WO2022233321A1 - 冠状病毒抗体及其应用 - Google Patents

冠状病毒抗体及其应用 Download PDF

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WO2022233321A1
WO2022233321A1 PCT/CN2022/091204 CN2022091204W WO2022233321A1 WO 2022233321 A1 WO2022233321 A1 WO 2022233321A1 CN 2022091204 W CN2022091204 W CN 2022091204W WO 2022233321 A1 WO2022233321 A1 WO 2022233321A1
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seq
antibody
antigen
sequence
binding fragment
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PCT/CN2022/091204
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English (en)
French (fr)
Inventor
黄贤明
张慧
苏紫琪
苏华飞
黄皓晖
汪志炜
陈俊有
郑丹丹
李嘉萍
朱圣花
李婵
李胜峰
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百奥泰生物制药股份有限公司
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Publication of WO2022233321A1 publication Critical patent/WO2022233321A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • 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
    • C07K2317/50—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to coronavirus antibodies and applications thereof.
  • Coronaviruses are single-stranded positive-stranded RNA viruses that are not segmented. According to serotype and genome characteristics, the coronavirus subfamily is divided into four genera: ⁇ , ⁇ , ⁇ , and ⁇ . Protrusion, shaped like a corolla named.
  • the new coronavirus (SARS-CoV-2 or 2019-nCoV) discovered in 2019 belongs to the new coronavirus of the genus ⁇ . Current research shows that SARS-CoV-2 has a high degree of homology with SARS-CoV.
  • the novel coronavirus pneumonia COVID-19 is mainly transmitted through the respiratory tract, but it may also be transmitted through contact.
  • the population is generally susceptible, the elderly and those with underlying diseases are more seriously ill after infection, and children and infants are also affected. Based on current epidemiological investigations, the incubation period of new coronaviruses is generally 1-14 days, and most are 3-7 days.
  • the main clinical symptoms of infected persons are fever, fatigue, and dry cough, while upper respiratory symptoms such as nasal congestion and runny nose are rare.
  • the total number of white blood cells in patients was normal or decreased, or the number of lymphocytes decreased, and some patients had increased liver enzymes, muscle enzymes and myoglobin.
  • Chest imaging showed that the patient presented with multiple small patchy shadows and interstitial changes in the early stage, which were obvious in the outer pulmonary band; then developed into multiple ground-glass shadows and infiltrative shadows in both lungs. In severe cases, lung consolidation may occur, and breathing difficulties gradually appeared. Acute respiratory distress syndrome (ARDS), shock, and various tissue damage and dysfunction of lung tissue, heart, and kidney occur in patients. Most patients with mild infection have a good prognosis, and severe patients are often critically ill and even die.
  • ARDS Acute respiratory distress syndrome
  • the present invention provides antibodies or antigen-binding fragments with high affinity to the spike proteins of SARS-CoV and SARS-CoV-2 to include fusion proteins of the antibodies or antigen-binding fragments.
  • the present invention provides antibodies or antigen-binding fragments with high affinity for spike proteins of SARS-CoV and SARS-CoV-2. These antibodies can specifically bind to spike proteins, prevent viral particles from binding to cells, and mediate immune cell phagocytosis and clearance of viral particles. These antibodies can be used to treat or ameliorate SARS and COVID-19, and can also be used to diagnose SARS and COVID-19.
  • SARS-CoV or SARS-CoV-2 Through the binding of the spike protein (S protein or spike protein) on its surface to an angiotensin-converting enzyme 2 (ACE2) on the surface of lung epithelial cells, SARS-CoV or SARS-CoV-2 enters the cell, and The cell is used to synthesize new virus particles for it; the new virus particles are released outside the cell, and in the same way, the virus infects the surrounding normal cells.
  • Anti-spike protein antibody can block the binding of spike protein to ACE2, thereby blocking the entry of virus into cells and exerting anti-viral effect.
  • Antibodies of the present invention can also mediate phagocytosis and clearance of viruses by immune cells.
  • HCDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 1 or 2, or a variant thereof having a single site substitution, deletion or insertion
  • HCDR2 comprising as shown in SEQ ID NO: 3 or The amino acid sequence shown in 4, or a variant thereof with a single site substitution, deletion or insertion
  • HCDR3 comprising the amino acid sequence shown in any one of SEQ ID NOs: 5-42, or variants thereof with a single site substitution, deletion or insertion.
  • the antibody or antigen-binding fragment specifically binds a spike protein and comprises:
  • HCDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 1 or 2, or a variant thereof having a single site substitution, deletion or insertion
  • HCDR2 comprising as shown in SEQ ID NO: 3 or The amino acid sequence shown in 4, or a variant thereof with a single site substitution, deletion or insertion
  • HCDR3 comprising the amino acid sequence shown in any one of SEQ ID NOs: 5-42, or its Variants with single site substitutions, deletions or insertions.
  • the antibody or antigen-binding fragment specifically binds a spike protein and comprises:
  • HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1 or 2
  • HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 3 or 4
  • HCDR3 comprising The amino acid sequence shown in any one of SEQ ID NOs: 5-42.
  • the antibody or antigen-binding fragment specifically binds a spike protein and comprises:
  • HCDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 1 or 2, or a variant thereof having a single site substitution, deletion or insertion
  • HCDR2 comprising as shown in SEQ ID NO: 3 or The amino acid sequence shown in 4, or a variant thereof with a single site substitution, deletion or insertion
  • HCDR3 comprising the amino acid sequence shown in any one of SEQ ID NOs: 5-42, or a variant thereof with Single-site substitution, deletion or insertion variant
  • LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 43 or 44, or a single-site substitution, deletion or insertion variant thereof
  • LCDR2 which comprises the amino acid sequence as shown in SEQ ID NO:45 or 46, or a variant thereof with a single site substitution, deletion or insertion
  • LCDR3 which comprises as SEQ ID NO:47 or the amino acid sequence shown in 48, or a variant thereof with a single site substitution, deletion or insertion.
  • the antibody or antigen-binding fragment specifically binds a spike protein and comprises:
  • HCDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 1 or 2, or a variant thereof having a single site substitution, deletion or insertion
  • HCDR2 comprising as shown in SEQ ID NO: 3 or The amino acid sequence shown in 4, or a variant thereof with a single site substitution, deletion or insertion
  • HCDR3 comprising the amino acid sequence shown in any one of SEQ ID NOs: 5-42, or a variant thereof with Single-site substitution, deletion or insertion variant
  • LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 43 or 44, or a single-site substitution, deletion or insertion variant thereof
  • LCDR2 which comprises the amino acid sequence as shown in SEQ ID NO:45 or 46, or a variant thereof with a single site substitution, deletion or insertion
  • LCDR3 which comprises as SEQ ID NO:47 or 48 The indicated amino acid sequence, or a variant thereof with a single site substitution, deletion or insertion.
  • the substitution variant is a conservative amino acid substitution variant.
  • the antibody or antigen-binding fragment specifically binds a spike protein
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO: 1 or 2, or HCDR1 as set forth in SEQ ID NO: 3 or 4 HCDR2 shown in any one of SEQ ID NOs: 5-42, LCDR1 shown in SEQ ID NO: 43 or 44, LCDR2 shown in SEQ ID NO: 45 or 46 and LCDR2 shown in SEQ ID NO: 45 or 46 One, two, three, four, five or all of the LCDR3 shown in NO: 47 or 48.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:5, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:6, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:7, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:8, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:9, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:10, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:11, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:12, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:13, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:14, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:15, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:16, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:17, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:18, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:19, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:20, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:21, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:22, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:23, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:24, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:25, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:26, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:27, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:28, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:29, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:30, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:31, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:32, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:33, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:34, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:35, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:36, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:37, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:38, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:39, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:40, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:41, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:2, HCDR2 as set forth in SEQ ID NO:4, HCDR3 as set forth in SEQ ID NO:42, LCDR1 shown in ID NO:44, LCDR2 shown in SEQ ID NO:46 and LCDR3 shown in SEQ ID NO:48.
  • the antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2, and/or HCDR3 described herein, the light chain variable region
  • the variable regions comprise LCDR1, LCDR2, and/or LCDR3 as described herein.
  • the framework regions of the heavy chain variable region of the antibody or antigen-binding fragment further comprise heavy chain FR1, heavy chain FR2, heavy chain FR3, and heavy chain FR4.
  • the heavy chain FR1 comprises the sequence set forth in SEQ ID NO: 49 or 50, or a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 49 or 50, or the sequence set forth in SEQ ID NO: 49 or 50 :
  • the sequence shown at 49 or 50 is compared to the amino acid sequence with one or more conservative amino acid substitutions.
  • the heavy chain FR2 comprises the sequence set forth in SEQ ID NO: 51 or 52, or a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 51 or 52, or the sequence set forth in SEQ ID NO: 51 or 52 :
  • the sequence shown in 51 or 52 is compared to the amino acid sequence with one or more conservative amino acid substitutions.
  • the heavy chain FR3 comprises the sequence set forth in SEQ ID NO: 53 or 54, or a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 53 or 54, or the sequence set forth in SEQ ID NO: 53 or 54 :
  • the sequence shown in 53 or 54 is compared to the amino acid sequence with one or more conservative amino acid substitutions.
  • the heavy chain FR4 comprises the sequence set forth in SEQ ID NO:55, or a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO:55, or the sequence set forth in SEQ ID NO:55 Sequences are compared to amino acid sequences with one or more conservative amino acid substitutions.
  • the heavy chain FR1 comprises the sequence set forth in SEQ ID NO: 49 or 50, or a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 49 or 50, or the sequence set forth in SEQ ID NO: 49 or 50 : an amino acid sequence with one or more conservative amino acid substitutions compared to the sequence shown in 49 or 50;
  • the heavy chain FR2 comprises the sequence shown in SEQ ID NO: 51 or 52, or with the sequence shown in SEQ ID NO: 51 or 52 A sequence having at least 90% identity, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 51 or 52;
  • the heavy chain FR3 comprises the sequence shown in SEQ ID NO: 53 or 54 The sequence shown, or a sequence with at least 90% identity to the sequence shown in SEQ ID NO: 53 or 54, or an amino acid sequence with one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 53 or 54
  • the heavy chain FR4 comprises the sequence shown in SEQ ID NO
  • the heavy chain FR1 comprises the sequence set forth in SEQ ID NO:49
  • the heavy chain FR2 comprises the sequence set forth in SEQ ID NO:51
  • the heavy chain FR3 comprises the sequence set forth in SEQ ID NO:53
  • the sequence shown, the heavy chain FR4 comprises the sequence shown in SEQ ID NO:55.
  • the heavy chain FR1 comprises the sequence set forth in SEQ ID NO:50
  • the heavy chain FR2 comprises the sequence set forth in SEQ ID NO:52
  • the heavy chain FR3 comprises the sequence set forth in SEQ ID NO:54
  • the heavy chain FR4 comprises the sequence shown in SEQ ID NO:55.
  • the heavy chain variable region comprises the structure heavy chain FR1-HCDR1-heavy chainFR2-HCDR2-heavy chainFR3-HCDR3-heavy chainFR4.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises heavy chain FRl as set forth in SEQ ID NO:49, HCDRl as set forth in SEQ ID NO:1, as SEQ ID NO:51 Heavy chain FR2 as shown, HCDR2 as shown in SEQ ID NO:3, heavy chain FR3 as shown in SEQ ID NO:53, HCDR3 as shown in any one of SEQ ID NOs:5-41 and as shown in SEQ ID NO:53 Heavy chain FR4 shown in ID NO:55.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises heavy chain FRl as set forth in SEQ ID NO:50, HCDRl as set forth in SEQ ID NO:2, as in SEQ ID NO:52 Heavy chain FR2 as shown, HCDR2 as shown in SEQ ID NO:4, heavy chain FR3 as shown in SEQ ID NO:54, HCDR3 as shown in SEQ ID NO:42 and as shown in SEQ ID NO:55 heavy chain FR4.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 56 or 57, or is at least 80% identical to the sequence set forth in SEQ ID NO: 56 or 57 , or an amino acid sequence with one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 56 or 57.
  • the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 58 or 59, or is at least 80% identical to the sequence set forth in SEQ ID NO: 58 or 59 , or an amino acid sequence with one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 58 or 59.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:56
  • the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:58 the sequence shown.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:57
  • the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:59 the sequence shown.
  • the antibody or antigen-binding fragment further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.
  • the light chain constant region is a kappa or lambda chain constant region.
  • the antibody or fragment thereof is one of the isotypes of IgG, IgM, IgA, IgE, or IgD.
  • the isotype is IgGl, IgG2, IgG3, or IgG4.
  • the antibody or antigen-binding fragment is a chimeric antibody, a humanized antibody, or a fully human antibody. In one aspect, the antibody or antigen-binding fragment is a humanized antibody.
  • the Fc is a variant Fc region.
  • the variant Fc region has one or more amino acid modifications, such as substitutions, deletions or insertions, relative to the parent Fc region.
  • the amino acid modification of the Fc region alters effector function activity relative to the activity of the parental Fc region.
  • variant Fc regions may have altered (ie, increased or decreased) antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), phagocytosis, opsonization, or cell binding .
  • the Fc region amino acid modifications can alter the affinity of the variant Fc region for Fc ⁇ Rs (Fc ⁇ receptors) relative to the parent Fc region.
  • the Fc region is derived from IgGl or IgG4. In some embodiments, the Fc region mutation is N297A. In some embodiments, the Fc region mutation is N297A, L234A, or L235A (Eu numbering). In some embodiments, the Fc region mutation is E345R or S440Y (Eu numbering).
  • the antibody or antigen-binding fragment is an isolated antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is scFV, Fab, F(ab) 2 , or IgG. In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody.
  • the heavy chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 60 or 61, or at least 80% the same as the sequence set forth in SEQ ID NO: 60 or 61 An identical sequence, or an amino acid sequence with one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 60 or 61.
  • the light chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:62, or a sequence that is at least 80% identical to the sequence set forth in SEQ ID NO:62 , or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 62.
  • the heavy chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 60 or 61, and/or the light chain constant region of the antibody or antigen-binding fragment comprises The amino acid sequence is the sequence shown in SEQ ID NO:62.
  • the heavy chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence as set forth in SEQ ID NO: 60, and the light chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence as SEQ ID NO: 60 The sequence shown in NO:62.
  • the heavy chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence as set forth in SEQ ID NO:61
  • the light chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence as SEQ ID NO: 61 The sequence shown in NO:62.
  • the antibody or antigen-binding fragment comprises a heavy chain comprising a heavy chain variable region and a heavy chain constant region described herein and/or a light chain comprising a light chain described herein chain variable region and light chain constant region.
  • the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 65 or 66, or a sequence that is at least 80% identical to the sequence set forth in SEQ ID NO: 65 or 66, or An amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 65 or 66; and/or
  • the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 67 or 68, or a sequence with at least 80% identity to the sequence shown in SEQ ID NO: 67 or 68, or with SEQ ID NO: 67 or amino acid sequence with one or more conservative amino acid substitutions compared to the sequence shown in 68.
  • the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 65 or 66, and/or the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 67 or 68 the sequence of.
  • the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:65 and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:67. In some embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:66 and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:68.
  • the antibody comprises 2 heavy chains with the same sequence and 2 light chains with the same sequence.
  • the antibody or antigen-binding fragment can specifically bind to spike protein and block the binding of SARS-CoV or SARS-CoV-2 viral particles to cells, as well as mediate immune cell phagocytosis and clearance of viral particles. In some embodiments, the antibody or antigen-binding fragment can specifically bind to the S1 subunit of the spike protein.
  • the present invention also provides fusion proteins comprising antibodies or antigen-binding fragments that can specifically bind to spike proteins, such as the antibodies or antigen-binding fragments described herein, and 6-HB interfering polypeptides that bind to the antibodies or antigen-binding fragments.
  • the antibodies or antigen-binding fragments in the fusion protein have high affinity for the spike proteins of SARS-CoV and SARS-CoV-2, and these antibodies or antigen-binding fragments can specifically bind to the spike proteins; Helical bundle (6-HB)" formation.
  • the fusion protein of the present invention can prevent the fusion of virus particles and cell membranes and can mediate the phagocytosis and removal of virus particles by immune cells. These fusion proteins are used to treat or ameliorate SARS and COVID-19, as well as to diagnose SARS and COVID-19.
  • the virus particle first interacts with a receptor-binding domain (RBD) in the S1 subunit of the spike protein (S protein or spike protein) on its surface and an enzyme called angiotensin-converting enzyme 2 (ACE2) on the surface of lung epithelial cells. combine. After the virus binds to the receptor and is hydrolyzed by protease, the S2 subunit located at the N-terminus of the S protein is exposed and embedded in the serosa or endosome membrane.
  • RBD receptor-binding domain
  • ACE2 angiotensin-converting enzyme 2
  • the HR2 (heptad repeated 2) domain in the S2 subunit combines with the HR1 (heptadrepeated 1) domain in the S2 subunit to form the 6-HB fusion core, resulting in fusion of the viral coat with the cell membrane, SARS-CoV or SARS-CoV- 2 Enter the cell, and use the cell to synthesize new virus particles for it; the new virus particles are released outside the cell, and then use the same method to infect surrounding normal cells.
  • the antibody in the fusion protein can block the combination of the spike protein and ACE2, and the 6-HB interfering polypeptide in the fusion protein prevents the fusion of the virus shell and the cell membrane, thereby blocking the virus from entering the cell and exerting an antiviral effect; the antibody part of the fusion protein also It can mediate the phagocytosis and clearance of viruses by immune cells.
  • the fusion protein comprises an antibody or antigen-binding fragment that specifically binds a spike protein and a 6-HB interfering polypeptide, and comprises: (a) HCDR1 comprising as SEQ ID NO: The amino acid sequence shown in 1 or 2, or a variant with a single site substitution, deletion or insertion; (b) HCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 3 or 4, or a single site Variants of site substitution, deletion or insertion; and/or (c) HCDR3 comprising the amino acid sequence shown in any one of SEQ ID NOs: 5-42, or a single site substitution, deletion or insertion thereof Variants;
  • At least one C-terminus or N-terminus of the antibody or antigen-binding fragment is linked to the 6-HB interfering polypeptide through a linker.
  • the linker is a polypeptide comprising glycine and serine.
  • the fusion protein comprises an antibody or antigen-binding fragment that specifically binds a spike protein and a 6-HB interfering polypeptide, and comprises: (a) HCDR1 comprising as SEQ ID NO: The amino acid sequence shown in 1 or 2, or a variant with a single site substitution, deletion or insertion; (b) HCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 3 or 4, or a single site Variants of site substitution, deletion or insertion; and (c) HCDR3 comprising the amino acid sequence shown in any one of SEQ ID NOs: 5-42, or a variant thereof with a single site substitution, deletion or insertion ;
  • At least one C-terminus or N-terminus of the antibody or antigen-binding fragment is linked to the 6-HB interfering polypeptide through a linker.
  • the linker is a polypeptide comprising glycine and serine.
  • the fusion protein comprises an antibody or antigen-binding fragment that specifically binds a spike protein and a 6-HB interfering polypeptide, and comprises:
  • HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1 or 2
  • HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 3 or 4
  • HCDR3 comprising The amino acid sequence shown in any one of SEQ ID NOs: 5-42;
  • At least one C-terminus or N-terminus of the antibody or antigen-binding fragment is linked to the 6-HB interfering polypeptide via a linker.
  • the linker is a polypeptide comprising glycine and serine.
  • the fusion protein comprises an antibody or antigen-binding fragment that specifically binds a spike protein and a 6-HB interfering polypeptide, and comprises: (a) HCDR1 comprising as SEQ ID NO: The amino acid sequence shown in 1 or 2, or a variant with a single site substitution, deletion or insertion; (b) HCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 3 or 4, or a single site A variant of site substitution, deletion or insertion; (c) HCDR3 comprising the amino acid sequence shown in any one of SEQ ID NOs: 5-42, or a variant thereof with a single site substitution, deletion or insertion; (d) LCDR1 comprising an amino acid sequence as set forth in SEQ ID NO: 43 or 44, or a variant thereof having a single site substitution, deletion or insertion; (e) LCDR2 comprising an amino acid sequence as set forth in SEQ ID NO: 45 or The amino acid sequence shown in 46, or a variant thereof with a single site substitution, deletion or insertion
  • At least one C-terminus or N-terminus of the antibody or antigen-binding fragment is linked to the 6-HB interfering polypeptide through a linker.
  • the linker is a polypeptide comprising glycine and serine.
  • the fusion protein comprises an antibody or antigen-binding fragment that specifically binds a spike protein and a 6-HB interfering polypeptide, and comprises: (a) HCDR1 comprising as SEQ ID NO : the amino acid sequence shown in 1 or 2, or a variant thereof with a single site substitution, deletion or insertion; (b) HCDR2, which comprises the amino acid sequence shown in SEQ ID NO: 3 or 4, or a variant with a single site substitution, deletion or insertion; A variant of site substitution, deletion or insertion; (c) HCDR3 comprising the amino acid sequence shown in any one of SEQ ID NOs: 5-42, or a variant thereof with a single site substitution, deletion or insertion (d) LCDR1, which comprises the amino acid sequence shown in SEQ ID NO:43 or 44, or a variant thereof with a single site substitution, deletion or insertion; (e) LCDR2, which comprises as SEQ ID NO:45 or the amino acid sequence shown in 46, or a variant thereof with a single site substitution, deletion
  • At least one C-terminus or N-terminus of the antibody or antigen-binding fragment is linked to the 6-HB interfering polypeptide through a linker.
  • the linker is a polypeptide comprising glycine and serine.
  • the substitution variant is a conservative amino acid substitution variant.
  • the sequence of the linker is (GmS) n , wherein each m is independently 2, 3, 4, or 5, and n is independently 1, 2, 3, 4, or 5. In some embodiments, the sequence of the linker is (GGGGS) n , where n is independently 1, 2, 3, 4, or 5. In some embodiments, the linker is GGGGS. In some embodiments, the linker is (GGGGS) 2 . In some embodiments, the linker is (GGGGS) 3 . In some embodiments, the linker is (GGGGS) 4 , as set forth in SEQ ID NO:64. In some embodiments, the linker is (GGGGS) 5 .
  • the 6-HB interfering polypeptide comprises the sequence set forth in SEQ ID NO:63, or a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO:63, or the sequence set forth in SEQ ID NO:63
  • the sequences shown at 63 are compared to amino acid sequences with one or more conservative amino acid substitutions.
