其抗原结合片段的轻链可变区选自如SEQ ID NOs：2，4，58，60，159，160，165和166任一项所示的轻链可变区。
  12. 权利要求1-11任一项所述的抗体或其抗原结合片段，其中，所述抗体包含如权利要求11所定义的重链可变区和如权利要求12所定义的轻链可变区。
  13. 权利要求1-12任一项所述的抗体或其抗原结合片段，其中：
    (a)所述抗体或其抗原结合片段的重链可变区选自如SEQ ID NOs：1，57，157和158任一项所示的重链可变区；并且，所述抗体或其抗原结合片段的轻链可变区选自如SEQ ID NOs：2，58，159和160任一项所示的轻链可变区；或
    (b)所述抗体或其抗原结合片段的重链可变区选自如SEQ ID NOs：3，59，163和164任一项所示的重链可变区；并且，所述抗体或其抗原结合片段的轻链可变区选自如SEQ ID NOs：4，60，165和166任一项所示的轻链可变区。
  14. 权利要求1-13任一项所述的抗体或其抗原结合片段，其中，所述抗体包含：
    (1)如SEQ ID NO：1所示的VH和如SEQ ID NO：2所示的VL；
    (2)如SEQ ID NO：3所示的VH和如SEQ ID NO：4所示的VL；
    (3)如SEQ ID NO：5所示的VH和如SEQ ID NO：4所示的VL；
    (4)如SEQ ID NO：57所示的VH和如SEQ ID NO：58所示的VL；
    (5)如SEQ ID NO：59所示的VH和如SEQ ID NO：60所示的VL；
    (6)如SEQ ID NO：157所示的VH和如SEQ ID NO：58所示的VL；
    (7)如SEQ ID NO：158所示的VH和如SEQ ID NO：58所示的VL；
    (8)如SEQ ID NO：57所示的VH和如SEQ ID NO：159所示的VL；
    (9)如SEQ ID NO：57所示的VH和如SEQ ID NO：160所示的VL；
    (10)如SEQ ID NO：163所示的VH和如SEQ ID NO：60所示的VL；
    (11)如SEQ ID NO：164所示的VH和如SEQ ID NO：60所示的VL；
    (12)如SEQ ID NO：59所示的VH和如SEQ ID NO：165所示的VL；或
    (13)如SEQ ID NO：59所示的VH和如SEQ ID NO：166所示的VL。
  15. 权利要求1-14任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段选自scFv、Fab、Fab’、(Fab’) 2、Fv片段、双抗体(diabody)、双特异性抗体、多特异性抗体、嵌合抗体或人源化抗体；优选地，所述抗体为嵌合抗体或人源化抗体。
  16. 权利要求1-15任一项所述的抗体或其抗原结合片段，其中，所述抗体为IgG类别(例如，IgG1、IgG2、IgG3或IgG4类别)。
  17. 权利要求1-16任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段能够特异性结合HBsAg，中和HBV的毒力，和/或降低HBV DNA和/或HBsAg在受试者体内的血清水平。
  18. 权利要求1-17任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段带有标记；优选地，所述抗体或其抗原结合片段带有可检测的标记，例如放射性同位素，荧光物质，发光物质，有色物质或酶(例如辣根过氧化物酶)。
  19. 分离的核酸分子，其编码权利要求1-18任一项所述的抗体或其抗原结合片段，或其重链可变区和/或轻链可变区。
  20. 载体，其包含权利要求19所述的核酸分子；优选地，所述载体为克隆载体或表达载体。
  21. 宿主细胞，其包含权利要求19所述的核酸分子或权利要求20所述的载体。
  22. 制备权利要求1-18任一项所述的抗体或其抗原结合片段的方法，其包括，在允许所述抗体或其抗原结合片段表达的条件下，培养权利要求21所述的宿主细胞，和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段。
  23. 药物组合物，其含有权利要求1-18任一项所述的抗体或其抗原结合片段，以及药学上可接受的载体和/或赋形剂。
  24. 权利要求1-18任一项所述的抗体或其抗原结合片段或权利要求23所述的药物组合物在制备药物中的用途，所述药物用于预防或治疗受试者(例如人)的HBV感染或与HBV感染相关的疾病(例如乙肝)，用于体外或在受试者(例如人)体内中和HBV的毒力，和/或用于在受试者(例如人)体内降低HBV DNA和/或HBsAg的血清水平。
  25. 权利要求1-18任一项所述的抗体或其抗原结合片段或权利要求23所述的药物组合物，其用于预防或治疗受试者(例如人)的HBV感染或与HBV感染相关的疾病(例如乙肝)，用于体外或在受试者(例如人)体内中和HBV的毒力，和/或用于在受试者(例如人)体内降低HBV DNA和/或HBsAg的血清水平。
  26. 一种方法，其用于预防或治疗受试者的HBV感染或与HBV感染相关的疾病(例如乙肝)，用于在受试者(例如人)体内中和HBV的毒力，和/或用于在受试者(例如人)体内降低HBV DNA和/或HBsAg的血清水平，所述方法包括，给有此需要的受试者施用有效量的权利要求1-18任一项所述的抗体或其抗原结合片段，或者权利要求23所述的药物组合物。
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JP2022534080A (ja) * 2019-05-23 2022-07-27 シァメン・ユニヴァーシティ 新規の抗ｂ型肝炎ウイルス抗体及びその使用
CN115677852A (zh) * 2021-07-29 2023-02-03 东莞市朋志生物科技有限公司 一种抗HBeAg抗体及其应用
RU2814471C2 (ru) * 2019-05-23 2024-02-29 Сямэнь Юниверсити Новые антитела против вируса гепатита b и их применение

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