  • the fusion protein comprises an antibody or antigen-binding fragment and a 6-HB interfering polypeptide, and the fusion protein comprises the following characteristics:
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO: 1 or 2, HCDR2 as set forth in SEQ ID NO: 3 or 4, as set forth in any of SEQ ID NO: 5-42 HCDR3, LCDR1 as SEQ ID NO:43 or 44, LCDR2 as SEQ ID NO:45 or 46 and LCDR3 as SEQ ID NO:47 or 48, one, two, three, four, five or all; and/or
  • At least one C-terminus of the antibody or antigen-binding fragment is connected to the 6-HB interfering polypeptide through a linker, and the C-terminus is the C-terminus of the heavy chain portion or the C-terminus of the light chain portion of the antibody or antigen-binding fragment. - terminal; and/or
  • the sequence of the linker is (GGGGS) n , and the n is independently 1, 2, 3, 4 or 5; and/or
  • the 6-HB interfering polypeptide comprises the sequence shown in SEQ ID NO:63, or a sequence with at least 90% identity to the sequence shown in SEQ ID NO:63, or compared with the sequence shown in SEQ ID NO:63 An amino acid sequence with one or more conservative amino acid substitutions.
  • the fusion protein comprises an antibody or antigen-binding fragment and a 6-HB interfering polypeptide, and the fusion protein comprises the following characteristics:
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO: 1 or 2, HCDR2 as set forth in SEQ ID NO: 3 or 4, as set forth in any of SEQ ID NO: 5-42 HCDR3, LCDR1 as SEQ ID NO:43 or 44, LCDR2 as SEQ ID NO:45 or 46 and LCDR3 as SEQ ID NO:47 or 48; and/or
  • At least one C-terminus of the antibody or antigen-binding fragment is connected to the 6-HB interfering polypeptide through a linker, and the C-terminus is the C-terminus of the heavy chain portion or the C-terminus of the light chain portion of the antibody or antigen-binding fragment. - terminal; and/or
  • the sequence of the linker is (GGGGS) n , and the n is independently 1, 2, 3, 4 or 5; and/or
  • the 6-HB interfering polypeptide comprises the sequence shown in SEQ ID NO:63, or a sequence with at least 90% identity to the sequence shown in SEQ ID NO:63, or compared with the sequence shown in SEQ ID NO:63 An amino acid sequence with one or more conservative amino acid substitutions.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:5, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:6, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:7, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:8, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:9, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:10, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:11, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:12, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:13, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:14, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:15, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:16, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:17, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:18, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:19, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:20, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:21, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:22, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:23, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:24, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:25, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:26, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:27, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:28, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:29, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:30, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:31, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:32, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:33, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:34, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:35, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:36, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:37, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:38, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:39, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:40, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:3, HCDR3 as set forth in SEQ ID NO:41, LCDR1 shown in ID NO:43, LCDR2 shown in SEQ ID NO:45, and LCDR3 shown in SEQ ID NO:47.
  • the antibody or antigen-binding fragment comprises at least HCDR1 as set forth in SEQ ID NO:2, HCDR2 as set forth in SEQ ID NO:4, HCDR3 as set forth in SEQ ID NO:42, LCDR1 shown in ID NO:44, LCDR2 shown in SEQ ID NO:46 and LCDR3 shown in SEQ ID NO:48.
  • the antibody or antigen-binding fragment comprises a heavy chain variable region, and a light chain variable region.
  • the framework regions of the heavy chain variable region comprise heavy chain FR1, heavy chain FR2, heavy chain FR3, and heavy chain FR4.
  • the heavy chain FR1 comprises the sequence set forth in SEQ ID NO: 49 or 50, or a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 49 or 50, or the sequence set forth in SEQ ID NO: 49 or 50 :
  • the sequence shown at 49 or 50 is compared to the amino acid sequence with one or more conservative amino acid substitutions.
  • the heavy chain FR2 comprises the sequence set forth in SEQ ID NO: 51 or 52, or a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 51 or 52, or the sequence set forth in SEQ ID NO: 51 or 52 :
  • the sequence shown in 51 or 52 is compared to the amino acid sequence with one or more conservative amino acid substitutions.
  • the heavy chain FR3 comprises the sequence set forth in SEQ ID NO: 53 or 54, or a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 53 or 54, or the sequence set forth in SEQ ID NO: 53 or 54 :
  • the sequence shown in 53 or 54 is compared to the amino acid sequence with one or more conservative amino acid substitutions.
  • the heavy chain FR4 comprises the sequence set forth in SEQ ID NO:55, or a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO:55, or the sequence set forth in SEQ ID NO:55 Sequences are compared to amino acid sequences with one or more conservative amino acid substitutions.
  • the heavy chain FR1 comprises the sequence set forth in SEQ ID NO: 49 or 50, or a sequence that is at least 90% identical to the sequence set forth in SEQ ID NO: 49 or 50, or the sequence set forth in SEQ ID NO: 49 or 50 : an amino acid sequence with one or more conservative amino acid substitutions compared to the sequence shown in 49 or 50;
  • the heavy chain FR2 comprises the sequence shown in SEQ ID NO: 51 or 52, or with the sequence shown in SEQ ID NO: 51 or 52 A sequence having at least 90% identity, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 51 or 52;
  • the heavy chain FR3 comprises the sequence shown in SEQ ID NO: 53 or 54 The sequence shown, or a sequence with at least 90% identity to the sequence shown in SEQ ID NO: 53 or 54, or an amino acid sequence with one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 53 or 54
  • the heavy chain FR4 comprises the sequence shown in SEQ ID NO
  • the heavy chain FR1 comprises the sequence set forth in SEQ ID NO:49
  • the heavy chain FR2 comprises the sequence set forth in SEQ ID NO:51
  • the heavy chain FR3 comprises the sequence set forth in SEQ ID NO:53
  • the sequence shown, the heavy chain FR4 comprises the sequence shown in SEQ ID NO:55.
  • the heavy chain FR1 comprises the sequence set forth in SEQ ID NO:50
  • the heavy chain FR2 comprises the sequence set forth in SEQ ID NO:52
  • the heavy chain FR3 comprises the sequence set forth in SEQ ID NO:54
  • the heavy chain FR4 comprises the sequence shown in SEQ ID NO:55.
  • the heavy chain variable region comprises the structure heavy chain FR1-HCDR1-heavy chainFR2-HCDR2-heavy chainFR3-HCDR3-heavy chainFR4.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises heavy chain FRl as set forth in SEQ ID NO:49, HCDRl as set forth in SEQ ID NO:1, as SEQ ID NO:51 Heavy chain FR2 as shown, HCDR2 as shown in SEQ ID NO:3, heavy chain FR3 as shown in SEQ ID NO:53, HCDR3 as shown in any one of SEQ ID NOs:5-41 and as shown in SEQ ID NO:53 Heavy chain FR4 shown in ID NO:55.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises heavy chain FRl as set forth in SEQ ID NO:50, HCDRl as set forth in SEQ ID NO:2, as in SEQ ID NO:52 Heavy chain FR2 as shown, HCDR2 as shown in SEQ ID NO:4, heavy chain FR3 as shown in SEQ ID NO:54, HCDR3 as shown in SEQ ID NO:42 and as shown in SEQ ID NO:55 heavy chain FR4.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 56 or 57, or is at least 80% identical to the sequence set forth in SEQ ID NO: 56 or 57 , or an amino acid sequence with one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 56 or 57.
  • the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO: 58 or 59, or is at least 80% identical to the sequence set forth in SEQ ID NO: 58 or 59 , or an amino acid sequence with one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 58 or 59.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:56
  • the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:58 the sequence shown.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:57
  • the light chain variable region of the antibody or antigen-binding fragment comprises the sequence set forth in SEQ ID NO:59 the sequence shown.
  • the antibody or antigen-binding fragment further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.
  • the light chain constant region is a kappa or lambda chain constant region.
  • the antibody or fragment thereof is one of the isotypes of IgG, IgM, IgA, IgE, or IgD.
  • the isotype is IgGl, IgG2, IgG3, or IgG4.
  • the C-terminus of the heavy chain constant region in the antibody or antigen-binding fragment is truncated.
  • the amino acid residues G and K are missing from the C-terminus of the heavy chain constant region in an IgGl or IgG4 type antibody.
  • the antibody or antigen-binding fragment is a chimeric antibody, a humanized antibody, or a fully human antibody. In one aspect, the antibody or antigen-binding fragment is a humanized antibody.
  • the antibody or antigen-binding fragment is an isolated antibody or antigen-binding fragment. In some embodiments, the antibody or antigen-binding fragment is scFV, Fab, F(ab) 2 , or IgG. In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody.
  • the antibody or antigen-binding fragment comprises a heavy chain comprising a heavy chain variable region and a heavy chain constant region described herein, and a light chain comprising a light chain variable region described herein Variable and light chain constant regions.
  • the heavy chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth as amino acid 1 to amino acid 328 in SEQ ID NO: 60 or 61, or the same as SEQ ID NO: A sequence that is at least 80% identical to the sequence shown in amino acid 1 to amino acid 328 in 60 or 61, or compared to the sequence shown in amino acid 1 to amino acid 328 in SEQ ID NO: 60 or 61 amino acid sequence with one or more conservative amino acid substitutions; and/or
  • the light chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence of the sequence shown in SEQ ID NO: 62, or a sequence with at least 80% identity to the sequence shown in SEQ ID NO: 62, or a sequence with SEQ ID NO: 62 :
  • the sequence shown in 62 is compared to the amino acid sequence with one or more conservative amino acid substitutions.
  • the heavy chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in amino acid 1 to amino acid 328 in SEQ ID NO: 60, and the antibody or antigen-binding fragment has a
  • the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:62.
  • the heavy chain constant region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in amino acid 1 to amino acid 328 of SEQ ID NO: 61, and the antibody or antigen-binding fragment has a
  • the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:62.
  • the C-terminus or N-terminus of the heavy chain or fragment thereof is linked to the 6-HB interfering polypeptide by a linker. In some embodiments, the C-terminus of the light chain or fragment thereof is linked to the 6-HB interfering polypeptide via a linker.
  • fusion protein comprising an antibody and a 6-HB interfering polypeptide, the fusion protein comprising the following characteristics:
  • the heavy chain of the antibody comprises the amino acid sequence as shown in amino acid 1 to amino acid 450 in SEQ ID NO: 65 or 66 or as shown in amino acid 1 to amino acid 451 in SEQ ID NO: 66 , or with at least 80% of the sequence shown in amino acids 1 to 450 in SEQ ID NO: 65 or 66 or the sequence shown in amino acids 1 to 451 in SEQ ID NO: 66 A sequence of identity, or the sequence shown in amino acid 1 to amino acid 450 in SEQ ID NO: 65 or 66 or the sequence shown in amino acid 1 to amino acid 451 in SEQ ID NO: 66 an amino acid sequence with one or more conservative amino acid substitutions; and/or
  • the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 67 or 68, or a sequence with at least 80% identity to the sequence shown in SEQ ID NO: 67 or 68, or with SEQ ID NO: 67 Or an amino acid sequence with one or more conservative amino acid substitutions compared to the sequence shown in 68; and/or
  • the C-terminus of the heavy chain or the C-terminus of the light chain of the antibody is covalently linked to a 6-HB interfering polypeptide comprising the SEQ ID NO: 64 linker shown in SEQ ID NO:
  • the sequence shown in 63 or a sequence with at least 90% identity or at least 95% identity with the sequence shown in SEQ ID NO: 63, or one or more conserved amino acids compared with the sequence shown in SEQ ID NO: 63 Substituted amino acid sequence.
  • the fusion protein comprises an antibody and a 6-HB interfering polypeptide linked by a linker, the heavy chain of the antibody comprising the amino acid sequence as shown in amino acids 1 to 450 of SEQ ID NO:65
  • the sequence shown, the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 67;
  • the linker shown in SEQ ID NO:64 is covalently linked to a 6-HB interfering polypeptide comprising the sequence shown in SEQ ID NO:63.
  • the fusion protein comprises an antibody and a 6-HB interfering polypeptide linked by a linker, the heavy chain of the antibody comprising the amino acid sequence as shown in amino acids 1 to 451 of SEQ ID NO:66
  • the sequence shown, the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 68;
  • the linker shown in SEQ ID NO:64 is covalently linked to a 6-HB interfering polypeptide comprising the sequence shown in SEQ ID NO:63.
  • the fusion protein comprises two first polypeptides with the same sequence and two fourth polypeptides with the same sequence.
  • the first polypeptide comprises or consists of a heavy chain of an antibody or antigen-binding fragment or a fragment thereof and the fourth polypeptide comprises a light chain of an antibody or antigen-binding fragment or a fragment thereof and its A linker, a 6-HB interfering polypeptide, or consisting of a linked linker in sequence.
  • the fusion protein comprises two second polypeptides that are identical in sequence and two third polypeptides that are identical in sequence.
  • the third polypeptide comprises or consists of a heavy chain of an antibody or antigen-binding fragment or a fragment thereof and a linker, a 6-HB interfering polypeptide in turn attached thereto, and the second polypeptide comprises A light chain or a fragment thereof, or consisting of it.
  • the fusion protein comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 69; the fusion protein further comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 67.
  • the antibody portion of the fusion protein can specifically bind to the spike protein and prevent the binding of SARS-CoV or SARS-CoV-2 viral particles to cells, as well as mediate immune cell phagocytosis and clearance of viral particles.
  • the antibody or antigen-binding fragment of the fusion protein can specifically bind to the S1 subunit of the spike protein.
  • the present invention also provides nucleic acids encoding the antibodies, antigen-binding fragments or fusion proteins thereof.
  • the nucleic acid is an isolated nucleic acid.
  • the present invention also provides a vector comprising the nucleic acid.
  • the vector is an isolated vector.
  • the vector comprising the nucleic acid is a nucleic acid fragment, plasmid, phage or virus.
  • the vector is an isolated plasmid.
  • the present invention also provides host cells comprising the nucleic acid or the vector.
  • the host cell is an isolated host cell.
  • the host cells are CHO cells, HKE293 cells, Cos1 cells, Cos7 cells, CV1 cells, and murine L cells.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody, antigen-binding fragment or fusion protein, and a pharmaceutically acceptable adjuvant.
  • the present invention also provides methods and uses of treatment.
  • a method for preventing, treating or ameliorating SARS or COVID-19 is provided, the method comprising administering to a patient an effective dose of the antibody, antigen-binding fragment or fusion protein.
  • use of the antibody, antigen-binding fragment or fusion protein or pharmaceutical composition for preventing, treating or ameliorating SARS or COVID-19 is provided.
  • use of the antibody, antigen-binding fragment or fusion protein or pharmaceutical composition in the manufacture of a medicament for preventing, treating or ameliorating SARS or COVID-19 is provided.
  • the present invention also provides diagnostic methods and uses.
  • methods are provided for detecting SARS-CoV or SARS-CoV-2 expression in a sample, contacting the sample with the antibody, antigen-binding fragment or fusion protein such that the antibody, antigen-binding fragment or fusion
  • the antibody in the protein binds to the spike protein, and its binding is detected, that is, the content of the spike protein in the sample.
  • use of the antibody, antigen-binding fragment or fusion protein in the preparation of a kit for diagnosing SARS or COVID-19 is provided.
  • diagnostic kits comprising the antibodies, antigen-binding fragments or fusion proteins are provided.
  • the invention provides a coronavirus antibody and application thereof.
  • the 6-HB interfering polypeptide in the fusion protein and the antibody cooperate to prevent the fusion of SARS-CoV or SARS-CoV-2 virus particles and cells, as well as mediate immune cells to phagocytose and remove virus particles, preventing , treatment of SARS or COVID-19;
  • the antibodies in the antibodies, antigen-binding fragments or fusion proteins of the present invention can also be used to diagnose and detect whether a patient is infected with SARS-CoV or SARS-CoV-2.
  • Figure 1 shows that the antibody blocks the binding of spike RBD to ACE2; the ACE2 control group has no antibody added.
  • an entity refers to one or more of such entities, eg "an antibody” should be understood to mean one or more antibodies, thus the term “an” (or “an” ), “one or more” and “at least one” are used interchangeably herein.
  • compositions, methods and the like include the recited elements, such as components or steps, but do not exclude others.
  • Consisting essentially of means that the compositions and methods exclude other elements that have an essential effect on the characteristics of the combination, but do not exclude elements that have no essential effect on the compositions or methods.
  • Consisting of means excluding elements not specifically recited.
  • polypeptide is intended to encompass the singular “polypeptide” as well as the plural “polypeptide”, and refers to a molecule composed of amino acid monomers linked linearly by amide bonds (also known as peptide bonds).
  • polypeptide refers to any single chain or chains of two or more amino acids, and does not refer to a particular length of the product.
  • the definition of “polypeptide” includes a peptide, dipeptide, tripeptide, oligopeptide, "protein”, “amino acid chain” or any other term used to refer to two or more amino acid chains, and the term “polypeptide” may Used in place of, or used interchangeably with, any of the above terms.
  • polypeptide is also intended to refer to the product of post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage or non-native Amino acid modifications that occur.
  • a polypeptide may be derived from a natural biological source or produced by recombinant techniques, but it need not be translated from a given nucleic acid sequence, and it may be produced by any means including chemical synthesis.
  • Amino acid refers to an organic compound containing both an amino group and a carboxyl group, such as an alpha-amino acid, which can be encoded by a nucleic acid directly or in a precursor form.
  • a single amino acid is encoded by a nucleic acid consisting of three nucleotides, so-called codons or base triplets. Each amino acid is encoded by at least one codon. The same amino acid is encoded by different codons called “degeneracy of the genetic code”.
  • Amino acids include natural amino acids and unnatural amino acids.
  • Natural amino acids include alanine (three-letter code: ala, one-letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine Amino acid (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I) ), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y) and valine (val, V).
  • alanine three-letter code: ala, one-letter code: A
  • arginine arg, R
  • asparagine asparag
  • Constant amino acid substitution refers to the replacement of one amino acid residue by another amino acid residue containing a side chain (R group) of similar chemical properties (eg, charge or hydrophobicity). In general, conservative amino acid substitutions will not substantially alter the functional properties of the protein.
  • amino acid classes containing chemically similar side chains include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine 3) Amide-containing side chains: asparagine and glutamine; 4) Aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) Basic side chains: lysine, Arginine and histidine; 6) Acidic side chains: aspartic acid and glutamic acid.
  • the number of amino acids in "conservative amino acid substitutions of VL and VH" is about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15 conservative amino acid substitutions, or a range (including endpoints) between any two of these values, or any value therein.
  • the number of amino acids in the "heavy chain constant region, light chain constant region, heavy or light chain, conservative amino acid substitutions of the first polypeptide or the second polypeptide of the fusion protein" is about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15, about 18, about 19, about 22 , about 24, about 25, about 29, about 31, about 35, about 38, about 41, about 45 conservative amino acid substitutions, or a range between any two of these values (including endpoints) or any value in it.
  • isolated refers to other components in the cell's natural environment, such as DNA or RNA, respectively of one or more of the isolated molecules.
  • isolated refers to a nucleic acid or peptide that is substantially free of cellular material, viral material or cell culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • isolated nucleic acid is intended to include nucleic acid fragments that do not, and would not exist in, their natural state.
  • isolated is also used herein to refer to cells or polypeptides that are separated from other cellular proteins or tissues. Isolated polypeptides are intended to include purified and recombinant polypeptides. Isolated polypeptides, antibodies, etc. are typically prepared by at least one purification step. In some embodiments, the isolated nucleic acid, polypeptide, antibody, etc. is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99% pure, or any of these values The range between any two values of , including the endpoint, or any value therein.
  • recombinant refers to a polypeptide or polynucleotide and means a form of the polypeptide or polynucleotide that does not occur in nature, non-limiting examples may be combined to produce polynucleotides that do not normally exist or peptide.
  • Homology refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions within each sequence that can be aligned. A molecule is homologous when a position in the sequences being compared is occupied by the same base or amino acid. The degree of homology between sequences is a function of the number of matches or homologous positions shared by the sequences.
  • At least 80% identity is about 80% identity, about 81% identity, about 82% identity, about 83% identity, about 85% identity, about 86% identity, about 87% identity, about 88% identity, about 90% identity, about 91% identity, about 92% identity, about 94% identity, about 95% identity, about 98% identity, about 99% identity, or these A range (including endpoints) between any two values in a numerical value or any value therein.
  • At least 90% identity is about 90% identity, about 91% identity, about 92% identity, about 93% identity, about 94% identity, about 95% identity, about 92% identity, About 96% identity, about 99% identity, or a range (including endpoints) between any two of these values or any value therein.
  • a polynucleotide or polynucleotide sequence (or polypeptide or antibody sequence) has a certain percentage (eg, 90%, 95%, 98% or 99%) "identity” or “sequence identity” to another sequence "Sex” means that when the sequences are aligned, the percentage of bases (or amino acids) in the two sequences being compared are identical.
  • the percent alignment or sequence identity can be determined using visual inspection or software programs known in the art, such as those described by Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology. Alignments are preferably performed using default parameters.
  • Biologically equivalent polynucleotides are polynucleotides that have the above-specified percentages of identity and encode polypeptides having the same or similar biological activity.
  • a polynucleotide is a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), or when polynucleotides For RNA, thymine is replaced by uracil (U).
  • a "polynucleotide sequence” can be represented by the letters of the polynucleotide molecule. This letter representation can be entered into a database in a computer with a central processing unit and used for bioinformatics applications, eg for functional genomics and homology searches.
  • polynucleotide and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown.
  • polynucleotides genes or gene fragments (eg probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribose Somatic RNA, ribozyme, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
  • Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • nucleotides can be made before or after assembly of the polynucleotide.
  • sequence of nucleotides can be interrupted by non-nucleotide components.
  • the polynucleotide can be further modified after polymerization, for example by conjugation to a labeling component.
  • the term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, embodiments of any polynucleotide of the present disclosure include the double-stranded form and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.
  • encoding when applied to a polynucleotide refers to a polynucleotide referred to as “encoding” a polypeptide which, in its natural state or when manipulated by methods well known to those skilled in the art, can be produced by transcription and/or translation the polypeptide and/or fragments thereof.
  • Antibody refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen.
  • Antibodies can be whole antibodies and any antigen-binding fragments thereof or single chains thereof.
  • the term “antibody” thus includes any protein or peptide in the molecule that contains at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen.
  • Antibodies and antigen-binding fragments include, but are not limited to, the complementarity determining regions (CDRs), heavy chain variable regions (VH), and light chain variable regions (VL) of heavy or light chains or their ligand-binding portions as described in the Examples , a heavy chain constant region (CH), a light chain constant region (CL), a framework region (FR) or any portion thereof, or at least a portion of a binding protein.
  • the CDR regions include the CDR regions of the light chain (LCDR1-3) and the CDR regions of the heavy chain (HCDR1-3).
  • the variable region may comprise the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • antibody fragment refers to a portion of an antibody, eg, F(ab') 2 , F(ab) 2 , Fab', Fab, Fv, scFv, and the like. Regardless of their structure, antibody fragments bind to the same antigen that is recognized by the intact antibody.
  • antibody fragment includes aptamers, Spiegelmers and diabodies.
  • antiigen-binding fragment also includes any synthetic or genetically engineered protein that functions as an antibody by binding to a specific antigen to form a complex.
  • Single-chain variable fragment refers to a fusion protein of the heavy chain variable region (VH) and light chain variable region (VL) of an immunoglobulin. In some aspects, these regions are linked to short linker peptides of 10 to about 25 amino acids. Linkers can be rich in glycine to increase flexibility, and serine or threonine to increase solubility, and can link the N-terminus of VH and the C-terminus of VL, and vice versa. Although the constant region has been removed and the linker introduced, the protein retains the specificity of the original immunoglobulin. ScFv molecules are generally known in the art and are described, for example, in US Pat. No. 5,892,019.
  • antibody includes a wide variety of biochemically distinguishable polypeptides. Those of skill in the art will appreciate that classes of heavy chains include gamma, mu, alpha, delta, or epsilon (gamma, mu, alpha, delta, epsilon), with some subclasses (eg, gamma1-gamma4). The nature of this chain determines the "class” of the antibody as IgG, IgM, IgA, IgG or IgE, respectively. Immunoglobulin subclasses (isotypes), eg, IgGl, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and the functional specificities conferred are known.
  • the immunoglobulin molecule is of the IgG class.
  • IgG typically comprises two identical light chain polypeptides of molecular weight about 23,000 Daltons and two identical heavy chain polypeptides of molecular weight about 53,000-70,000.
  • the four chains are connected by disulfide bonds in a "Y" configuration, where the light chain begins at the "Y" mouth and continues through the variable region surrounding the heavy chain.
  • Antibodies, antigen-binding fragments, or derivatives disclosed herein include, but are not limited to, polyclonal, monoclonal, multispecific, fully human, humanized, primatized, chimeric antibodies, single-chain antibodies, epitopes Binding fragments such as Fab, Fab' and F(ab') 2 , Fd, Fvs, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), fragments comprising VK or VH domains, or expression libraries from Fab Fragments and anti-idiotype (anti-Id) antibodies produced.
  • Binding fragments such as Fab, Fab' and F(ab') 2 , Fd, Fvs, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), fragments comprising VK or VH domains, or expression libraries from Fab Fragments and anti-idiotype (anti-Id) antibodies produced.
  • the immunoglobulin or antibody molecules disclosed herein can be of any type (eg, IgG, IgE, IgM, IgD, IgA, and IgY) or class (eg, IgGl, IgG2, IgG3, IgG4, IgA1, and IgA2) or class of immunoglobulins, or subclass.
  • Light chains can be classified as kappa ( ⁇ ) or lambda ( ⁇ ). Each heavy chain can bind to a kappa or lambda light chain.
  • ⁇ kappa
  • ⁇ lambda
  • Each heavy chain can bind to a kappa or lambda light chain.
  • immunoglobulins are produced by hybridomas, B cells or genetically engineered host cells, their light and heavy chains are joined by covalent bonds, and the "tail" portion of the two heavy chains is joined by a covalent disulfide bond or non-covalent bond.
  • the amino acid sequence extends from the N-terminus of the forked terminus in the Y configuration to the C-terminus at the bottom of each chain.
  • the variable region of immunoglobulin kappa light chain is V ⁇ ; the variable region of immunoglobulin lambda light chain is V ⁇ .
  • Both light and heavy chains are divided into regions of structural and functional homology.
  • the terms "constant” and “variable” are used according to function.
  • the light chain variable region (VL) and heavy chain variable region (VH) determine antigen recognition and specificity.
  • the constant regions of the light and heavy chains confer important biological properties such as secretion, transplacental movement, Fc receptor binding, complement fixation, and the like. By convention, the numbering of constant regions increases as they become further from the antigen binding site or amino terminus of the antibody.
  • the N-terminal portion is the variable region and the C-terminal portion is the constant region; the CH3 and CL domains actually comprise the carboxy-terminus of the heavy and light chains, respectively.
  • variable regions enable antibodies to selectively recognize and specifically bind epitopes on an antigen.
  • a subset of the VL and VH domains or complementarity determining regions (CDRs) of an antibody combine to form variable regions that define a three-dimensional antigen-binding site.
  • This antibody quaternary structure forms the antigen binding site present at the end of each arm of Y. More specifically, the antigen binding site is defined by three CDRs (ie, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) in the VH and VL chains, respectively.
  • the complete immunoglobulin molecule may consist of only heavy chains and no light chains . See, eg, Hamers-Casterman et al., Nature, 363:446-448 (1993).
  • each antigen-binding domain In naturally occurring antibodies, the six “complementarity determining regions" or “CDRs” present in each antigen-binding domain are the short, A non-contiguous amino acid sequence that specifically binds an antigen. The remaining other amino acids in the antigen binding domain, referred to as the "framework” region, show less inter-molecular variability.
  • the framework regions mostly adopt a ⁇ -sheet conformation, and the CDRs form loop structures to which they are attached, or in some cases form part of a ⁇ -sheet structure. Thus, the framework regions position the CDRs in the correct orientation by forming a scaffold through non-covalent interchain interactions.
  • An antigen binding domain with CDRs at specific positions forms a surface complementary to an epitope on an antigen that facilitates non-covalent binding of the antibody to its antigenic epitope.
  • an antigen binding domain with CDRs at specific positions forms a surface complementary to an epitope on an antigen that facilitates non-covalent binding of the antibody to its antigenic epitope.
  • amino acids comprising CDRs and framework regions can be identified by known methods (see Kabat, E., et al., U.S. Department of Health and Human Services, Sequences of Proteins of Immunological Interest, (1983) and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).
  • CDR complementarity determining region
  • CDRs as defined by Kabat and Chothia include overlaps or subsets of amino acid residues when compared to each other. Nonetheless, it is within the scope of the invention to apply either definition to refer to the CDRs of an antibody or variant thereof.
  • the exact residue numbers encompassing a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can usually determine which specific residues the CDRs contain based on the amino acid sequence of the variable region of the antibody.
  • Kabat et al. also define a numbering system applicable to variable region sequences of any antibody.
  • One of ordinary skill in the art can apply this "Kabat numbering" system to any variable region sequence independent of experimental data other than the sequence itself.
  • Kabat Numbering means the numbering system proposed by Kabat et al., U.S. Dept. of Health and Human Services in "Sequence of Proteins of Immunological Interest” (1983).
  • Antibodies may also use the EU numbering system.
  • the antibodies or antigen-binding fragments disclosed herein can be derived from any animal, including birds and mammals.
  • the antibody is of human, murine, donkey, rabbit, goat, camel, llama, horse or chicken origin.
  • the variable regions may be of condricthoid origin (eg, from sharks).
  • Heavy chain constant regions include amino acid sequences derived from immunoglobulin heavy chains.
  • a heavy chain constant region-containing polypeptide includes at least one of a CH1 domain, a hinge (eg, upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment.
  • the antibodies or antigen-binding fragments disclosed herein comprise a CH1 domain; comprise a CH1 domain, at least a portion of the hinge region and a CH2 domain; comprise a CH1 domain and a CH3 domain; comprise a CH1 domain and at least a portion of the hinge region and CH3 domain; or comprises a CH1 domain, at least a portion of the hinge region, and a CH2 domain and a CH3 domain.
  • the antibodies or antigen-binding fragments of the present disclosure comprise a CH3 domain.
  • the antibodies or antigen-binding fragments used in the present invention may lack part or all of the CH2 domain.
  • heavy chain constant regions can be modified so as to alter the amino acid sequence of their naturally occurring immunoglobulin molecules.
  • the heavy chain constant regions of antibodies can be derived from different immunoglobulin molecules.
  • the heavy chain constant region of a polypeptide can include a CH1 domain derived from an IgG 1 molecule and a hinge region derived from an IgG 3 molecule.
  • the heavy chain constant region may comprise a hinge region derived in part from an IgG 1 molecule and in part from an IgG 3 molecule.
  • a part of the heavy chain may comprise a chimeric hinge region derived in part from an IgG 1 molecule and in part from an IgG 4 molecule.
  • a “light chain constant region” includes the amino acid sequence from the light chain of an antibody.
  • the light chain constant region comprises at least one of a constant kappa domain or a constant lambda domain.
  • a “light chain-heavy chain pair” refers to a collection of light and heavy chains that can dimerize through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.
  • the "VH domain” includes the amino-terminal variable domain of an immunoglobulin heavy chain
  • the "CH1 domain” includes the first (mostly amino-terminal) constant region of an immunoglobulin heavy chain.
  • the CH1 domain is adjacent to the VH domain and is the amino terminus of the hinge region of the immunoglobulin heavy chain molecule.
  • the CH2 domains are not tightly paired with other domains, but rather two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the intact native IgG molecule.
  • the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and contains approximately 108 residues.
  • the "hinge region” includes a portion of the heavy chain region connecting the CH1 domain and the CH2 domain.
  • the hinge region comprises about 25 residues and is resilient, allowing the two N-terminal antigen binding regions to move independently.
  • the hinge region can be subdivided into three distinct domains: upper, middle and lower hinge domains (Roux et al., J. Immunol. 161:4083 (1998)).
  • Disulfide bond refers to a covalent bond formed between two sulfur atoms.
  • the thiol group of cysteine can form a disulfide bond or bridge with a second thiol group.
  • the CH1 and CL regions are connected by a disulfide bond, and the two heavy chains are connected by two disulfide bonds at positions 239 and 242 in the Kabat numbering system (EU numbering system positions 226 and 229) Connected everywhere.
  • Chimeric antibody refers to any antibody whose variable regions are obtained or derived from a first species and whose constant regions (which may be intact, partial or modified) are derived from a second species.
  • the variable regions are derived from a non-human source (eg, mouse or primate), and the constant regions are derived from a human source.
  • Specifically binds or “specifically for” generally refers to the formation of a relatively stable complex of an antibody or antigen-binding fragment with a specific antigen through complementary binding of its antigen-binding domain to an epitope.
  • Specificity can be expressed in terms of the relative affinity with which an antibody or antigen-binding fragment binds to a particular antigen or epitope. For example, if antibody “A” has a greater relative affinity for the same antigen than antibody "B”, antibody “A” can be considered to be more specific for that antigen than antibody "B”.
  • Specific binding can be described by the equilibrium dissociation constant (KD), a smaller KD means tighter binding.
  • KD equilibrium dissociation constant
  • An antibody that "specifically binds" a spike protein includes an equilibrium dissociation constant KD of less than or equal to about 100 nM, less than or equal to about 10 nM, less than or equal to about 5 nM, less than or equal to about 1 nM with the spike protein.
  • Treatment means both therapeutic treatment and prophylactic or prophylactic measures, the purpose of which is to prevent, slow, ameliorate, or stop an undesirable physiological change or disorder, such as the progression of a disease, including but not limited to the following whether detectable or undetectable As a result, remission of symptoms, reduction of disease severity, stabilization of disease state (ie, no worsening), delay or slowdown of disease progression, improvement, alleviation, alleviation or disappearance of disease state (whether in part or in whole), prolongation and Expected duration of survival when not receiving treatment, etc.
  • Patients in need of treatment include patients already suffering from a condition or disorder, a patient susceptible to a condition or disorder, or a patient in need of prevention of such a condition or disorder, which may or may be expected from administration of the antibodies or pharmaceutical compositions disclosed herein for detection , patients who benefit from the diagnostic process and/or treatment.
  • Patient refers to any mammal in need of diagnosis, prognosis, or treatment, including humans, dogs, cats, rabbits, rats, mice, horses, cattle, and the like.
  • the present invention provides antibodies or antigen-binding fragments thereof with high affinity to spike proteins.
  • Antibodies or antigen-binding fragments exhibit potent binding activity and can be used for therapeutic and diagnostic purposes.
  • these antibodies or antigen-binding fragments can prevent the fusion of SARS-CoV and SARS-CoV-2 viral particles and cell membranes, as well as mediate immune cell phagocytosis and clearance of viral particles.
  • the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 65 or 66, and/or the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 67 or 68 the sequence of.
  • the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:65 and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:67.
  • the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:65, excluding the Fc region, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:67.
  • the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:66 and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:68. In some embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:66, excluding the Fc region, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO:68.
  • the present invention also provides fusion proteins.
  • Fusion proteins comprise antibodies or antigen-binding fragments and 6-HB interfering polypeptides.
  • the antibodies or antigen-binding fragments in the fusion protein have high affinity for the spike proteins of SARS-CoV and SARS-CoV-2, and these antibodies or antigen-binding fragments can specifically bind to the spike proteins; Helical bundle (6-HB)" formation.
  • Fusion proteins exhibit potent binding activity and can be used for therapeutic and diagnostic applications.
  • these antibodies or antigen-binding fragments in the fusion protein can prevent the fusion of SARS-CoV and SARS-CoV-2 viral particles and cell membranes, and mediate immune cell phagocytosis and clearance of viral particles.
  • the C-terminus (ie, the CH3 terminus) of the heavy chain of the antibody is covalently linked to the 6-HB interfering polypeptide through a linker.
  • the light chain C-terminus (ie, the CL terminus) of the antibody is covalently linked to the 6-HB interfering polypeptide through a linker.
  • the fusion proteins disclosed herein comprise a 6-HB interfering polypeptide and an antibody or antigen-binding fragment, the C-terminus of the heavy chain (or heavy chain fragment) of the antibody or antigen-binding fragment or the C-terminus of the light chain passing through
  • the linker (as shown in SEQ ID NO:64) is covalently linked to the 6-HB interfering polypeptide (as shown in SEQ ID NO:63).
  • the fusion protein comprises two first polypeptides with the same sequence and two fourth polypeptides with the same sequence, the fourth polypeptide comprising the light chain of an antibody or antigen-binding fragment or a fragment thereof linked in sequence , linker and 6-HB interfering polypeptide.
  • the first polypeptide comprises or consists of a heavy chain of an antibody or antigen-binding fragment or a fragment thereof; the fourth polypeptide comprises a light chain of an antibody or antigen-binding fragment or a fragment thereof and its A linker, a 6-HB interfering polypeptide, or consisting of a linked linker in sequence.
  • the fusion protein comprises 2 second polypeptides with the same sequence and 2 third polypeptides with the same sequence, the third polypeptide comprising the heavy chain of an antibody or antigen-binding fragment or a fragment thereof linked in sequence , linker and 6-HB interfering polypeptide.
  • the third polypeptide comprises or consists of a heavy chain of an antibody or antigen-binding fragment or a fragment thereof and a linker, a 6-HB interfering polypeptide in turn attached thereto, and the second polypeptide comprises A light chain or a fragment thereof, or consisting of it.
  • the fusion protein comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 69; the fusion protein further comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 67.
  • the antibody, or fusion protein eg, wherein the antibody may also be linked to an amino acid sequence or one or more modifying groups.
  • the antibodies disclosed herein, or fusion proteins such as wherein the antibody may comprise a flexible linker sequence, or may be modified to add functional groups (eg, PEG, drug, toxin, or tag).
  • antibodies can be glycosylated, acetylated, pegylated, phosphorylated, amidated, derivatized by known protecting/blocking groups, proteolytically cleaved, linked to cellular ligands or other proteins, etc. Any of a number of chemical modifications can be performed by existing techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like.
  • the antibody, or fusion protein eg, wherein the antibody can be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG.
  • Antibodies, or fusion proteins such as wherein the antibody can be conjugated or fused to a therapeutic agent, which can include a detectable label, such as a radiolabel, immunomodulatory agent, hormone, enzyme, oligonucleotide, photosensitizing therapeutic agent, diagnostic agent , cytotoxic agents, ultrasound enhancers, non-radioactive labels and compositions thereof, and other such agents known in the art.
  • a detectable label such as a radiolabel, immunomodulatory agent, hormone, enzyme, oligonucleotide, photosensitizing therapeutic agent, diagnostic agent , cytotoxic agents, ultrasound enhancers, non-radioactive labels and compositions thereof, and other such agents known in the art.
  • Antibodies, or fusion proteins such as wherein the antibody can be detectably labeled by conjugating it to a chemiluminescent compound.
  • the presence of the chemiluminescent labeled antibody is then determined by detecting the luminescence that occurs during the chemical reaction.
  • chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridine esters, imidazoles, acridine salts, and oxalate esters.
  • the present invention also discloses polynucleotides or nucleic acid molecules encoding the antibodies, antigen-binding fragments or fusion proteins or derivatives thereof of the present invention.
  • the polynucleotides disclosed in the present invention can encode heavy chain, light chain, heavy chain variable region, light chain variable region, Fc region, part of heavy chain variable region or part of light chain variable region or fusion protein. Methods of making antibodies and fusion proteins are well known in the art and described in the present invention.
  • the variable and constant regions of the antibodies, antigen-binding fragments disclosed herein are fully human. Fully human antibodies and antigen-binding fragments can be prepared using techniques disclosed in the art and described herein.
  • Fully human antibodies directed against a particular antigen can be prepared by administering the antigen to transgenic animals that have been modified to produce fully human antibodies in response to challenge with the antigen.
  • Exemplary techniques that can be used to make such antibodies are found in US Pat. Nos. 6,458,592; 6,420,140, the entire contents of which are incorporated herein by reference.
  • the antibodies, fusion proteins produced do not elicit an adverse immune response in the animal (eg, human) to be treated.
  • the antibodies, antigen-binding fragments, fusion proteins or derivatives disclosed herein are modified to reduce their immunogenicity using art-recognized techniques.
  • antibodies can be humanized, primatized, deimmunized or chimeric antibodies can be prepared. These types of antibodies are derived from non-human antibodies, usually murine or primate antibodies, that retain or substantially retain the antigen-binding properties of the parent antibody but are less immunogenic in humans.
  • CDRs complementarity determining regions
  • framework residues in the human framework regions will be replaced by corresponding residues from the CDR donor antibody, eg, residues that improve antigen binding.
  • framework substitutions can be identified by methods well known in the art, such as by modeling the interaction of CDRs and framework residues to identify framework residues that are important for antigen binding and by sequence alignment to identify framework residues that are aberrant at specific positions. (See US Pat. No. 5,585,089; the entire contents of which are incorporated herein by reference).
  • Antibodies can be humanized using a variety of techniques known in the art, such as CDR grafting (EP 239,400; WO 91/09967; US Pat. Nos. 5,225,539, 5,530,101 and 5,585,089), repair or surface rearrangement (EP 592,106; EP 519,596) , and Rearrangement of Chains (US Pat. No. 5,565,332), the entire contents of which are incorporated herein by reference.
  • Deimmunization can also be used to reduce the immunogenicity of antibodies.
  • the term "deimmunization” includes altering antibodies to modify T cell epitopes (see eg WO/9852976A1 and WO/0034317A2).
  • the heavy and light chain variable region sequences from the starting antibody are analyzed and a "map" of human T cell epitopes from each variable region is generated, showing the epitopes relative to complementarity determining regions (CDRs) and the positions of other key residues within the sequence.
  • CDRs complementarity determining regions
  • a series of alternative heavy chain variable region sequences and light chain variable region sequences comprising combinations of amino acid substitutions are designed, and these sequences are subsequently incorporated into a series of binding polypeptides.
  • the genes containing the modified variable and human constant regions of the intact heavy and light chains are then cloned into expression vectors, and the plasmids are subsequently transformed into cell lines to produce intact antibodies.
  • the optimal antibodies are then identified by comparing the antibodies in appropriate biochemical and biological experiments.
  • the binding specificity of the disclosed antibodies, or antibodies in fusion proteins can be detected by in vitro assays such as co-immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • in vitro assays such as co-immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • scFvs single chain Fvs
  • antibodies include those described in US Pat. Nos. 4,946,778 and 5,258,498.
  • Chimeric antibodies are a class of molecules in which different portions of the antibody are derived from different animal species, eg, antibodies having the variable regions of murine monoclonal antibodies and the constant regions of human immunoglobulins. Methods of producing chimeric antibodies are known in the art, see US Pat. Nos. 5,807,715, 4,816,567 and 4,816,397, the entire contents of which are incorporated herein by reference.
  • Antibodies can be prepared by a variety of methods known in the art, including phage display methods using antibody libraries from immunoglobulin sequences. See also U.S. Patent Nos. 4,444,887 and 4,716,111, and PCT Publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741, each The entire contents of the patents are incorporated herein by reference.
  • Fully human antibodies are particularly desirable for treating human patients.
  • Fully human antibodies can be prepared by a variety of methods known in the art, such as the production of human antibodies by transgenic mice that are incapable of expressing functional endogenous immunoglobulins but capable of expressing human immunoglobulin genes.
  • human heavy and light chain immunoglobulin gene complexes can be introduced randomly or by homologous recombination into mouse embryonic stem cells.
  • human variable, constant and diversity regions can be introduced into mouse embryonic stem cells in addition to human heavy and light chain genes.
  • the immunoglobulin genes of the mouse heavy and light chains can be rendered inoperative by homologous recombination separately or simultaneously by introduction of human immunoglobulin loci.
  • homozygous deletion of the JH region can prevent the production of endogenous antibodies.
  • the modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. Chimeric mice are then bred to generate homozygous offspring expressing the human antibody.
  • Transgenic mice are immunized in a conventional manner with an antigen of choice, eg, all or part of the polypeptide target of interest.
  • Antigen-targeted monoclonal antibodies can be obtained from immunized transgenic mice using conventional hybridoma technology. Human immunoglobulin transgenes carried by transgenic mice rearrange during B cell differentiation, followed by class switching and somatic mutation.
  • IgG, IgA, IgM and IgE antibodies can be generated using this technique.
  • a related review of this technique for producing fully human antibodies can be found in Lonberg and Huszar, Int. Rev. Immunol. 73:65-93 (1995).
  • a technique known as "guided selection” can also be used to produce fully human antibodies that recognize selective epitopes.
  • selected non-human monoclonal antibodies eg, mouse antibodies
  • are used to guide the screening of fully human antibodies recognizing the same epitope see US Pat. No. 5,565,332, which is incorporated herein by reference in its entirety).
  • DNA encoding the desired monoclonal antibody can be isolated and subjected to Sequencing.
  • Isolated and subcloned hybridoma cells can serve as a source of such DNA.
  • the DNA can be placed in an expression vector and then transfected into prokaryotic or eukaryotic host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma that does not produce other immunoglobulins in cells.
  • Isolated DNA (which may be synthetic as described herein) can also be used to prepare the sequences of the constant and variable regions of antibodies, as described in US Pat. No. 5,658,570, which is incorporated herein by reference in its entirety. This method extracts RNA from selected cells and converts it into cDNA, which is then amplified by PCR techniques using Ig-specific primers. Suitable probes for this purpose are also mentioned in US Pat. No. 5,658,570.
  • one or more CDRs of the antibodies of the invention can be inserted into framework regions, eg, into human framework regions, to construct humanized, non-fully human antibodies.
  • Framework regions can be naturally occurring or consensus framework regions, preferably human framework regions (see Chothia et al., J. Mol. Biol. 278:457-479 (1998), which lists a list of human framework regions).
  • Some polynucleotides may encode an antibody that specifically binds to at least one epitope of an antigen of interest resulting from a combination of framework regions and CDRs.
  • One or more amino acid substitutions may be made within the framework regions, and amino acid substitutions may be selected to improve binding of the antibody to its antigen.
  • substitution or deletion of cysteine residues in one or more variable regions involved in interchain disulfide bond formation can be performed using this method, resulting in antibody molecules lacking one or more interchain disulfide bonds.
  • Other changes to polynucleotides that are within the skill in the art are also encompassed by the present invention.
  • Antibody-producing cell lines can be selected, constructed and cultured using techniques well known to those skilled in the art. These techniques are described in various laboratory manuals and major publications. In this regard, the techniques described below suitable for use in the present invention refer to Current Protocols in Immunology, Coligan et al., Eds., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991), the entire contents of which include Supplementary content is incorporated by reference in its entirety.
  • DNA encoding the antibody, antigen-binding fragment, or fusion protein can be designed and synthesized according to the amino acid sequence of the antibody, antigen-binding fragment, or fusion protein described herein, placed into an expression vector, and then transfected according to conventional methods. Host cells. The transfected host cells are grown in culture to produce antibodies, antigen-binding fragments or fusion proteins.
  • the expression vector includes at least one promoter element, an antibody, antigen-binding fragment or fusion protein coding sequence, a transcription termination signal and a polyA tail. Other elements include enhancers, Kozak sequences, and donor and acceptor sites for RNA splicing flanking the inserted sequence.
  • Efficient transcription can be obtained by the early and late promoters of SV40, long terminal repeats from retroviruses such as RSV, HTLV1, HIVI, and the early promoter of cytomegalovirus, and other cellular promoters such as muscle can be used.
  • Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or pLNCX, pcDNA3.1(+/-), pcDNA/Zeo(+/-), pcDNA3.1/Hygro(+/-), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI and pCS2 etc.
  • Commonly used mammalian cells include HEK293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells and CHO cells.
  • the inserted gene fragment needs to contain a selectable marker.
  • selectable markers include dihydrofolate reductase, glutamine synthase, neomycin resistance, hygromycin resistance and other selection genes, so as to facilitate transfection Screening isolation of successful cells.
  • the constructed plasmid is transfected into host cells without the above-mentioned genes, and cultured in selective medium, the successfully transfected cells grow in large quantities to produce the desired target protein.
  • mutations can be introduced into the nucleotide sequences encoding the antibodies, antigen-binding fragments or fusion proteins of the invention using standard techniques known to those of skill in the art, including but not limited to site-directed mutagenesis and PCR-mediated amino acid substitutions mutation.
  • Variants include derivatives
  • substitution of amino acids, substitution of less than 4 amino acids, substitution of less than 3 amino acids, or substitution of less than 2 amino acids can be introduced randomly along all or part of the coding sequence, for example by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity.
  • substitutions described herein are conservative amino acid substitutions.
  • the present invention also provides methods and uses of treatment.
  • a method for treating or ameliorating SARS or COVID-19 comprising administering to a patient an effective dose of the antibody, antigen-binding fragment or fusion protein.
  • use of the antibody, antigen-binding fragment or fusion protein in the treatment or amelioration of SARS or COVID-19 is provided.
  • use of the antibody, antigen-binding fragment or fusion protein in the preparation of a medicament for the treatment or amelioration of SARS or COVID-19 is provided.
  • the patient is a patient suspected of being infected with SARS-CoV or SARS-CoV-2 virus.
  • the patient is a patient in contact with a SARS-CoV or SARS-CoV-2 virus carrier. In some embodiments, the patient is a patient diagnosed with SARS-CoV or SARS-CoV-2 virus. In some embodiments, the patient is a mildly symptomatic patient. In some embodiments, the patient is a severely symptomatic patient. In some embodiments, the patient has fever, cough, hypotension, hypoxia, and/or acute respiratory distress syndrome (ARDS).
  • ARDS acute respiratory distress syndrome
  • the specific dosage and treatment regimen for any particular patient will depend on various factors, including the particular antibody, antigen-binding fragment or fusion protein or derivative used, the patient's age and weight, general health, sex and diet, and the amount of Duration of medication, frequency of excretion, combination of medications, and severity of the specific disease being treated. These factors are left to the judgment of health care professionals, including those within the purview of those of ordinary skill in the art.
  • the dosage will also depend on the individual patient to be treated, the route of administration, the type of formulation, the nature of the compound used, the severity of the disease, and the effect desired.
  • the dose to be used can be determined by pharmacological and pharmacokinetic principles well known in the art.
  • Methods of administration of the antibody, antigen-binding fragment, fusion protein or derivative include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, epidural, and oral injection.
  • the pharmaceutical compositions can be administered by any convenient route, such as by infusion or bolus injection, absorbed through epithelia or mucocutaneous (eg, oral mucosa, rectal and intestinal mucosa, etc.), and can be co-administered with other biologically active agents.
  • the pharmaceutical composition containing the antibody, antigen-binding fragment or fusion protein of the present invention can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, externally (eg, by powder, ointment, drops or transdermal patches), by mouth or by oral or nasal spray.
  • parenteral refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
  • the mode of administration can be systemic or topical.
  • compositions of the present invention comprise a nucleic acid or polynucleotide encoding a protein that can be administered in vivo to facilitate expression of the protein encoded thereby by constructing it as part of a suitable nucleic acid expression vector, Parts of the vectors described above are then administered to make them intracellular, for example, by the use of retroviral vectors (see US Pat. No. 4,980,286), or by direct injection, or by the use of particle bombardment (eg, a gene gun; Biolistic, Dupont) , or coated with lipids or cell surface receptors or transfection reagents, or administered by linkage to homeobox peptoids known to enter the nucleus (see e.g.
  • nucleic acid can be introduced into a cell by homologous recombination and integrated into the host cell DNA for expression.
  • the antibody, antigen-binding fragment or fusion protein of the invention is administered to a patient at a dose of 0.01 mg/kg to 100 mg/kg of the patient's body weight, or 0.1 mg/kg to 20 mg/kg of the patient's body weight.
  • a second or more doses of the antibody, antigen-binding fragment, or fusion protein may be administered subsequent to the initial dose at about the same or less than the initial dose, wherein the subsequent doses may be separated by at least 1 to 3 days; or at least a week.
  • the uptake and tissue penetration (eg, into the brain) of the antibody, antigen-binding fragment or fusion protein can be enhanced by modifications such as lipidation, thereby reducing the dose and frequency of administration of the antibody, antigen-binding fragment or fusion protein of the invention .
  • Various known delivery systems can be used to administer the antibodies, antigen-binding fragments or fusion proteins or derivatives thereof of the invention or polynucleotides encoding them, eg, encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compounds , receptor-mediated endocytosis (see, eg, Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of nucleic acids as part of retroviral or other vectors, and the like.
  • the antibodies, antigen-binding fragments, or fusion proteins of the invention can be combined with other therapeutic or prophylactic regimens, including administration of one or more antibodies, antigen-binding fragments, or fusion proteins of the invention and one or more other Therapeutic agents or methods are used together or in combination.
  • the antibody, antigen-binding fragment or fusion protein can be administered simultaneously or separately from the other therapeutic agent.
  • the antibody, antigen-binding fragment or fusion protein of the invention can be administered before or after administration of the other other therapeutic agent.
  • the therapeutic agent administered in combination with the antibody, antigen-binding fragment or fusion protein of the present invention is at least one of the following: HIV drugs, antimalarial drugs, RNA polymerase inhibitors, antiviral drugs, and monoclonal antibodies medicine.
  • HIV drugs include lopinavir/ritonavir, ASC09/ritonavir, and darunavir.
  • antimalarial drugs include chloroquine, hydroxychloroquine, and chloroquine phosphate.
  • the antiviral drug includes abidol, favipiravir.
  • the monoclonal antibody includes BDB-001.
  • the antiviral drug is an antibody or antigen-binding fragment in the fusion protein of the present invention.
  • the antibody, antigen-binding fragment or fusion protein of the present invention can be combined with adalimumab (adalimumab, such as and its biosimilars, such as Abrilada TM (adalimumab-afzb), Amjevita (adalimumab-att), Cyltezo TM (adalimumab-adbm), Hyrimoz TM (adalimumab-adaz), Hulio TM , ( BAT1406)) or tochilizumab, such as and its biosimilars, such as BAT1806) in combination therapy, which can slow down the inflammatory response caused by the up-regulation of TNF- ⁇ expression.
  • adalimumab such as and its biosimilars, such as Abrilada TM (adalimumab-afzb), Amjevita (adalimumab-att), Cyltezo TM (adalim
  • the patients treated by the present methods are diagnosed with novel coronavirus infection and have one or more cytokines (including tumor necrosis factor alpha (TNF- ⁇ ), IFN- ⁇ , IL-1 ⁇ , IL-2, IL -4, IL-7, IL-8, IL-10, IL-12p70, IL-13, granulocyte colony stimulating factor (GSCF), interferon-inducible protein-10 (IP-10), monocyte chemotactic protein -1 (MCP1), macrophage inflammatory protein 1 ⁇ (MIP1A)) increased.
  • the patients treated by the present methods have elevated TNF-alpha.
  • the one or more cytokines are at least 50% above normal levels.
  • the one or more cytokines are at least 2, 3, or 4 times normal levels.
  • the patient has fever, hypotension, hypoxia, and/or acute respiratory distress syndrome (ARDS) prior to treatment in the present method.
  • ARDS acute respiratory distress syndrome
  • the patient has lungs filled with inflammatory fluid (so-called "white lung") prior to treatment in the present methods.
  • the patient has Cytokine Release Syndrome (CRS) due to cytokine storm prior to treatment in the present method.
  • CRS Cytokine Release Syndrome
  • the antibodies, antigen-binding fragments or fusion proteins of the invention are used in conjunction with ICU therapy. In some embodiments, the antibodies, antigen-binding fragments or fusion proteins of the invention bind to in vitro ECMO and/or IMV therapy. In some embodiments, the antibodies, antigen-binding fragments or fusion proteins of the invention bind to oxygen therapy. In some embodiments, the antibodies, antigen-binding fragments or fusion proteins of the invention bind to NIV/HFNC therapy. In some embodiments, after treatment, the patient's one or more cytokines are at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% lower than before treatment . In some embodiments, the method heals the patient.
  • spike proteins are observed in certain samples, and patients with cells expressing spike proteins may respond to treatment with the antibodies, antigen-binding fragments or fusion proteins of the invention. Accordingly, the antibodies, antigen-binding fragments or fusion proteins of the present invention can also be used for diagnosis and prognosis.
  • a sample containing cells can be obtained from a patient. After selective pretreatment of the sample, the sample can be incubated with an antibody (or fusion protein) of the invention under conditions that allow the antibody (or fusion protein) to interact with spike proteins that may be present in the sample.
  • the presence of spike proteins in a sample can be detected using methods such as ELISA using antibodies or antibodies in fusion proteins.
  • the presence (eg, amount or concentration) of spike protein in a sample can be used to diagnose a related disease, as an indication that a patient is eligible for antibody or fusion protein therapy, or as an indication that a patient has (or has not) responded to treatment for a disorder.
  • one, two, or more tests can be performed at specific stages at the initiation of disease treatment to indicate the progress of treatment.
  • compositions comprise an effective dose of the antibody or fusion protein and pharmaceutically acceptable excipients.
  • the term "pharmaceutically acceptable” refers to a substance approved by a regulatory agency of the government or listed in other generally recognized pharmacopeias for use in animals, particularly in humans.
  • pharmaceutically acceptable adjuvants generally refer to any type of non-toxic solid, semi-solid or liquid fillers, diluents, encapsulating materials or formulation aids and the like.
  • adjuvant refers to a diluent, adjuvant, excipient or carrier with which the active ingredient can be administered to a patient.
  • Such pharmaceutical carriers can be sterile liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is the preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerin, Propylene, ethylene glycol, water, ethanol, etc.
  • the pharmaceutical compositions may also contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents such as acetates, citrates, or phosphates, if desired.
  • Antibacterial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as EDTA, and tonicity adjusting agents such as sodium chloride or dextrose are also contemplated.
  • These pharmaceutical compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like.
  • the pharmaceutical composition can be formulated as a suppository with traditional binders and carriers such as triglycerides.
  • Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.
  • compositions will contain a clinically effective dose of the antibody or antigen-binding fragment or fusion protein, preferably in purified form, together with a suitable amount of carrier to provide a form suitable for administration to the patient.
  • the formulation should be suitable for the mode of administration.
  • the formulation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions are formulated according to conventional procedures into pharmaceutical compositions suitable for intravenous injection into humans.
  • Compositions for intravenous administration are usually solutions in sterile isotonic aqueous buffer.
  • the pharmaceutical composition may also contain a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site.
  • the active ingredients are supplied in unit dosage form either individually or mixed together, eg, as a dry lyophilized powder or anhydrous concentrate in a hermetically sealed container (eg, an ampule or sachet) indicative of the amount of active agent.
  • the composition may be dispensed in an infusion bottle containing sterile pharmaceutical grade water or saline.
  • ampoules of sterile water for injection or saline can be used so that the active ingredient can be mixed prior to administration.
  • the compounds of the present invention may be formulated in neutral or salt form.
  • Pharmaceutically acceptable salts include those derived from anions such as hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like, and those derived from, for example, sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2 - Salts formed from cations of ethylaminoethanol, histidine, procaine, etc.
  • ECMO refers to extracorporeal membrane oxygenation (Extracorporeal Membrane Oxygenation, ECMO), which is a medical emergency technical equipment, mainly used to provide continuous extracorporeal respiration and circulation to patients with severe cardiopulmonary failure to maintain their lives.
  • ICU refers to an intensive care unit (Intensive Care Unit), where treatment, nursing, and rehabilitation can be carried out simultaneously, providing isolation places and equipment for critically ill or comatose patients, providing optimal care, comprehensive treatment, combination of medical care, and surgery. Post-early rehabilitation, joint care exercise therapy and other services.
  • IMV intermittent mandatory ventilation
  • intermittent mandatory ventilation implements periodic volume or pressure ventilation according to a preset time interval or time trigger. This period allows the patient to breathe spontaneously at any set basal pressure level during mandatory ventilation.
  • spontaneous breathing the patient can breathe spontaneously with continuous airflow support, or the machine will open the valve on demand to allow spontaneous breathing.
  • Most ventilators can provide pressure support during spontaneous breathing.
  • HFNC high-flow nasal cannula oxygen therapy, which is an oxygen therapy method in which a certain oxygen concentration of air and oxygen mixed with high-flow gas is directly delivered to the patient through a nasal cannula that does not need to be sealed.
  • a form of non-invasive respiratory support that rapidly improves oxygenation. At present, it can be used in patients with acute hypoxic respiratory failure, patients after surgery, patients with respiratory failure without endotracheal intubation, immunosuppressed patients, and patients with cardiac insufficiency.
  • NMV non-invasive ventilation
  • Non-invasine Ventilation refers to non-invasive mechanical ventilation other than tracheal intubation and tracheotomy.
  • EC50 the concentration for 50% of maximal effect (EC50), refers to the concentration that elicits 50% of the maximal effect.
  • IC50 means the 50% inhibitory concentration, that is, the concentration of drug or inhibitor required to inhibit half the indicated biological process.
  • the "parental Fc region” can be a naturally occurring Fc region, and the gene encoding the Fc region can be from human, mouse, rabbit, camel, monkey, preferably human and mouse; for example, the parental Fc region is SEQ ID NO:60 , the Fc region in SEQ ID NO:61 or SEQ ID NO:66.
  • Antibodies and fusion proteins can be prepared by the following methods or other known methods: sequence optimization is carried out according to the codon preference characteristics of CHO of host cells, and DNA sequences are obtained from amino acid sequences. The optimized and synthesized sequences were cloned into the vector respectively, and then a large number of plasmids were extracted respectively to construct stable expression cell lines: the linearized expression vector was mixed with CHO cells, and then added to a 0.4 cm electroporation cup for electroporation; after electroporation was completed, press 1200 cells per well were plated into a 96-well cell culture plate, and after about 2-3 weeks, the parent clone with high expression was selected for cell expansion and expression detection from 96 wells to 24 wells to 6 wells to shake flasks, and the shake flasks were selected.
  • the clones with high expression levels were sub-cloned, sub-cloning was expanded and expressed to identify the same parent clone, and the monoclonal stable cell line was selected according to the expression level and cell line stability, and the supernatant was harvested for about 12 days of suspension culture and proA affinity capture , Anion and cation chromatography to obtain antibodies or fusion proteins with a purity greater than 95%.
  • VH and CH constitute the heavy chain of the antibody
  • VL and CL constitute the light chain of the antibody
  • antibody 2F8 contains two heavy chains with the same sequence (as shown in SEQ ID NO: 66) and two light chains with the same sequence (as shown in SEQ ID NO: 68); antibody anti-19 contains two sequences with the same sequence
  • the fusion protein 20 comprises two polypeptide A with the same sequence (as shown in SEQ ID NO: 69 shown) and two identical sequences of polypeptide B (shown in SEQ ID NO: 67); polypeptide A includes a heavy chain fragment, a linker and a 6-HB interfering polypeptide, and polypeptide B includes a light chain; in Tables 2 and 3
  • the Fc region of the heavy chain is marked with a single underline, and the linker of polypeptide A and the 6-HB interfering polypeptide in Table 3 are marked with a single underline and a double underline, respectively.
  • the purified antibody and fusion protein were sequenced to confirm the sequence as described above.
  • Elisa detection was carried out for the above-mentioned antibodies.
  • the detection method was as follows: coat a 96-well plate (Corning, 9018) with spike-RBD-mFC (sino biologicals), seal it with tape and store it; put the plate in washing buffer PBST (PBS, 0.05 % Tween 20) for 3 times, then add blocking solution (200 ⁇ L of 10 mg/ml BSA per well, the solvent is washing buffer); after incubation (1 h, 37 °C), the plate is washed 3 times with washing buffer, then Add 100 ⁇ L of gradient diluted samples to each well; after incubation (1.5h, 37°C), wash the plate with washing buffer, then add anti-human kappa light chain antibody-peroxidase conjugate (diluted in blocking solution) to 1:2000, 100 ⁇ L/well); the plate was washed with wash buffer, and the test samples were incubated (1 h, 37°C) before adding 100 ⁇ L of TMB (Tetramethylbenz
  • EC 50 was calculated by absorbance value, and the EC 50 values of various antibodies binding to SARS-CoV-2 spike protein are shown in Table 5.
  • BiaCore Biomolecular Interaction Analysis: The antibody was coated on a protein A chip at a concentration of 10 ⁇ g/mL, the binding kinetics were measured using BiaCore at 25°C, and the probe was equilibrated; recombinant spike S1 RBD protein (Acrobiosystems, SPD) -C52H3) or a trimeric spike trimer (Acrobiosystems, SPN-C52H8) at 10-400 nM concentrations through antibody-coated chips; flow-through buffer only (PBS buffer containing 0.05% Tween-20) was used as a control minus Background and nonspecific binding signals were removed; kinetic constants were calculated using the 1:1 Langmuir binding model on BiaCore Data Analysis software (ka is the on-rate, kd is the dissociation rate, and kD is the binding-dissociation equilibrium constant).
  • antibody anit-19 and fusion protein 20 have good binding ability to trimeric spike trimer.
  • Embodiment 4 Detection of anti-spike protein antibody binding activity
  • the antibody 2F8 was detected by ELISA.
  • the detection method was as follows: the spike RBD protein (Acrobiosytems, SPD-C52H3) was diluted to 2 ⁇ g/ml, and 100 ⁇ l per well was placed in a 96-well plate (Corning, 9018) and coated overnight at 4°C; The 96-well plate was washed 3 times in washing buffer PBST (PBS buffer containing 0.05% Tween-20), then blocking solution (200 ⁇ L of 3 mg/ml BSA per well, the solvent was washing buffer) was added, and incubated at 37°C for 2 h; Then, the 96-well plate was washed 3 times with washing buffer, 100 ⁇ L of gradient diluted antibody solution was added to each well, and incubated at 37°C for 1.5 h; the 96-well plate was washed 5 times with washing buffer, and 100 ⁇ L of anti-human kappa light was added.
  • PBST PBS buffer containing 0.05% T
  • TMB Tetramethylbenzidine, Biopanda TMB-S
  • SPR Surface Plasmon Resonance
  • 100nM biotinylated spike RBD protein (Acrobiosystems, SPD-C82E9) was first combined with streptavidin probe, and the probe combined with biotinylated spike RBD was incubated with 100nM antibody solution.
  • Antibody probes were incubated with 100 nM ACE2 (Inshore Bio, C419) protein to detect whether the antibody-bound spike RBD protein could also bind to ACE2 in solution.
  • antibody 2F8 blocked the binding of ACE2 to spike RBD.
  • ACE2 + 293F cells were used to detect the ability of the antibody to inhibit the infection of cells by the SARS-CoV-2 pseudovirus carrying the luciferase gene.
  • the main principle is: using ACE2 + 293F cells as susceptible cells, and incubating different concentrations of antibodies with the SARS-CoV-2-Fluc pseudovirus system; when the antibody is incubated with the pseudovirus, it will block virus infection from entering ACE2 + 293F cells; the pseudovirus cannot effectively infect the cells, and the luciferase reporter gene on its genome cannot be expressed in the cells and generate a fluorescent signal; since the signal value of the fluorescent signal is negatively correlated with the concentration of the added antibody, the antibody can be detected.
  • the mutant strain D614G and D936Y have the article number GM-0220PV19-96T (Jiman Bio), the mutant strain D839Y has the article number GM-0220PV6-480T (Jiman Bio), and the mutant strain V483A has the article number GM-0220PV17-480T (Jiman Biological).
  • Man Bio mutant strain D614G, the product number of A831V is GM-0220PV24-480T (Jiman Bio), and the mutant strain B.1.351/501Y.V2 is GM-0220PV32-96T (Ji Man Bio).
  • the detection method of pseudovirus inhibitory ability is as follows: the antibody is diluted to 6 ⁇ g/ml, then 4-fold gradient dilution, and transferred to a 96-well detection plate according to the volume of 50 ⁇ l per well, for use; The DMEM medium of FBS was diluted, and the diluted pseudovirus solution was transferred to the above-mentioned 96-well plate containing antibodies according to 25 ⁇ l per well.
  • inhibition rate [1-(sample group-blank control group)/(negative) Control group-blank control group)] ⁇ 100%; wherein, the negative control group was added with pseudovirus solution and no antibody was added, and the blank control group was not added with pseudovirus solution.
  • ACE2 + 293F cells The construction method of ACE2 + 293F cells is as follows: HEK293F cells are cultured in DMEM complete medium containing 10% FBS, and lipofectamine 2000 transfection reagent (Thermo Fisher, 11668019) is used for transfection of ACE2 expression plasmid (Yiqiao Shenzhou, HG10108-M). After staining, cells were further expanded to select PE-positive cells by press screening with hygromycin (200 ⁇ g/ml) and flow sorting (using 10 ⁇ g/ml anti-ACE2 and PE-coupled Anti-Human IgG-Fc). Monoclones with a rate of >90% were further amplified to screen out HEK293F cells expressing ACE2, namely ACE2 + 293F cells.
  • antibody 2F8 can effectively inhibit pseudovirus infection of cells.
  • the supernatant of African green monkey kidney cell line Vero E6 cells was taken, and 50 ⁇ l of the incubated virus-antibody mixture was transferred to a Vero E6 cell plate, placed in a cell incubator at 37°C, and incubated for 1 hour; aspirated Discard the supernatant from the Vero E6 cell plate, add 100 ⁇ l of pre-warmed DMEM medium (containing 1.6% CMC (carboxymethyl cellulose)) at 37°C, and place it in a 37°C cell incubator for 24 hours; after 24 hours, take out the cell plate, Add 200 ⁇ l of 4% PFA (paraformaldehyde solution), incubate at 4°C overnight to fix the inactivated virus and cells; the next day, remove the supernatant and replace with fresh 4% PFA, and bring the 96-well cell plate into the BSL-2 experiment Use FRNT50 (plaque/focus reduction neutralization test (P/FRNT)) experiment for detection: SARS-CoV
  • the cytopathic effect (CPE, cytopathic effect) reduction method was used to detect the preventive effect of the fusion protein.
  • the main method of the test is: add the antibody or fusion protein to an equal volume of 100PFU COVID-19 virus solution (Genebank accession no.MT123290.1), mix well and incubate at 37°C for 1 h; add the mixture to Vero E6 cells cultured in monolayer, Incubate at 37°C for 1 h; remove the virus solution, wash once with PBS, and add 200 ⁇ L of medium to each well. Equal volume of culture medium was added to each well of blank control group and normal cell group, cultured at 37°C for 4 days, and the degree of cytopathic changes was observed and recorded under microscope.

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Abstract

本发明提供特异性结合SARS-CoV-2或SARS-CoV的spike蛋白的抗体、融合蛋白及其应用。

Description

冠状病毒抗体及其应用 技术领域
本发明属于生物技术领域，尤其涉及冠状病毒抗体及其应用。
背景
冠状病毒为不分节段的单股正链RNA病毒，根据血清型和基因组特点冠状病毒亚科被分为α、β、γ和δ四个属，由于病毒包膜上有向四周伸出的突起，形如花冠而得名。2019年发现的新型冠状病毒(SARS-CoV-2或2019-nCoV)属于β属的新型冠状病毒，有包膜，颗粒呈圆形或椭圆形，常为多形性，直径60-140nm。目前研究显示，SARS-CoV-2与SARS-CoV具有高度同源性。
新型冠状病毒肺炎COVID-19主要通过呼吸道传染，其也可能通过接触传播。人群普遍易感，老年人及有基础疾病者感染后病情较重，儿童及婴幼儿也有发病。基于目前的流行病学调查，新型冠状病毒的潜伏期一般为1-14天，大多数在3-7天。感染者的主要临床症状是发热、乏力、干咳，而鼻塞、流涕等上呼吸道症状少见。在发病早期，患者的白细胞总数正常或降低，或淋巴细胞数目减少，部分患者出现肝酶、肌酶和肌红蛋白增高的现象。胸部影像显示患者早期呈现多发小斑片影及间质改变，以肺外带明显；进而发展为双肺多发磨玻璃影、浸润影，严重者可出现肺实变，并逐渐出现呼吸困难，严重者发生急性呼吸窘迫综合征(ARDS)、休克以及肺组织、心脏、肾脏多种组织损伤和功能障碍。多数轻度感染患者预后良好，重度患者病情常常危重，甚至死亡。
发明内容
本发明提供对SARS-CoV和SARS-CoV-2的spike蛋白具有高亲和力的抗体或抗原结合片段以包含所述抗体或抗原结合片段的融合蛋白。
本发明提供对SARS-CoV和SARS-CoV-2的spike蛋白具有高亲和力的抗体或抗原结合片段。这些抗体可以特异性结合spike蛋白，阻止病毒颗粒和细胞结合以及可以介导免疫细胞吞噬、清除病毒颗粒。这些抗体可用于治疗或改善SARS和COVID-19，也可以用于诊断SARS和COVID-19。
通过其表面的spike蛋白(S蛋白或棘突蛋白)与肺上皮细胞表面的一种称为血管紧张素转化酶2(ACE2)进行结合，SARS-CoV或SARS-CoV-2进入细胞内，并利用 细胞为其合成新的病毒颗粒；新的病毒颗粒释放到细胞外，再利用同样的方式，病毒感染周围正常的细胞。抗spike蛋白抗体能阻断spike蛋白与ACE2的结合，进而阻断了病毒进入细胞，发挥抗病毒作用。本发明抗体还可介导免疫细胞吞噬和清除病毒。
一些实施方案提供了抗体或抗原结合片段，所述抗体或抗原结合片段特异性结合spike蛋白，并且包含：
(a)HCDR1，其包含如SEQ ID NO:1或2所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；(b)HCDR2，其包含如SEQ ID NO:3或4所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；和/或(c)HCDR3，其包含如SEQ ID NO:5-42中任一项所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体。
一些实施方案中，所述抗体或抗原结合片段特异性结合spike蛋白，并且包含：
(a)HCDR1，其包含如SEQ ID NO:1或2所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；(b)HCDR2，其包含如SEQ ID NO:3或4所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；和(c)HCDR3，其包含如SEQ ID NO:5-42中任一项所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体。
一些实施方案中，所述抗体或抗原结合片段特异性结合spike蛋白，并且包含：
(a)HCDR1，其包含如SEQ ID NO:1或2所示的氨基酸序列；(b)HCDR2，其包含如SEQ ID NO:3或4所示的氨基酸序列；和(c)HCDR3，其包含如SEQ ID NO:5-42中任一项所示的氨基酸序列。
一些实施方案中，所述抗体或抗原结合片段特异性结合spike蛋白，并且包含：
(a)HCDR1，其包含如SEQ ID NO:1或2所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；(b)HCDR2，其包含如SEQ ID NO:3或4所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；(c)HCDR3，其包含如SEQ ID NO:5-42中任一项所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；(d)LCDR1，其包含如SEQ ID NO:43或44所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；(e)LCDR2，其包含如SEQ ID NO:45或46所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；和/或(f)LCDR3，其包含如SEQ ID NO:47或48所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体。
在一些实施方案中，所述抗体或抗原结合片段特异性结合spike蛋白，并且包含：
(a)HCDR1，其包含如SEQ ID NO:1或2所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；(b)HCDR2，其包含如SEQ ID NO:3或4所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；(c)HCDR3，其包含如SEQ ID NO:5-42中任一项所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；(d)LCDR1，其包含如SEQ ID NO:43或44所示的氨基酸序列，或其有单一位点取代、缺失或插入 的变体；(e)LCDR2，其包含如SEQ ID NO:45或46所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；和(f)LCDR3，其包含如SEQ ID NO:47或48所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体。
在一些实施方案中，所述取代变体为保守氨基酸取代变体。
在一些实施方案中，所述抗体或抗原结合片段特异性结合spike蛋白，所述抗体或抗原结合片段至少包含如SEQ ID NO:1或2所示的HCDR1、如SEQ ID NO:3或4所示的HCDR2、如SEQ ID NO:5-42中任一项所示的HCDR3、如SEQ ID NO:43或44所示的LCDR1、如SEQ ID NO:45或46所示的LCDR2和如SEQ ID NO:47或48所示的LCDR3中的一个、两个、三个、四个、五个或全部。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:5所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:6所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:7所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:8所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:9所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:10所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的 HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:11所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:12所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:13所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:14所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:15所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:16所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:17所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:18所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:19所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:20所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:21所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:22所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:23所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:24所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:25所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:26所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:27所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:28所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示 的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:29所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:30所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:31所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:32所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:33所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:34所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:35所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:36所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:37所示的HCDR3、如SEQ  ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:38所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:39所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:40所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:41所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:2所示的HCDR1、如SEQ ID NO:4所示的HCDR2、如SEQ ID NO:42所示的HCDR3、如SEQ ID NO:44所示的LCDR1、如SEQ ID NO:46所示的LCDR2和如SEQ ID NO:48所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段包含重链可变区和轻链可变区，所述重链可变区包含本文所述HCDR1、HCDR2、和/或HCDR3，所述轻链可变区包含本文所述LCDR1、LCDR2、和/或LCDR3。
在一些实施方案中，所述抗体或抗原结合片段的重链可变区的框架区还包含重链FR1、重链FR2、重链FR3和重链FR4。
在一些实施方案中，所述重链FR1包含SEQ ID NO:49或50所示的序列，或与SEQ ID NO:49或50所示序列具有至少90％同一性的序列，或与SEQ ID NO:49或50所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述重链FR2包含SEQ ID NO:51或52所示的序列，或与SEQ ID NO:51或52所示序列具有至少90％同一性的序列，或与SEQ ID NO:51或52所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述重链FR3包含SEQ ID NO:53或54所示的序列，或与 SEQ ID NO:53或54所示序列具有至少90％同一性的序列，或与SEQ ID NO:53或54所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述重链FR4包含SEQ ID NO:55所示的序列，或与SEQ ID NO:55所示序列具有至少90％同一性的序列，或与SEQ ID NO:55所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述重链FR1包含SEQ ID NO:49或50所示的序列，或与SEQ ID NO:49或50所示序列具有至少90％同一性的序列，或与SEQ ID NO:49或50所示序列相比具有一或多个保守氨基酸取代的氨基酸序列；所述重链FR2包含SEQ ID NO:51或52所示的序列，或与SEQ ID NO:51或52所示序列具有至少90％同一性的序列，或与SEQ ID NO:51或52所示序列相比具有一或多个保守氨基酸取代的氨基酸序列；所述重链FR3包含SEQ ID NO:53或54所示的序列，或与SEQ ID NO:53或54所示序列具有至少90％同一性的序列，或与SEQ ID NO:53或54所示序列相比具有一或多个保守氨基酸取代的氨基酸序列；所述重链FR4包含SEQ ID NO:55所示的序列，或与SEQ ID NO:55所示序列具有至少90％同一性的序列，或与SEQ ID NO:55所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。在一些实施方案中，一或多个保守氨基酸取代为约1个、约2个或约3个保守氨基酸取代。
在一些实施方案中，所述重链FR1包含SEQ ID NO:49所示的序列，所述重链FR2包含SEQ ID NO:51所示的序列，所述重链FR3包含SEQ ID NO:53所示的序列，所述重链FR4包含SEQ ID NO:55所示的序列。在一些实施方案中，所述重链FR1包含SEQ ID NO:50所示的序列，所述重链FR2包含SEQ ID NO:52所示的序列，所述重链FR3包含SEQ ID NO:54所示的序列，所述重链FR4包含SEQ ID NO:55所示的序列。在一些实施方案中，所述重链可变区包含结构重链FR1-HCDR1-重链FR2-HCDR2-重链FR3-HCDR3-重链FR4。
在一些实施方案中，所述抗体或抗原结合片段的重链可变区包含如SEQ ID NO:49所示的重链FR1、如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:51所示的重链FR2、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:53所示的重链FR3、如SEQ ID NO:5-41中任一项所示的HCDR3和如SEQ ID NO:55所示的重链FR4。
在一些实施方案中，所述抗体或抗原结合片段的重链可变区包含如SEQ ID NO:50所示的重链FR1、如SEQ ID NO:2所示的HCDR1、如SEQ ID NO:52所示的重链FR2、如SEQ ID NO:4所示的HCDR2、如SEQ ID NO:54所示的重链FR3、如SEQ ID NO:42所示的HCDR3和如SEQ ID NO:55所示的重链FR4。
在一些实施方案中，所述抗体或抗原结合片段的重链可变区包含SEQ ID NO:56或57所示的序列，或与SEQ ID NO:56或57所示序列具有至少80％同一性的序列， 或与SEQ ID NO:56或57所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO:58或59所示的序列，或与SEQ ID NO:58或59所示序列具有至少80％同一性的序列，或与SEQ ID NO:58或59所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述抗体或抗原结合片段的重链可变区包含SEQ ID NO:56所示的序列，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO:58所示的序列。
在一些实施方案中，所述抗体或抗原结合片段的重链可变区包含SEQ ID NO:57所示的序列，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO:59所示的序列。
在一些实施方案中，抗体或抗原结合片段还包含重链恒定区、轻链恒定区、Fc区或其组合。在一些实施方案中，轻链恒定区是κ或λ链恒定区。在一些实施方案中，抗体或其片段是IgG、IgM、IgA、IgE或IgD其中一种同种型。在一些实施方案中，同种型是IgG1、IgG2、IgG3或IgG4。没有限制地，抗体或抗原结合片段是嵌合抗体、人源化抗体或是全人源抗体。在某一方面，抗体或抗原结合片段是人源化抗体。
在一些实施方案中，Fc是变体Fc区。在一些实施方案中，相对于亲本Fc区，变体Fc区具有一个或多个氨基酸修饰，如取代、缺失或插入。在一些实施方案中，相对于亲本Fc区活性，Fc区的氨基酸修饰改变了效应功能活性。在一些实施方案中，变体Fc区可以具有改变的(即，增加的或降低的)抗体依赖性细胞毒性(ADCC)、补体介导的细胞毒性(CDC)、吞噬作用、调理作用或细胞结合。在一些实施方案中，相对于亲本Fc区，Fc区氨基酸修饰可以改变变体Fc区对FcγR(Fcγ受体)的亲和力。在一些实施方案中，所述Fc区来源于IgG1或IgG4。在一些实施方案中，Fc区突变是N297A。在一些实施方案中，Fc区突变是N297A、L234A或L235A(Eu编号)。在一些实施方案中，Fc区突变是E345R或S440Y(Eu编号)。
在一些实施方案中，所述抗体或抗原结合片段为分离的抗体或抗原结合片段。在一些实施方案中，所述抗体或抗原结合片段为scFV、Fab、F(ab) 2或IgG。在一些实施方案中，所述抗体或抗原结合片段为单克隆抗体。
在一些实施方案中，所述抗体或抗原结合片段的重链恒定区包含氨基酸序列如SEQ ID NO:60或61所示的序列，或与SEQ ID NO:60或61所示序列具有至少80％同一性的序列，或与SEQ ID NO:60或61所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述抗体或抗原结合片段的轻链恒定区包含氨基酸序列如SEQ ID NO:62所示的序列，或与SEQ ID NO:62所示序列具有至少80％同一性的序列，或与SEQ ID NO:62所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述抗体或抗原结合片段的重链恒定区包含氨基酸序列如SEQ ID NO:60或61所示的序列，和/或所述抗体或抗原结合片段的轻链恒定区包含氨基酸序列如SEQ ID NO:62所示的序列。在一些实施方案中，所述抗体或抗原结合片段的重链恒定区包含氨基酸序列如SEQ ID NO:60所示的序列，所述抗体或抗原结合片段的轻链恒定区包含氨基酸序列如SEQ ID NO:62所示的序列。在一些实施方案中，所述抗体或抗原结合片段的重链恒定区包含氨基酸序列如SEQ ID NO:61所示的序列，所述抗体或抗原结合片段的轻链恒定区包含氨基酸序列如SEQ ID NO:62所示的序列。
在一些实施方案中，所述抗体或抗原结合片段包含重链和/或轻链，所述重链包含本文所述重链可变区和重链恒定区，所述轻链包含本文所述轻链可变区和轻链恒定区。
在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO:65或66所示的序列，或与SEQ ID NO:65或66所示序列具有至少80％同一性的序列，或与SEQ ID NO:65或66所示序列相比具有一或多个保守氨基酸取代的氨基酸序列；和/或
所述抗体的轻链包含氨基酸序列如SEQ ID NO:67或68所示的序列，或与SEQ ID NO:67或68所示序列具有至少80％同一性的序列，或与SEQ ID NO:67或68所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO:65或66所示的序列，和/或所述抗体的轻链包含氨基酸序列如SEQ ID NO:67或68所示的序列。
在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO:65所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO:67所示的序列。在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO:66所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO:68所示的序列。
在一些实施方案中，抗体包含2条序列相同的重链和2条序列相同的轻链。
在一些实施方案中，所述抗体或抗原结合片段体可以特异性结合spike蛋白，并阻断SARS-CoV或SARS-CoV-2病毒颗粒和细胞结合，以及介导免疫细胞吞噬、清除病毒颗粒。在一些实施方案中，所述抗体或抗原结合片段体可以特异性结合spike蛋白中S1亚基。
本发明还提供融合蛋白，包含可以特异性结合spike蛋白的抗体或抗原结合片段，如本文所述抗体或抗原结合片段，及与该抗体或抗原结合片段结合的6-HB干扰多肽。融合蛋白中抗体或抗原结合片段对SARS-CoV和SARS-CoV-2的spike蛋白具有高亲和力，这些抗体或抗原结合片段可以特异性结合spike蛋白；融合蛋白中6-HB干扰多肽可干扰“六螺旋束(6-HB)”的形成。本发明融合蛋白可阻止病毒颗粒和细胞膜融合以及可以介导免疫细胞吞噬、清除病毒颗粒。这些融合蛋白用于治疗或改善SARS 和COVID-19，也用于诊断SARS和COVID-19。
病毒颗粒首先通过其表面的spike蛋白(S蛋白或棘突蛋白)的S1亚基中的受体结合域(RBD)与肺上皮细胞表面的一种称为血管紧张素转化酶2(ACE2)进行结合。病毒与受体结合并被蛋白酶水解之后，位于S蛋白N端的S2亚基暴露，并嵌入浆膜或者内吞体膜中。S2亚基中的HR2(heptad repeated 2)结构域与S2亚基中的HR1(heptadrepeated 1)结构域结合，形成6-HB融合核心，导致病毒外壳与细胞膜融合，SARS-CoV或SARS-CoV-2进入细胞内，并利用细胞为其合成新的病毒颗粒；新的病毒颗粒释放到细胞外，再利用同样的方式侵染周围正常的细胞。融合蛋白中抗体能阻断spike蛋白与ACE2的结合，融合蛋白中6-HB干扰多肽阻止了病毒外壳与细胞膜的融合，进而阻断了病毒进入细胞，发挥抗病毒作用；融合蛋白中抗体部分还可介导免疫细胞吞噬和清除病毒。
一些实施方案中，所述融合蛋白包含抗体或抗原结合片段以及6-HB干扰多肽，所述抗体或抗原结合片段特异性结合spike蛋白，并且包含：(a)HCDR1，其包含如SEQ ID NO:1或2所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；(b)HCDR2，其包含如SEQ ID NO:3或4所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；和/或(c)HCDR3，其包含如SEQ ID NO:5-42中任一项所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；
所述抗体或抗原结合片段的至少一个C-末端或N-末端通过连接子与6-HB干扰多肽进行连接。在一些实施方案中，所述连接子为包含甘氨酸和丝氨酸的多肽。
一些实施方案中，所述融合蛋白包含抗体或抗原结合片段以及6-HB干扰多肽，所述抗体或抗原结合片段特异性结合spike蛋白，并且包含：(a)HCDR1，其包含如SEQ ID NO:1或2所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；(b)HCDR2，其包含如SEQ ID NO:3或4所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；和(c)HCDR3，其包含如SEQ ID NO:5-42中任一项所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；
所述抗体或抗原结合片段的至少一个C-末端或N-末端通过连接子与6-HB干扰多肽进行连接。在一些实施方案中，所述连接子为包含甘氨酸和丝氨酸的多肽。
一些实施方案中，所述融合蛋白包含抗体或抗原结合片段以及6-HB干扰多肽，所述抗体或抗原结合片段特异性结合spike蛋白，并且包含：
(a)HCDR1，其包含如SEQ ID NO:1或2所示的氨基酸序列；(b)HCDR2，其包含如SEQ ID NO:3或4所示的氨基酸序列；和(c)HCDR3，其包含如SEQ ID NO:5-42中任一项所示的氨基酸序列；
所述抗体或抗原结合片段的至少一个C-末端或N-末端通过连接子与6-HB干扰多 肽进行连接。在一些实施方案中，所述连接子为包含甘氨酸和丝氨酸的多肽。
一些实施方案中，所述融合蛋白包含抗体或抗原结合片段以及6-HB干扰多肽，所述抗体或抗原结合片段特异性结合spike蛋白，并且包含：(a)HCDR1，其包含如SEQ ID NO:1或2所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；(b)HCDR2，其包含如SEQ ID NO:3或4所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；(c)HCDR3，其包含如SEQ ID NO:5-42中任一项所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；(d)LCDR1，其包含如SEQ ID NO:43或44所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；(e)LCDR2，其包含如SEQ ID NO:45或46所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；和/或(f)LCDR3，其包含如SEQ ID NO:47或48所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；
所述抗体或抗原结合片段的至少一个C-末端或N-末端通过连接子与6-HB干扰多肽进行连接。在一些实施方案中，所述连接子为包含甘氨酸和丝氨酸的多肽。
在一些实施方案中，所述融合蛋白包含抗体或抗原结合片段以及6-HB干扰多肽，所述抗体或抗原结合片段特异性结合spike蛋白，并且包含：(a)HCDR1，其包含如SEQ ID NO:1或2所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；(b)HCDR2，其包含如SEQ ID NO:3或4所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；(c)HCDR3，其包含如SEQ ID NO:5-42中任一项所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；(d)LCDR1，其包含如SEQ ID NO:43或44所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；(e)LCDR2，其包含如SEQ ID NO:45或46所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；和(f)LCDR3，其包含如SEQ ID NO:47或48所示的氨基酸序列，或其有单一位点取代、缺失或插入的变体；
所述抗体或抗原结合片段的至少一个C-末端或N-末端通过连接子与6-HB干扰多肽进行连接。在一些实施方案中，所述连接子为包含甘氨酸和丝氨酸的多肽。
在一些实施方案中，所述取代变体为保守氨基酸取代变体。
在一些实施方案中，所述连接子的序列为(G mS) n，其中每个m独立为2、3、4或5，n独立为1、2、3、4或5。在一些实施方案中，所述连接子的序列为(GGGGS) n，所述n独立为1、2、3、4或5。在一些实施方案中，所述连接子为GGGGS。在一些实施方案中，所述连接子为(GGGGS) 2。在一些实施方案中，所述连接子为(GGGGS) 3。在一些实施方案中，所述连接子为(GGGGS) 4，如SEQ ID NO:64所示。在一些实施方案中，所述连接子为(GGGGS) 5。
在一些实施方案中，所述6-HB干扰多肽包含如SEQ ID NO:63所示的序列，或与 SEQ ID NO:63所示序列具有至少90％同一性的序列，或与SEQ ID NO:63所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述融合蛋白包含抗体或抗原结合片段以及6-HB干扰多肽，所述融合蛋白包含以下特征：
所述抗体或抗原结合片段至少包含如SEQ ID NO:1或2所示的HCDR1、如SEQ ID NO:3或4所示的HCDR2、如SEQ ID NO:5-42中任一项所示的HCDR3、如SEQ ID NO:43或44所示的LCDR1、如SEQ ID NO:45或46所示的LCDR2和如SEQ ID NO:47或48所示的LCDR3中的一个、两个、三个、四个、五个或全部；和/或
所述抗体或抗原结合片段的至少一个C-末端通过连接子与6-HB干扰多肽进行连接，所述C-末端为抗体或抗原结合片段中重链部分的C-末端或轻链部分的C-末端；和/或
所述连接子的序列为(GGGGS) n，所述n独立为1、2、3、4或5；和/或
所述6-HB干扰多肽包含如SEQ ID NO:63所示的序列，或与SEQ ID NO:63所示序列具有至少90％同一性的序列，或与SEQ ID NO:63所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述融合蛋白包含抗体或抗原结合片段以及6-HB干扰多肽，所述融合蛋白包含以下特征：
所述抗体或抗原结合片段至少包含如SEQ ID NO:1或2所示的HCDR1、如SEQ ID NO:3或4所示的HCDR2、如SEQ ID NO:5-42中任一项所示的HCDR3、如SEQ ID NO:43或44所示的LCDR1、如SEQ ID NO:45或46所示的LCDR2和如SEQ ID NO:47或48所示的LCDR3；和/或
所述抗体或抗原结合片段的至少一个C-末端通过连接子与6-HB干扰多肽进行连接，所述C-末端为抗体或抗原结合片段中重链部分的C-末端或轻链部分的C-末端；和/或
所述连接子的序列为(GGGGS) n，所述n独立为1、2、3、4或5；和/或
所述6-HB干扰多肽包含如SEQ ID NO:63所示的序列，或与SEQ ID NO:63所示序列具有至少90％同一性的序列，或与SEQ ID NO:63所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:5所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:6所示的HCDR3、如SEQ ID  NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:7所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:8所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:9所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:10所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:11所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:12所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:13所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:14所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的 HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:15所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:16所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:17所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:18所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:19所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:20所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:21所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:22所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:23所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:24所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:25所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:26所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:27所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:28所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:29所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:30所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:31所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:32所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示 的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:33所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:34所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:35所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:36所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:37所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:38所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:39所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:40所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:41所示的HCDR3、如SEQ  ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段至少包含如SEQ ID NO:2所示的HCDR1、如SEQ ID NO:4所示的HCDR2、如SEQ ID NO:42所示的HCDR3、如SEQ ID NO:44所示的LCDR1、如SEQ ID NO:46所示的LCDR2和如SEQ ID NO:48所示的LCDR3。
在一些实施方案中，所述抗体或抗原结合片段包含重链可变区，和轻链可变区。在一些实施方案中，所述重链可变区的框架区包含重链FR1、重链FR2、重链FR3和重链FR4。
在一些实施方案中，所述重链FR1包含SEQ ID NO:49或50所示的序列，或与SEQ ID NO:49或50所示序列具有至少90％同一性的序列，或与SEQ ID NO:49或50所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述重链FR2包含SEQ ID NO:51或52所示的序列，或与SEQ ID NO:51或52所示序列具有至少90％同一性的序列，或与SEQ ID NO:51或52所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述重链FR3包含SEQ ID NO:53或54所示的序列，或与SEQ ID NO:53或54所示序列具有至少90％同一性的序列，或与SEQ ID NO:53或54所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述重链FR4包含SEQ ID NO:55所示的序列，或与SEQ ID NO:55所示序列具有至少90％同一性的序列，或与SEQ ID NO:55所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述重链FR1包含SEQ ID NO:49或50所示的序列，或与SEQ ID NO:49或50所示序列具有至少90％同一性的序列，或与SEQ ID NO:49或50所示序列相比具有一或多个保守氨基酸取代的氨基酸序列；所述重链FR2包含SEQ ID NO:51或52所示的序列，或与SEQ ID NO:51或52所示序列具有至少90％同一性的序列，或与SEQ ID NO:51或52所示序列相比具有一或多个保守氨基酸取代的氨基酸序列；所述重链FR3包含SEQ ID NO:53或54所示的序列，或与SEQ ID NO:53或54所示序列具有至少90％同一性的序列，或与SEQ ID NO:53或54所示序列相比具有一或多个保守氨基酸取代的氨基酸序列；所述重链FR4包含SEQ ID NO:55所示的序列，或与SEQ ID NO:55所示序列具有至少90％同一性的序列，或与SEQ ID NO:55所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。在一些实施方案中，一或多个保守氨基酸取代为约1个、约2个或约3个保守氨基酸取代。
在一些实施方案中，所述重链FR1包含SEQ ID NO:49所示的序列，所述重链FR2包含SEQ ID NO:51所示的序列，所述重链FR3包含SEQ ID NO:53所示的序列，所述 重链FR4包含SEQ ID NO:55所示的序列。在一些实施方案中，所述重链FR1包含SEQ ID NO:50所示的序列，所述重链FR2包含SEQ ID NO:52所示的序列，所述重链FR3包含SEQ ID NO:54所示的序列，所述重链FR4包含SEQ ID NO:55所示的序列。在一些实施方案中，所述重链可变区包含结构重链FR1-HCDR1-重链FR2-HCDR2-重链FR3-HCDR3-重链FR4。
在一些实施方案中，所述抗体或抗原结合片段的重链可变区包含如SEQ ID NO:49所示的重链FR1、如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:51所示的重链FR2、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:53所示的重链FR3、如SEQ ID NO:5-41中任一项所示的HCDR3和如SEQ ID NO:55所示的重链FR4。
在一些实施方案中，所述抗体或抗原结合片段的重链可变区包含如SEQ ID NO:50所示的重链FR1、如SEQ ID NO:2所示的HCDR1、如SEQ ID NO:52所示的重链FR2、如SEQ ID NO:4所示的HCDR2、如SEQ ID NO:54所示的重链FR3、如SEQ ID NO:42所示的HCDR3和如SEQ ID NO:55所示的重链FR4。
在一些实施方案中，所述抗体或抗原结合片段的重链可变区包含SEQ ID NO:56或57所示的序列，或与SEQ ID NO:56或57所示序列具有至少80％同一性的序列，或与SEQ ID NO:56或57所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO:58或59所示的序列，或与SEQ ID NO:58或59所示序列具有至少80％同一性的序列，或与SEQ ID NO:58或59所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述抗体或抗原结合片段的重链可变区包含SEQ ID NO:56所示的序列，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO:58所示的序列。
在一些实施方案中，所述抗体或抗原结合片段的重链可变区包含SEQ ID NO:57所示的序列，所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO:59所示的序列。
在一些实施方案中，抗体或抗原结合片段还包含重链恒定区、轻链恒定区、Fc区或其组合。在一些实施方案中，轻链恒定区是κ或λ链恒定区。在一些实施方案中，抗体或其片段是IgG、IgM、IgA、IgE或IgD其中一种同种型。在一些实施方案中，同种型是IgG1、IgG2、IgG3或IgG4。在一些实施方案中，抗体或抗原结合片段中重链恒定区的C末端被截短。在一些实施方案中，IgG1或IgG4型抗体中重链恒定区的C末端缺少氨基酸残基G和K。没有限制地，抗体或抗原结合片段是嵌合抗体、人源化抗体或是全人源抗体。在某一方面，抗体或抗原结合片段是人源化抗体。
在一些实施方案中，所述抗体或抗原结合片段为分离的抗体或抗原结合片段。在一些实施方案中，所述抗体或抗原结合片段为scFV、Fab、F(ab) 2或IgG。在一些实施 方案中，所述抗体或抗原结合片段为单克隆抗体。
在一些实施方案中，所述抗体或抗原结合片段包含重链和轻链，所述重链包含本文所述重链可变区和重链恒定区，所述轻链包含本文所述轻链可变区和轻链恒定区。在一些实施方案中，所述抗体或抗原结合片段的重链恒定区包含氨基酸序列如SEQ ID NO:60或61中第1位氨基酸至第328位氨基酸所示的序列，或与SEQ ID NO:60或61中第1位氨基酸至第328位氨基酸所示序列具有至少80％同一性的序列，或与SEQ ID NO:60或61中第1位氨基酸至第328位氨基酸所示序列相比具有一或多个保守氨基酸取代的氨基酸序列；和/或
所述抗体或抗原结合片段的轻链恒定区包含氨基酸序列如SEQ ID NO:62所示的序列，或与SEQ ID NO:62所示序列具有至少80％同一性的序列，或与SEQ ID NO:62所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
在一些实施方案中，所述抗体或抗原结合片段的重链恒定区包含氨基酸序列如SEQ ID NO:60中第1位氨基酸至第328位氨基酸所示的序列，所述抗体或抗原结合片段的轻链恒定区包含氨基酸序列如SEQ ID NO:62所示的序列。
在一些实施方案中，所述抗体或抗原结合片段的重链恒定区包含氨基酸序列如SEQ ID NO:61中第1位氨基酸至第328位氨基酸所示的序列，所述抗体或抗原结合片段的轻链恒定区包含氨基酸序列如SEQ ID NO:62所示的序列。
在一些实施方案中，所述重链或其片段的C-末端或N-末端通过连接子与6-HB干扰多肽进行连接。在一些实施方案中，所述轻链或其片段的C-末端通过连接子与6-HB干扰多肽进行连接。
在一些实施方案中提供了一种融合蛋白，所述融合蛋白包含抗体以及6-HB干扰多肽，所述融合蛋白包含以下特征：
所述抗体的重链包含氨基酸序列如SEQ ID NO:65或66中第1位氨基酸至第450位氨基酸所示的序列或如SEQ ID NO:66中第1位氨基酸至第451位氨基酸所示的序列，或与SEQ ID NO:65或66中第1位氨基酸至第450位氨基酸所示的序列或如SEQID NO:66中第1位氨基酸至第451位氨基酸所示的序列具有至少80％同一性的序列，或与SEQ ID NO:65或66中第1位氨基酸至第450位氨基酸所示的序列或如SEQ ID NO:66中第1位氨基酸至第451位氨基酸所示的序列相比具有一或多个保守氨基酸取代的氨基酸序列；和/或
所述抗体的轻链包含氨基酸序列如SEQ ID NO:67或68所示的序列，或与SEQ ID NO:67或68所示序列具有至少80％同一性的序列，或与SEQ ID NO:67或68所示序列相比具有一或多个保守氨基酸取代的氨基酸序列；和/或
抗体的重链的C-末端或轻链C-末端通过如SEQ ID NO:64所示的连接子与6-HB 干扰多肽进行共价连接，所述6-HB干扰多肽包含如SEQ ID NO:63所示的序列，或与SEQ ID NO:63所示序列具有至少90％同一性或至少95％同一性的序列，或与SEQ ID NO:63所示序列相比具有一或多个保守氨基酸取代的的氨基酸序列。
在一些实施方案中，所述融合蛋白包含抗体以及通过连接子连接的6-HB干扰多肽，所述抗体的重链包含氨基酸序列如SEQ ID NO:65中第1位氨基酸至第450位氨基酸所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO:67所示的序列；抗体的重链的C-末端(即CH3末端)或轻链C-末端(即CL末端)通过如SEQ ID NO:64所示的连接子与6-HB干扰多肽进行共价连接，所述6-HB干扰多肽包含如SEQ ID NO:63所示的序列。
在一些实施方案中，所述融合蛋白包含抗体以及通过连接子连接的6-HB干扰多肽，所述抗体的重链包含氨基酸序列如SEQ ID NO:66中第1位氨基酸至第451位氨基酸所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO:68所示的序列；抗体的重链的C-末端(即CH3末端)或轻链C-末端(即CL末端)通过如SEQ ID NO:64所示的连接子与6-HB干扰多肽进行共价连接，所述6-HB干扰多肽包含如SEQ ID NO:63所示的序列。
在一些实施方案中，融合蛋白包含2条序列相同的第一多肽和2条序列相同的第四多肽。在一些实施方案中，所述第一多肽包含抗体或抗原结合片段的重链或其片段，或由其组成，所述第四多肽包含抗体或抗原结合片段的轻链或其片段以及与其依次连接的连接子、6-HB干扰多肽，或由其组成。
在一些实施方案中，融合蛋白包含2条序列相同的第二多肽和2条序列相同的第三多肽。在一些实施方案中，所述第三多肽包含抗体或抗原结合片段的重链或其片段以及与其依次连接的连接子、6-HB干扰多肽，或由其组成，所述第二多肽包含轻链或其片段，或由其组成。
在一些实施方案中，融合蛋白包含氨基酸序列如SEQ ID NO:69所示的序列，或由其组成；融合蛋白还包含氨基酸序列如SEQ ID NO:67所示的序列，或由其组成。
在一些实施方案中，所述融合蛋白中抗体部分可以特异性结合spike蛋白，并阻止SARS-CoV或SARS-CoV-2病毒颗粒和细胞结合，以及介导免疫细胞吞噬、清除病毒颗粒。在一些实施方案中，所述融合蛋白中抗体或抗原结合片段体可以特异性结合spike蛋白中S1亚基。
本发明还提供了编码所述的抗体、抗原结合片段或其融合蛋白的核酸。在一些实施方案中，所述核酸为分离的核酸。
本发明还提供了包含所述的核酸的载体。在一些实施方案中，所述载体为分离的 载体。在一些实施方案中，包含所述核酸的载体为核酸片段、质粒、噬菌体或病毒。在一些实施方案中，所述载体为分离的质粒。
本发明还提供了包含所述核酸或所述载体的宿主细胞。在一些实施方案中，所述宿主细胞为分离的宿主细胞。在一些实施方案中，所述宿主细胞为CHO细胞、HKE293细胞、Cos1细胞、Cos7细胞、CV1细胞和鼠L细胞。
本发明还提供了药物组合物，所述药物组合物包含所述抗体、抗原结合片段或融合蛋白，以及药学上可接受的辅料。
本发明还提供了治疗方法和用途。在一些实施方案中，提供了用于预防、治疗或改善SARS或COVID-19的方法，所述方法包括向患者施用有效剂量的所述的抗体、抗原结合片段或融合蛋白。在一些实施方案中，提供了所述抗体、抗原结合片段或融合蛋白或药物组合物在预防、治疗或改善SARS或COVID-19中的应用。在一些实施方案中，提供了所述抗体、抗原结合片段或融合蛋白或药物组合物在制备用于预防、治疗或改善SARS或COVID-19的药物中的应用。
本发明还提供了诊断方法和用途。在一些实施方案中，提供了检测样品中SARS-CoV或SARS-CoV-2表达的方法，使样品与所述抗体、抗原结合片段或融合蛋白进行接触，使得所述抗体、抗原结合片段或融合蛋白中抗体结合spike蛋白，并检测其结合，即样品中spike蛋白的含量。在一些实施方案中，提供了所述抗体、抗原结合片段或融合蛋白在制备用于诊断SARS或COVID-19的试剂盒中的应用。在一些实施方案中，提供了包含所述抗体、抗原结合片段或融合蛋白的诊断试剂盒。
本发明提供了冠状病毒抗体及其应用，融合蛋白中6-HB干扰多肽与抗体协同阻止SARS-CoV或SARS-CoV-2病毒颗粒和细胞融合，以及介导免疫细胞吞噬、清除病毒颗粒，预防、治疗SARS或COVID-19；本发明抗体、抗原结合片段或融合蛋白中抗体还可以用于诊断检测患者是否感染SARS-CoV或SARS-CoV-2。
附图说明
图1示抗体阻断spike RBD与ACE2的结合；其中，ACE2对照组中未添加抗体。
术语
除非另作说明，否则下列的每一个术语应当具有下文所述的含义。
定义
应当注意的是，术语“一种”实体是指一种或多种该实体，例如“一种抗体”应当被理解为一种或多种抗体，因此，术语“一种”(或“一个”)、“一种或多种”和“至少一种”可以在本文中互换使用。
本文所用的术语“包含”或“包括”意味着组合物和方法等包括所列举的元素，例如组份或步骤，但不排除其它。“基本上由……组成”意味着组合物和方法排除对组合的特征有根本影响的其它元素，但不排除对组合物或方法无本质上影响的元素。“由……组成”意味着排除未特别列举的元素。
术语“多肽”旨在涵盖单数的“多肽”以及复数的“多肽”，并且是指由通过酰胺键(也称为肽键)线性连接的氨基酸单体构成的分子。术语“多肽”是指含两个或更多个氨基酸的任何单条链或多条链，并且不涉及产物的特定长度。因此，“多肽”的定义中包括肽、二肽、三肽、寡肽、“蛋白质”、“氨基酸链”或用于指两个或多个氨基酸链的任何其他术语，并且术语“多肽”可以用来代替上述任何一个术语，或者与上述任何一个术语交替使用。术语“多肽”也意在指多肽表达后修饰的产物，包括但不限于糖基化、乙酰化、磷酸化、酰胺化、通过已知的保护/封闭基团衍生化、蛋白水解切割或非天然发生的氨基酸修饰。多肽可以源自天然生物来源或通过重组技术产生，但其不必从指定的核酸序列翻译所得，它可能以包括化学合成的任何方式产生。
“氨基酸”是指既含氨基又含羧基的有机化合物，比如α-氨基酸，其可直接或以前体的形式由核酸编码。单个氨基酸由三个核苷酸(所谓的密码子或碱基三联体)组成的核酸编码。每一个氨基酸由至少一个密码子编码。相同氨基酸由不同密码子编码称为“遗传密码的简并性”。氨基酸包括天然氨基酸和非天然氨基酸。天然氨基酸包括丙氨酸(三字母代码：ala，一字母代码：A)、精氨酸(arg，R)、天冬酰胺(asn，N)、天冬氨酸(asp，D)、半胱氨酸(cys，C)、谷氨酰胺(gln，Q)、谷氨酸(glu，E)、甘氨酸(gly，G)、组氨酸(his，H)、异亮氨酸(ile，I)、亮氨酸(leu，L)、赖氨酸(lys，K)、甲硫氨酸(met，M)、苯丙氨酸(phe，F)、脯氨酸(pro，P)、丝氨酸(ser，S)、苏氨酸(thr，T)、色氨酸(trp，W)、酪氨酸(tyr，Y)和缬氨酸(val，V)。
“保守氨基酸取代”是指一个氨基酸残基被另一个含有化学性质(例如电荷或疏水性)相似的侧链(R基团)的氨基酸残基所取代。一般而言，保守氨基酸取代不大会在实质上改变蛋白质的功能性质。含有化学性质相似侧链的氨基酸类别的实例包括：1)脂族侧链：甘氨酸、丙氨酸、缬氨酸、亮氨酸和异亮氨酸；2)脂族羟基侧链：丝氨酸和苏氨酸；3)含酰胺的侧链：天冬酰胺和谷氨酰胺；4)芳族侧链：苯丙氨酸、酪氨酸和色氨酸；5)碱性侧链：赖氨酸、精氨酸和组氨酸；6)酸性侧链：天冬氨酸和谷氨酸。
“VL、VH的保守氨基酸取代”的氨基酸数目为约1个、约2个、约3个、约4个、约5个、约6个、约8个、约9个、约10个、约11个、约13个、约14个、约15个保守氨基酸取代，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。“重链恒定区、轻链恒定区、重链或轻链、融合蛋白第一多肽或第二多肽的保守氨基酸 取代”的氨基酸数目为约1个、约2个、约3个、约4个、约5个、约6个、约8个、约9个、约10个、约11个、约13个、约14个、约15个、约18个、约19个、约22个、约24个、约25个、约29个、约31个、约35个、约38个、约41个、约45个保守氨基酸取代，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。
本发明中关于细胞、核酸、多肽、抗体等所使用的术语“分离的”，例如“分离的”DNA、RNA、多肽、抗体是指分别于细胞天然环境中的其它组分如DNA或RNA中的一种或多种所分离的分子。本发明使用的术语“分离的”还指当通过重组DNA技术产生时基本上不含细胞材料、病毒材料或细胞培养基的核酸或肽，或化学合成时的化学前体或其他化学品。此外，“分离的核酸”意在包括不以天然状态存在的核酸片段，并且不会以天然状态存在。术语“分离的”在本发明中也用于指从其他细胞蛋白质或组织分离的细胞或多肽。分离的多肽意在包括纯化的和重组的多肽。分离的多肽、抗体等通常通过至少一个纯化步骤制备。在一些实施方案中，分离的核酸、多肽、抗体等的纯度至少为约50％、约60％、约70％、约80％、约90％、约95％、约99％，或这些数值中的任何两个值之间的范围(包括终点)或其中任何值。
术语“重组”涉及多肽或多聚核苷酸，意指天然不存在的多肽或多聚核苷酸的形式，不受限制的实施例可以通过组合产生通常并不存在的多聚核苷酸或多肽。
“同源性”或“同一性”或“相似性”是指两个肽之间或两个核酸分子之间的序列相似性。可以通过比较每个序列中可以比对的位置来确定同源性。当被比较的序列中的位置被相同的碱基或氨基酸占据时，则分子在该位置是同源的。序列之间的同源程度是由序列共有的匹配或同源位置的数目组成的一个函数。
“至少80％同一性”为约80％同一性、约81％同一性、约82％同一性、约83％同一性、约85％同一性、约86％同一性、约87％同一性、约88％同一性、约90％同一性、约91％同一性、约92％同一性、约94％同一性、约95％同一性、约98％同一性、约99％同一性，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。
“至少90％同一性”为约90％同一性、约91％同一性、约92％同一性、约93％同一性、约94％同一性、约95％同一性、约92％同一性、约96％同一性、约99％同一性，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值。多聚核苷酸或多聚核苷酸序列(或多肽或抗体序列)与另一序列有具有一定百分比(例如90％、95％、98％或者99％)的“同一性”或“序列同一性”是指当序列比对时，所比较的两个序列中该百分比的碱基(或氨基酸)相同。可以使用目测或本领域已知的软件程序来确定该比对同一性百分比或序列同一性，比如Ausubel et al.eds.(2007)在Current Protocols in Molecular Biology中所述的软件程序。优选使用默认参数进行比对。其中一种比对程 序是使用默认参数的BLAST，例如BLASTN和BLASTP，两者使用下列默认参数：Geneticcode＝standard；filter＝none；strand＝both；cutoff＝60；expect＝10；Matrix＝BLOSUM62；Descriptions＝50sequences；sortby＝HIGHSCORE；Databases＝non-redundant；GenBank+EMBL+DDBJ+PDB+GenBankCDStranslations+SwissProtein+SPupdate+PIR。生物学上等同的多聚核苷酸是具有上述指定百分比的同一性并编码具有相同或相似生物学活性的多肽的多聚核苷酸。
多聚核苷酸是由四个核苷酸碱基的特定序列组成：腺嘌呤(A)、胞嘧啶(C)、鸟嘌呤(G)、胸腺嘧啶(T)，或当多聚核苷酸是RNA时胸腺嘧啶换为尿嘧啶(U)。“多聚核苷酸序列”可以以多聚核苷酸分子的字母表示。该字母表示可以被输入到具有中央处理单元的计算机中的数据库中，并用于生物信息学应用，例如用于功能基因组学和同源性搜索。
术语“多聚核苷酸”和“寡核苷酸”可互换使用，是指任何长度的核苷酸的聚合形式，无论是脱氧核糖核苷酸还是核糖核苷酸或其类似物。多聚核苷酸可以具有任何三维结构并且可以执行已知或未知的任何功能。以下是不受限制的多聚核苷酸的实施例：基因或基因片段(例如探针、引物、EST或SAGE标签)、外显子、内含子、信使RNA(mRNA)、转运RNA、核糖体RNA、核糖酶、cDNA、dsRNA、siRNA、miRNA、重组多聚核苷酸、分支的多聚核苷酸、质粒、载体、任何序列的分离的DNA、任何序列的分离的RNA、核酸探针和引物。多聚核苷酸可以包含修饰的核苷酸，例如甲基化的核苷酸和核苷酸类似物。如果存在该修饰，则对核苷酸的结构修饰可以在组装多聚核苷酸之前或之后进行。核苷酸的序列可以被非核苷酸组分中断。聚合后可以进一步修饰多聚核苷酸，例如通过与标记组分缀合。这个术语也指双链和单链分子。除另有说明或要求外，本公开的任何多聚核苷酸的实施例包括双链形式和已知或预测构成双链形式的两种可互补单链形式中的每一种。
术语“编码”应用于多核苷酸时，是指被称为“编码”多肽的多核苷酸，在其天然状态或当通过本领域技术人员公知的方法操作时，经转录和/或翻译可以产生该多肽和/或其片段。
“抗体”、“抗原结合片段”是指特异性识别和结合抗原的多肽或多肽复合物。抗体可以是完整的抗体及其任何抗原结合片段或其单链。因此术语“抗体”包括分子中含有具有与抗原结合的生物学活性的免疫球蛋白分子的至少一部分的任何蛋白质或肽。抗体和抗原结合片段包括但不局限实施例所述的重链或轻链或其配体结合部分的互补决定区(CDR)、重链可变区(VH)、轻链可变区(VL)、重链恒定区(CH)、轻链恒定区(CL)、框架区(FR)或其任何部分，或结合蛋白的至少一部分。CDR区包括轻链的CDR区(LCDR1-3)和重链的CDR区(HCDR1-3)。可变区可包含结构 FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
术语“抗体片段”或“抗原结合片段”指抗体的一部分，例如F(ab’) 2、F(ab) 2、Fab'、Fab、Fv、scFv等。不管其结构如何，抗体片段与被完整抗体识别的同一抗原结合。术语“抗体片段”包括适体、镜像异构体和双价抗体。术语“抗原结合片段”还包括通过与特定抗原结合形成复合物起抗体作用的任何合成或基因工程蛋白质。
“单链可变片段”或“scFv”是指免疫球蛋白的重链可变区(VH)和轻链可变区(VL)的融合蛋白。在一些方面，这些区域与10个至约25个氨基酸的短接头肽连接。接头可以富含甘氨酸以增加柔韧性，以及富含丝氨酸或苏氨酸以增加溶解性，并且可以连接VH的N端和VL的C端，反之亦然。尽管该蛋白质被除去了恒定区和引入了接头，但其保留了原始免疫球蛋白的特异性。ScFv分子通常是本领域中已知的，例如在美国专利5,892,019中有相关描述。
术语“抗体”包括可以在生物化学上区分的各种广泛种类的多肽。本领域技术人员将会理解，重链的类别包括gamma、mu、alpha、delta或epsilon(γ、μ、α、δ、ε)，其中还有一些亚类(例如γ1-γ4)。该链的性质决定了抗体的“种类”分别为IgG、IgM、IgA、IgG或IgE。免疫球蛋白亚类(同种型)，例如IgG1、IgG2、IgG3、IgG4、IgG5等已被充分表征并且赋予的功能特异性也已知。所有的免疫球蛋白种类都在本发明公开的保护范围内。在一些实施方案中，免疫球蛋白分子为IgG种类。IgG通常包含分子量约23,000道尔顿的两条相同的轻链多肽和分子量约为53,000-70,000的两条相同的重链多肽。这四条链通过二硫键以“Y”构型连接，其中轻链从“Y”口开始并延续通过可变区包围重链。
本发明公开的抗体、抗原结合片段、或衍生物包括但不限于多克隆、单克隆、多特异性，全人源、人源化、灵长类化、嵌合抗体，单链抗体、表位结合片段例如Fab、Fab'和F(ab') 2、Fd、Fvs、单链Fvs(scFv)，二硫键连接的Fvs(sdFv)，包含VK或VH结构域的片段，或由Fab表达文库产生的片段和抗独特型(抗Id)抗体。本发明公开的免疫球蛋白或抗体分子可以是免疫球蛋白的任何类型(例如IgG、IgE、IgM、IgD、IgA和IgY)或种类(例如，IgG1、IgG2、IgG3、IgG4、IgA1和IgA2)或者亚类。
轻链可以分为kappa(κ)或lambda(λ)。每个重链可以与κ或λ轻链结合。一般来说，当由杂交瘤，B细胞或基因工程宿主细胞生产免疫球蛋白时，其轻链和重链通过共价键结合，两条重链的“尾巴”部分通过共价二硫键或非共价键结合。在重链中，氨基酸序列从Y构型的叉状末端的N末端延伸至每条链底部的C末端。免疫球蛋白κ轻链可变区为Vκ；免疫球蛋白λ轻链可变区为V λ。
轻链和重链都分成结构和功能同源性的区域。术语“恒定的”和“可变的”根据功能被使用。轻链可变区(VL)和重链可变区(VH)决定了抗原识别和特异性。轻链和重链的恒定区赋予重要的生物学性质，如分泌、经胎盘移动、Fc受体结合、补体结合等。按照惯例，恒定区的编号随着它们变得更远离抗体的抗原结合位点或氨基末端而增加。N端部分是可变区，C端部分是恒定区；CH3和CL结构域实际上分别包含重链和轻链的羧基端。
如上所述，可变区使得抗体能够选择性识别和特异性结合抗原上的表位。具体而言，抗体的VL结构域和VH结构域或互补决定区(CDR)的子集结合形成了限定三维抗原结合位点的可变区。该抗体四级结构形成存在于Y的每个臂末端的抗原结合位点。更具体地说，抗原结合位点由VH和VL链中各自的三个CDR(即HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3)定义。在某些情况下，例如某些来源于骆驼科动物的免疫球蛋白分子或基于骆驼科动物免疫球蛋白改造的免疫球蛋白分子，完整的免疫球蛋白分子可以仅由重链组成，没有轻链。例如参见Hamers-Casterman et al.，Nature，363:446-448(1993)。
在天然存在的抗体中，假设抗体在含水环境中呈现其三维构型时，存在于每个抗原结合域中的六个“互补决定区”或“CDR”是形成抗原结合结构域的短的、非连续的与抗原特异性结合的氨基酸序列。抗原结合结构域中被称为“构架”区域的剩余其它氨基酸显示出较小的分子间可变性。构架区大部分采用β-折叠构象，CDR形成与之连接的环状结构，或在某些情况下形成β折叠结构的一部分。因此，框架区通过形成支架从而通过链间非共价相互作用使CDR定位在正确的方位上。具有特定位置的CDR的抗原结合域形成了与抗原上的表位互补的表面，该互补表面促进抗体和其抗原表位的非共价结合。对于给定的重链或轻链可变区，本领域普通技术人员都可以通过已知方法鉴定出包含CDR和框架区的氨基酸(参见Kabat,E.,et al.,U.S.Department of Health and Human Services,Sequences of Proteins of Immunological Interest,(1983)和Chothia and Lesk,J.Mol.Biol.,196:901-917(1987))。
在本领域中使用和/或接受的术语有两个或多个定义的情况下，除非明确地对立指出，否则本文使用的术语的定义包括所有这些含义。一个具体的例子是使用“互补决定区”(“CDR”)一词来描述在重链和轻链多肽的可变区内发现的非连续的抗原结合位点。这一特定区域在Kabat et al.,U.S.Dept.of Health and Human Services,Sequences of Proteins of Immunological Interest(1983)和Chothia等在J.Mol.Biol.196:901-917(1987)有相关描述，其通过引用全部并入本文。
根据Kabat和Chothia定义的CDR包括相互比较时的氨基酸残基的重叠或子集。尽管如此，应用任一定义来指代抗体或其变体的CDR都在本发明范围内。包含特定 CDR的确切残基编号将根据CDR的序列和大小而变化。本领域技术人员通常可以根据抗体的可变区氨基酸序列确定出CDR包含哪些特定的残基。
Kabat等人还定义了适用于任何抗体的可变区序列的编号系统。本领域普通技术人员可以不依赖于序列本身以外的其他实验数据将该“Kabat编号”系统应用到任何可变区序列。“Kabat编号”是指由Kabat et al.,U.S.Dept.of Health and Human Services在“SequenceofProteinsof Immunological Interest”(1983)提出的编号系统。抗体还可以用EU编号系统。
本发明公开的抗体或抗原结合片段可以来源于任何动物，包括鸟类和哺乳动物。较佳地，抗体是人源、鼠源、驴源、兔源、山羊源、骆驼源、美洲驼源、马源或鸡源抗体。在另一实施方案中，可变区可以是软骨鱼纲(condricthoid)来源(例如来自鲨鱼)。
“重链恒定区”包括来源于免疫球蛋白重链的氨基酸序列。包含重链恒定区的多肽包括CH1结构域、铰链(例如上、中和/或下铰链区)结构域、CH2结构域、CH3结构域，或变体或片段中的至少一种。例如，本发明公开的抗体或抗原结合片段包含CH1结构域；包含CH1结构域、至少一部分铰链区以及CH2结构域；包含CH1结构域和CH3结构域；包含CH1结构域以及至少一部分铰链区以及CH3结构域；或包含CH1结构域、至少一部分铰链区以及CH2结构域和CH3结构域。在另一实施方案中，本发明公开的抗体或抗原结合片段包含CH3结构域。此外，本发明中使用的抗体或抗原结合片段可能缺部分或全部CH2结构域。如上所述，本领域普通技术人员应当理解，重链恒定区可以被修饰从而使得它们天然存在的免疫球蛋白分子的氨基酸序列发生变化。
抗体的重链恒定区可以来源于不同的免疫球蛋白分子。例如，多肽的重链恒定区可以包括源自IgG 1分子的CH1结构域和源自IgG 3分子的铰链区。在另一实施方案中，重链恒定区可以包括部分源自IgG 1分子和部分源自IgG 3分子的铰链区。在另一实施方案中，部分重链可以包括部分源自IgG 1分子和部分源自IgG 4分子的嵌合铰链区。
“轻链恒定区”包括来自抗体轻链的氨基酸序列。较佳地，轻链恒定区包含恒定κ结构域或恒定λ结构域中的至少一个。“轻链-重链对”是指可通过轻链的CL结构域和重链的CH1结构域之间的二硫键形成二聚体的轻链和重链的集合。
如上所述，各种免疫球蛋白种类的恒定区的亚基结构和三维构型是众所周知的。“VH结构域”包括免疫球蛋白重链的氨基末端可变结构域，“CH1结构域”包括免疫球蛋白重链的第一个(大部分氨基末端)恒定区。CH1结构域与VH结构域相邻，并且是免疫球蛋白重链分子铰链区的氨基端。CH2结构域不与其它结构域紧密配对，而是在 完整的天然IgG分子的两个CH2结构域之间插入两个N-连接的分支碳水化合物链。CH3结构域从CH2结构域开始延伸到IgG分子的C-末端，大约包含108个残基。“铰链区”包括连接CH1结构域和CH2结构域的部分重链区域。所述铰链区包含约25个残基并且是有韧性的，从而使得两个N端抗原结合区能够独立移动。铰链区可以被细分为三个不同的结构域：上、中和下铰链结构域(Roux et al.,J.Immunol.161:4083(1998))。
“二硫键”指两个硫原子之间形成的共价键。半胱氨酸的硫醇基团可以与第二个硫醇基团形成二硫键或桥接。在大多数天然存在的IgG分子中，CH1和CL区通过二硫键连接，两条重链通过两个二硫键在Kabat编号系统中对应的位置239和242(EU编号系统位置226和229)处相连接。
“嵌合抗体”指其可变区从第一个物种中获得或衍生，而其恒定区(可以是完整的、部分的或修饰过的)来源于第二个物种的任何抗体。某些实施方案中，可变区来自非人源(例如小鼠或灵长类动物)，而恒定区来自人源。
“特异性结合”或“对……具有特异性”通常是指抗体或抗原结合片段与特定抗原通过其抗原结合结构域与表位互补性结合形成相对稳定的复合物。“特异性”可以用抗体或抗原结合片段与特定抗原或表位结合的相对亲和力表达。例如，如果抗体“A”比抗体“B”与同一抗原的相对亲和力大，可以认为抗体“A”比抗体“B”对该抗原具有更高的特异性。特异性结合可以用平衡解离常数(KD)来描述，较小的KD意味着较紧密的结合。确定两个分子是否特异性结合的方法是本领域内众所周知的，并包括例如平衡透析、表面等离子共振、生物膜层光学干涉测量法等。“特异性结合”spike蛋白的抗体包括与spike蛋白平衡解离常数KD小于或等于约100nM、小于或等于约10nM、小于或等于约5nM、小于或等于约1nM。
“治疗”是指治疗性治疗和预防性或防治性措施，其目的是预防、减缓、改善或停止不良的生理改变或紊乱，例如疾病的进程，包括但不限于以下无论是可检测还是不可检测的结果，症状的缓解、疾病程度的减小、疾病状态的稳定(即不恶化)、疾病进展的延迟或减缓、疾病状态的改善、缓和、减轻或消失(无论是部分还是全部)、延长与不接受治疗时预期的生存期限等。需要治疗的患者包括已经患有病症或紊乱的患者，容易患有病症或紊乱的患者，或者需要预防该病症或紊乱的患者，可以或预期从施用本发明公开的抗体或药物组合物用于检测、诊断过程和/或治疗中受益的患者。
“患者”指需要诊断、预后或治疗的任何哺乳动物，包括人类、狗、猫、兔子、大鼠、小鼠、马、牛等。
本文提及出版物的相关描述均通过引用全部并入本文。
抗体和融合蛋白
本发明提供了对spike蛋白具有高亲和力的抗体或抗原其结合片段。抗体或抗原结合片段表现出有效的结合活性，并可用于治疗和诊断用途。比如，这些抗体或抗原结合片段可以阻止SARS-CoV和SARS-CoV-2病毒颗粒和细胞膜融合，以及介导免疫细胞吞噬、清除病毒颗粒。
在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO:65或66所示的序列，和/或所述抗体的轻链包含氨基酸序列如SEQ ID NO:67或68所示的序列。在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO:65所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO:67所示的序列。在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO:65中除Fc区以外的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO:67所示的序列。在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO:66所示的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO:68所示的序列。在一些实施方案中，所述抗体的重链包含氨基酸序列如SEQ ID NO:66中除Fc区以外的序列，所述抗体的轻链包含氨基酸序列如SEQ ID NO:68所示的序列。
本发明还提供了融合蛋白。融合蛋白包含抗体或抗原结合片段以及6-HB干扰多肽。融合蛋白中抗体或抗原结合片段对SARS-CoV和SARS-CoV-2的spike蛋白具有高亲和力，这些抗体或抗原结合片段可以特异性结合spike蛋白；融合蛋白中6-HB干扰多肽可干扰“六螺旋束(6-HB)”的形成。融合蛋白表现出有效的结合活性，并可用于治疗和诊断用途。比如，融合蛋白中这些抗体或抗原结合片段可以阻止SARS-CoV和SARS-CoV-2病毒颗粒和细胞膜融合，以及介导免疫细胞吞噬、清除病毒颗粒。
在一些实施方案中，抗体的重链的C-末端(即CH3末端)通过连接子与6-HB干扰多肽进行共价连接。在一些实施方案中，抗体的轻链C-末端(即CL末端)通过连接子与6-HB干扰多肽进行共价连接。
在一些实施方案中，本发明公开的融合蛋白包含6-HB干扰多肽以及抗体或抗原结合片段，抗体或抗原结合片段的重链(或重链片段)的C-末端或轻链C-末端通过连接子(如SEQ ID NO:64所示)与6-HB干扰多肽(如SEQ ID NO:63所示)进行共价连接。
在一些实施方案中，融合蛋白包含2条序列相同的第一多肽和2条序列相同的第四多肽，所述第四多肽包含依次连接的抗体或抗原结合片段的轻链或其片段、连接子和6-HB干扰多肽。在一些实施方案中，所述第一多肽包含抗体或抗原结合片段的重链或其片段，或由其组成；所述第四多肽包含抗体或抗原结合片段的轻链或其片段以及与其依次连接的连接子、6-HB干扰多肽，或由其组成。
在一些实施方案中，融合蛋白包含2条序列相同的第二多肽和2条序列相同的第三多肽，所述第三多肽包含依次连接的抗体或抗原结合片段的重链或其片段、连接子和6-HB干扰多肽。在一些实施方案中，所述第三多肽包含抗体或抗原结合片段的重链或其片段以及与其依次连接的连接子、6-HB干扰多肽，或由其组成，所述第二多肽包含轻链或其片段，或由其组成。
在一些实施方案中，融合蛋白包含氨基酸序列如SEQ ID NO:69所示的序列，或由其组成；融合蛋白还包含氨基酸序列如SEQ ID NO:67所示的序列，或由其组成。
在一些实施方案中，抗体，或融合蛋白例如其中抗体还可连接氨基酸序列或一个或多个修饰基团。例如，本发明公开的抗体，或融合蛋白例如其中抗体可以包含有韧性的接头序列，或者可以被修饰以添加功能性基团(例如PEG、药物、毒素或标签)。
本发明公开的抗体，或融合蛋白例如其中抗体还包含被修饰的衍生物，即通过任何类型的分子与抗体的共价连接进行修饰，其中共价连接不会阻止抗体与表位结合。包括但不限制以下实例，抗体可以被糖基化、乙酰化、聚乙二醇化、磷酸化、酰胺化、通过已知的保护/封闭基团衍生化、蛋白水解切割、连接至细胞配体或其他蛋白质等。众多化学修饰中的任一种修饰可以通过现有技术进行，包括但不限于特异性化学裂解、乙酰化、甲酰化、衣霉素的代谢合成等。
在一些实施方案中，抗体，或融合蛋白例如其中抗体可以与治疗剂、药物前体、肽、蛋白质、酶、病毒、脂类、生物反应调节剂、药剂或PEG进行缀合。
抗体，或融合蛋白例如其中抗体可以与治疗剂缀合或融合，所述治疗剂可包括可检测标记，如放射性标记、免疫调节剂、激素、酶、寡核苷酸、光敏治疗剂、诊断剂、细胞毒性剂、超声增强剂、非放射性标记物及其组合物，和本领域已知的其它此类试剂。
抗体，或融合蛋白例如其中抗体可通过将其偶联至化学发光化合物来被可检测地标记。然后通过检测在化学反应过程中出现的发光从而确定化学发光标记的抗体的存在。化学发光标记化合物的实例包括鲁米诺、异鲁米诺、芳香吖啶酯、咪唑、吖啶盐和草酸酯。
编码抗体、融合蛋白的多聚核苷酸和制备抗体、融合蛋白的方法
本发明还公开了编码本发明所述抗体、抗原结合片段或融合蛋白或其衍生物的多聚核苷酸或核酸分子。本发明公开的多聚核苷酸可以编码重链、轻链、重链可变区、轻链可变区、Fc区、部分重链可变区或部分轻链可变区或融合蛋白。制备抗体和融合蛋白的方法是本领域公知的并且在本发明中有所描述。在某些实施方案中，本发明公开的抗体、抗原结合片段的可变区和恒定区都是全人源的。全人源抗体和抗原结合片 段可以使用本领域中公开的技术和本发明所述的技术制备。例如，针对特定抗原的全人源抗体可以通过将抗原施用于转基因动物中来制备，所述转基因动物已经被改良过以响应抗原攻击而产生全人源抗体。可用于制备这种抗体的示例性技术参见美国专利6,458,592；6,420,140，其全部内容通过引用并入本文。
在某些实施方案中，制备的抗体、融合蛋白不会在待治疗的动物(例如人类)中引起有害的免疫应答。在一实施方案中，本发明公开的抗体、抗原结合片段、融合蛋白或衍生物使用本领域公认的技术修饰以降低其免疫原性。例如，抗体可以被人源化、灵长类化、去免疫化或者可以制备嵌合抗体。这些类型的抗体来源于非人抗体，通常是鼠类或灵长类抗体，其保留或基本保留亲本抗体的抗原结合特性但在人体中免疫原性较低。其可以通过多种方法来实现，包括(a)将整个非人源的可变区移植到人源的恒定区以产生嵌合抗体；(b)将一个或多个非人类互补决定区(CDR)的至少一部分移植到人源的框架和恒定区中，保留或不保留关键的框架残基；或(c)移植整个非人源的可变区，但通过用类人源的部分置换表面残基从而“隐藏”它们。通常人框架区中的框架残基将被来自CDR供体抗体的相应残基取代，比如能够改善抗原结合的残基。这些框架替换可以通过本领域公知的方法鉴定，例如通过模拟CDR和框架残基的相互作用以鉴定对抗原结合起重要作用的框架残基和通过序列对比以鉴定特定位置上异常的框架残基。(参考美国专利5,585,089；其全部内容通过引用并入本文)。可以使用本领域公知的多种技术使抗体人源化，例如CDR移植(EP 239,400；WO 91/09967；美国专利5,225,539,5,530,101和5,585,089)，修复或者表面重排(EP592,106；EP519,596)，以及链的重排(美国专利5,565,332)，其全部内容通过引用并入本文。
去免疫化也可用于降低抗体的免疫原性。在本发明中，术语“去免疫化”包括改变抗体以修饰T细胞表位(参见例如WO/9852976A1和WO/0034317A2)。例如，分析来自起始抗体的重链可变区序列和轻链可变区序列，并产生来自每个可变区的人T细胞表位“图谱”，显示表位相对于互补决定区(CDRs)和序列内其它关键残基的位置。分析来自T细胞表位图的单个T细胞表位，以鉴定具有较低改变抗体活性风险的可选择的氨基酸取代。设计包含氨基酸取代组合的一系列可选的重链可变区序列和轻链可变区序列，随后将这些序列掺入到一系列结合多肽中。然后将包含修饰过的可变区和人类恒定区的完整重链和轻链的基因克隆到表达载体中，随后将质粒转入细胞系以产生完整的抗体。然后利用合适的生物化学和生物学实验中比较抗体，鉴定出最佳的抗体。
本发明公开的抗体，或融合蛋白中抗体的结合特异性可以通过体外实验，例如免疫共沉淀、放射免疫实验(RIA)或酶联免疫吸附实验(ELISA)来检测。
或者，用于生产单链单元的技术(美国专利4,694,778)可适用于生产本发明公 开的单链单元。通过氨基酸桥接Fv区的重链和轻链片段形成单链单元，产生单链融合肽。也可以使用在大肠杆菌中组装功能性Fv片段的技术(Skerra et al.,Science 242:1038-1041(1988))。
可用于生产单链Fv(scFv)和抗体的技术的实例包括如美国专利4,946,778和5,258,498中所述。对于包括在人体内使用抗体和体外检测实验的某些用途，可以使用嵌合抗体、人源化抗体或全人源抗体。嵌合抗体是抗体的不同部分源自不同动物物种的一类分子，例如具有鼠源单克隆抗体的可变区和人源免疫球蛋白恒定区的抗体。生产嵌合抗体的方法是本领域已知的，参见美国专利5,807,715、4,816,567和4,816,397，其全部内容通过引用并入本文。
此外，在Newman,Biotechnology 10:1455-1460(1992)中公开了另一种生产重组抗体的高效方法，特别地，该技术能产生含有猴可变区和人恒定区序列的灵长类抗体，该参考文献的全部内容通过引用并入本文。此外，该技术也在共同转让的美国专利5,658,570、5,693,780和5,756,096中有所提及，每个专利的全部内容通过引用并入本文。
抗体可以通过本领域已知的多种方法制备，包括使用来自免疫球蛋白序列的抗体文库进行的噬菌体展示方法。也可参考美国专利4,444,887和4,716,111，以及PCT公布文本WO 98/46645、WO 98/50433、WO 98/24893、WO 98/16654、WO 96/34096、WO 96/33735和WO 91/10741，每个专利的全部内容通过引用并入本文。
对于治疗人类患者来说，全人源抗体是特别理想的。全人源抗体可以通过本领域已知的多种方法制备，比如可以转基因小鼠来生产人源抗体，所述小鼠不能表达功能性内源性免疫球蛋白但能表达人类免疫球蛋白基因。例如，人重链和轻链免疫球蛋白基因复合物可以随机引入或通过同源重组引入到小鼠胚胎干细胞。或者，除了人重链和轻链基因之外，还可以将人的可变区、恒定区和多样性区域引入小鼠胚胎干细胞中。小鼠重链和轻链的免疫球蛋白基因可以通过同源重组分别或同时通过引入人免疫球蛋白基因座而丧失功能。例如，JH区域的纯合缺失可以防止内源抗体的产生。将修饰过的胚胎干细胞扩增并显微注射进囊胚中以产生嵌合小鼠。然后培育嵌合小鼠以产生表达人源抗体的纯合后代。用选择出的抗原例如全部或部分目标多肽靶点以常规方式免疫转基因小鼠。可以使用常规杂交瘤技术从免疫的转基因小鼠获得靶向抗原的单克隆抗体。转基因小鼠携带的人免疫球蛋白转基因在B细胞分化过程中重排，随后发生类别转换和体细胞突变。因此，使用这种技术可以产生可用于治疗的IgG、IgA、IgM和IgE抗体。关于这种生产全人源抗体的技术相关综述，可以参见Lonberg and Huszar，Int.Rev.Immunol.73:65-93(1995)。关于生产全人源抗体和人单克隆抗体的该技术的详细讨论和生产这种抗体的步骤，参见PCT公布文本WO 98/24893、WO 96/34096、WO  96/33735，以及美国专利5,413,923、5,625,126、5,633,425、5,569,825、5,661,016、5,545,806、5,814,318和5,939,598，其全部内容通过引用并入本文。
也可以使用被称为“引导选择”的技术来生产识别选择性表位的全人源抗体。在该方法中，使用选择的非人单克隆抗体(例如小鼠抗体)来引导识别相同表位的全人源抗体的筛选(参见美国专利5,565,332，其全部内容通过引用并入本文)。
在另一实施方案中，使用常规方法(例如使用能够特异性结合编码鼠抗体重链和轻链的基因的寡核苷酸探针)，可以分离编码所需单克隆抗体的DNA并对其进行测序。分离的和亚克隆的杂交瘤细胞可以作为此类DNA的来源。一旦分离出来，DNA可以被置于表达载体中，然后被转染到原核或真核宿主细胞如大肠杆菌细胞、猿猴COS细胞、中国仓鼠卵巢(CHO)细胞或不产生其他免疫球蛋白的骨髓瘤细胞中。分离的DNA(如本文所述可以是合成的)也可用于制备抗体的恒定区和可变区的序列，如美国专利5,658,570中所述，其全部内容通过引用并入本文。该方法从所选细胞中提取RNA并转化成cDNA，然后使用Ig特异性引物通过PCR技术进行扩增。适于此目的的合适的探针在美国专利5,658,570中也有所提及。
此外，使用常规重组DNA技术，可将本发明的抗体的一个或多个CDR插入框架区，例如插入到人类框架区以构建人源化非全人源抗体。框架区可以是天然存在的或共有的框架区，优选人类框架区(参见Chothia et al.,J.Mol.Biol.278:457-479(1998)，其列出一系列人类框架区)。一些多核苷酸可以编码框架区和CDR组合产生的与目标抗原的至少一个表位特异性结合的抗体。在框架区内可以进行一个或多个氨基酸取代，可以选择能够改善抗体与其抗原结合的氨基酸取代。另外，可用此法进行参与链间二硫键形成的一个或多个可变区中半胱氨酸残基的取代或缺失，从而产生缺少一个或多个链间二硫键的抗体分子。本领域技术范围内的对多核苷酸进行的其他改变也涵盖于本发明中。
通过使用本领域技术人员公知的技术可以选择、构建和培养生产抗体的细胞系。这些技术在各种实验室手册和主要出版物中均有描述。在这方面，下文描述的适合本发明使用的技术参考Current Protocols in Immunology,Coligan et al.,Eds.,Green Publishing Associates and Wiley-Interscience,John Wiley and Sons,New York(1991)，其全部内容包括补充内容通过引用并入全文。
在一些实施方案中，可以按常规方法根据本文所述抗体、抗原结合片段或融合蛋白的氨基酸序列设计合成编码抗体、抗原结合片段或融合蛋白的DNA，将其置入表达载体中，然后转染宿主细胞，在培养基中培养被转染的宿主细胞产生抗体、抗原结合片段或融合蛋白。在一些实施方案中，表达载体包括至少一个启动子元件，抗体、抗原结合片段或融合蛋白编码序列，转录终止信号和polyA尾。其他元件包括增强子， Kozak序列及插入序列两侧RNA剪接的供体和受体位点。可以通过SV40的前期和后期启动子，来自逆转录病毒的长末端重复序列如RSV、HTLV1、HIVI及巨细胞病毒的早期启动子来获得高效的转录，也可应用其它一些细胞的启动子如肌动蛋白启动子。合适的表达载体可包括pIRES1neo，pRetro-Off，pRetro-On，PLXSN，或者pLNCX，pcDNA3.1(+/-)，pcDNA/Zeo(+/-)，pcDNA3.1/Hygro(+/-)，PSVL，PMSG，pRSVcat，pSV2dhfr，pBC12MI和pCS2等。常使用的哺乳动物细胞包括HEK293细胞、Cos1细胞、Cos7细胞、CV1细胞、鼠L细胞和CHO细胞等。
在一些实施方案中，插入基因片段需含有筛选标记，常见的筛选标记包括二氢叶酸还原酶，谷氨酰胺合成酶，新霉素抗性，潮霉素抗性等筛选基因，以便于转染成功的细胞的筛选分离。将构建好的质粒转染到无上述基因的宿主细胞，经过选择性培养基培养，转染成功的细胞大量生长，产生想要获得的目的蛋白。
此外，可以使用本领域技术人员已知的标准技术在编码本发明所述抗体、抗原结合片段或融合蛋白的核苷酸序列中引入突变，包括但不限于导致氨基酸取代的定点突变和PCR介导的突变。变体(包括衍生物)编码相对于原重链可变区VH CDR1、VH CDR2、VH CDR3和轻链可变区VL CDR1、VL CDR2或VL CDR3来说少于50个氨基酸的取代、少于40个氨基酸的替换、少于30个氨基酸的取代、少于25个氨基酸的取代、少于20个氨基酸的取代、少于15个氨基酸的取代、少于10个氨基酸的取代、少于5个氨基酸的取代、少于4个氨基酸的取代、少于3个氨基酸的取代或少于2个氨基酸的取代。或者可以沿着全部或部分编码序列时随机引入突变，例如通过饱和突变，以及可以筛选所得突变体的生物活性以鉴定保留活性的突变体。
在一些实施方案中，本文所述取代为保守氨基酸取代。
治疗方法
本发明还提供了治疗方法和用途。在一些实施方案中，提供了用于治疗或改善SARS或COVID-19的方法，所述方法包括向患者施用有效剂量的所述的抗体、抗原结合片段或融合蛋白。在一些实施方案中，提供了所述的抗体、抗原结合片段或融合蛋白在治疗或改善SARS或COVID-19中的应用。在一些实施方案中，提供了所述的抗体、抗原结合片段或融合蛋白在制备用于治疗或改善SARS或COVID-19的药物中的应用。在一些实施方案中，所述患者为疑似感染SARS-CoV或SARS-CoV-2病毒的患者。在一些实施方案中，所述患者为与SARS-CoV或SARS-CoV-2病毒携带者有接触的患者。在一些实施方案中，所述患者为确诊感染SARS-CoV或SARS-CoV-2病毒的患者。在一些实施方案中，所述患者为有轻微症状的患者。在一些实施方案中，所述患者为有严重症状的患者。在一些实施方案中，所述患者有发热，咳嗽，低血压，缺氧，和/或急性呼吸窘迫综合征(ARDS)。
对于任何特定患者的具体剂量和治疗方案将取决于各种因素，包括所使用的特定抗体、抗原结合片段或融合蛋白或衍生物、患者的年龄和体重、一般健康状况、性别和饮食，以及给药时间、排泄频率、药物组合，以及所治疗的特定疾病的严重程度。由包括在本领域普通技术人员范围内的医疗护理人员对这些因素进行判断。所述剂量还将取决于待治疗的个体患者、给药途径、制剂类型、所用化合物的特性、疾病的严重程度以及所需的效果。所用剂量可以通过本领域熟知的药理学和药代动力学原理确定。
抗体、抗原结合片段、融合蛋白或衍生物的施用方法包括但不限于真皮内、肌肉、腹腔、静脉、皮下、鼻腔、硬脊膜外和口服注射。药物组合物可以通过任何方便的途径施用，例如通过输注或推注，通过上皮或皮肤粘膜(例如口腔粘膜、直肠和肠粘膜等)吸收，并且可以与其他生物活性剂共同施用。因此，含有本发明的抗体、抗原结合片段或融合蛋白的药物组合物可以口服给药、直肠给药、肠胃外给药、脑池内给药、阴道内给药、腹腔内给药、外敷(如通过粉末，软膏，滴剂或透皮贴剂)、口腔给药或通过口服或鼻腔喷雾给药。
本发明使用的术语“肠胃外”是指包括静脉内、肌肉内、腹腔内、胸骨内、皮下和关节内注射和输注的施用方式。施用方式可以是全身施用或局部施用。
在一些实施方案中，本发明组合物包含编码蛋白质的核酸或多聚核苷酸，可以通过将其构建为合适的核酸表达载体的一部分来体内施用所述核酸以促进其编码的蛋白质的表达，然后通过下述方式施用上述部分载体使其变为胞内部分，例如通过使用逆转录病毒载体(参见美国专利4,980,286)，或通过直接注射，或通过使用微粒轰击(例如基因枪；Biolistic，Dupont)，或用脂质或细胞表面受体或转染试剂包被，或者通过与已知进入细胞核的同源异型盒类肽连接施用(参见例如Joliot et al.,1991,Proc.Natl.Acad.Sci.USA 88:1864-1868)等等。可选地，核酸可以通过同源重组在引入细胞内并整合至宿主细胞DNA中用于表达。
在一些实施方案中，本发明的抗体、抗原结合片段或融合蛋白施用于患者的剂量为0.01mg/kg至100mg/kg患者体重，或0.1mg/kg至20mg/kg患者的体重。在初始剂量之后可随后给予第二剂或多剂该抗体、抗原结合片段或融合蛋白，其剂量与初始剂量大致相同或较少，其中该随后的剂量可相隔至少1天至3天；或至少一星期。可以通过例如脂质化等修饰来增强抗体、抗原结合片段或融合蛋白的摄取和组织穿透能力(例如进入脑内)，从而减少本发明抗体、抗原结合片段或融合蛋白的施用的剂量和频率。
各种已知输送系统可用于施用本发明抗体、抗原结合片段或融合蛋白或衍生物或其编码多核苷酸，例如包封于脂质体、微粒、微胶囊、能够表达所述化合物的重组细 胞、受体介导的内吞作用(参见例如Wu and Wu,1987,J.Biol.Chem.262:4429-4432)、作为逆转录病毒或其它载体的一部分的核酸的构建等。
联合疗法
在一些实施方案中，本发明的抗体、抗原结合片段或融合蛋白可以结合其它治疗或预防方案，包括施用一种或多种本发明的抗体、抗原结合片段或融合蛋白以及一种或多种其它治疗剂或方法一起使用或联合使用。对于联合治疗，抗体、抗原结合片段或融合蛋白可以与其它治疗剂可同时或分开施用。当分开施用时，可以在施用另一种其它治疗剂之前或之后施用本发明的抗体、抗原结合片段或融合蛋白。
在一些实施方案中，与本发明抗体、抗原结合片段或融合蛋白联合用药的治疗剂至少为以下中的一种：HIV药物、抗疟药、RNA聚合酶抑制剂、抗病毒药物和单抗类药。在一些实施方案中，HIV药物包括洛匹那韦/利托那韦，ASC09/利托那韦和达芦那韦。在一些实施方案中，抗疟药包括氯喹，羟氯喹和磷酸氯喹。在一些实施方案中，抗病毒药物包括阿比多尔、法匹拉韦。在一些实施方案中，单抗类药物包括BDB-001。在一些实施方案中，所述抗病毒药物为本发明所述融合蛋白中的抗体或抗原结合片段。
一些新型冠状病毒肺炎重型或危重型患者存在细胞因子风暴现象，本发明抗体、抗原结合片段或融合蛋白可与阿达木单抗(adalimumab，例如
Figure PCTCN2022091204-appb-000001
及其生物类似物，如Abrilada TM(adalimumab-afzb)，Amjevita(adalimumab-att)，Cyltezo TM(adalimumab-adbm)，Hyrimoz TM(adalimumab-adaz)，Hulio TM，
Figure PCTCN2022091204-appb-000002
(
Figure PCTCN2022091204-appb-000003
BAT1406))或托珠单抗(tochilizumab,例如
Figure PCTCN2022091204-appb-000004
及其生物类似物，如BAT1806)联合用于治疗，其可以减缓TNF-α表达上调导致的炎症反应。在一些实施方案中，本方法治疗的患者被确诊感染新型冠状病毒并且有一种或多种细胞因子(包括肿瘤坏死因子α(TNF-α)，IFN-γ、IL-1β、IL-2、IL-4、IL-7、IL-8、IL-10、IL-12p70、IL-13、粒细胞集落刺激因子(GSCF)、干扰素诱导蛋白-10(IP-10)、单核细胞趋化蛋白-1(MCP1)、巨噬细胞炎性蛋白1α(MIP1A))增高。在一些实施方案中，本方法治疗的患者有TNF-α增高。在一些实施方案中，一种或多种细胞因子高于正常水平至少50％。在一些实施方案中，一种或多种细胞因子至少为正常水平的2倍、3倍或4倍。在一些实施方案中，本方法治疗前患者有发热，低血压，缺氧，和/或急性呼吸窘迫综合征(ARDS)。在一些实施方案中，本方法治疗前患者有肺部充满炎性液体(即所谓的“白肺”)。在一些实施方案中，本方法治疗前患者有细胞因子风暴引起的细胞因子释放综合症(Cytokine Release Syndrome，CRS)。
在一些实施方案中，本发明抗体、抗原结合片段或融合蛋白用于结合ICU治疗。在一些实施方案中，本发明抗体、抗原结合片段或融合蛋白结合体外ECMO和/或IMV治疗。在一些实施方案中，本发明抗体、抗原结合片段或融合蛋白结合氧疗。在 一些实施方案中，本发明抗体、抗原结合片段或融合蛋白结合NIV/HFNC治疗。在一些实施方案中，治疗后，患者的一种或多种细胞因子比治疗前至少降低20％、30％、40％、50％、60％、70％、80％、90％、或95％。在一些实施方案中，本方法使患者痊愈。
诊断方法
在某些样品中观察到spike蛋白的表达，并且具有spike蛋白表达的细胞的患者可能对使用本发明的抗体、抗原结合片段或融合蛋白的治疗有响应。因此，本发明的抗体、抗原结合片段或融合蛋白也可以用于诊断和预后。
包含细胞的样品可以从患者体内获得。在选择性地对样品进行预处理之后，可以在允许抗体(或融合蛋白)与可能存在于样品中的spike蛋白相互作用的条件下，将样品与本发明的抗体(或融合蛋白)一起孵育。可以使用诸如ELISA的方法，利用抗体或融合蛋白中的抗体来检测样品中spike蛋白的存在。
样品中spike蛋白的存在(比如含量或浓度)可以用于诊断相关疾病，作为患者适用抗体或融合蛋白治疗的指示，或作为患者已经(或没有)对病症治疗作出反应的指示。对于预后方法，可以在开始疾病治疗时在特定阶段进行一次、两次或更多次地检测，以指示治疗的进展。
药物组合物
本发明还提供了药物组合物。这样的组合物包含有效剂量的抗体或融合蛋白以及药学上可接受的辅料。
在一些实施方案中，术语“药学上可接受的”是指由政府的监管机构批准的或其他公认的药典中列出的用于动物(特别是用于人类)的物质。此外，“药学上可接受的辅料”通常指是任何类型的无毒固体、半固体或液体填充剂、稀释剂、包封材料或制剂助剂等。
术语“辅料”是指可以与活性成分一起施用于患者的稀释剂、佐剂、赋形剂或载体。这此类药物载体可以是无菌液体，如水和油，包括石油、动植物或合成来源的油，如花生油、大豆油、矿物油、芝麻油等。当药物组合物静脉内给药时，水是优选的载体。盐水溶液和葡萄糖水溶液和甘油溶液也可用作液体载体，特别是用于注射溶液。合适的药物赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、大米、面粉、白垩、硅胶、硬脂酸钠、单硬脂酸甘油酯、滑石、氯化钠、脱脂奶粉、甘油、丙烯、乙二醇、水、乙醇等。如有需要，药物组合物还可以含有少量的润湿剂、乳化剂，或pH缓冲剂如乙酸盐、柠檬酸盐或磷酸盐。抗菌剂如苯甲醇或对羟基苯甲酸甲酯、抗氧化剂如抗坏血酸或亚硫酸氢钠、螯合剂如乙二胺四乙酸，以及调节张力的试剂如氯化钠或右 旋葡萄糖也是可以预见的。这些药物组合物可以采取溶液、悬液、乳剂、片剂、丸剂、胶囊、散剂、缓释制剂等形式。该药物组合物可以用传统的粘合剂和载体如甘油三酯配制成栓剂。口服制剂可以包括标准载体，例如药物等级的甘露糖醇、乳糖、淀粉、硬脂酸镁、糖精钠、纤维素、碳酸镁等。合适的药物载体的实例在E.W.Martin的Remington's Pharmaceutical Sciences中有描述，在此通过引用并入本发明。此类组合物将含有临床有效剂量的抗体或抗原结合片段或融合蛋白，优选以纯化后的形式，连同合适数量的载体，以提供适合于患者的给药形式。该制剂应该适用于给药模式。制剂可以封装在安瓿瓶、一次性注射器或由玻璃或塑料制成的多剂量小瓶中。
在一些实施方案中，根据常规步骤将组合物配制成适合静脉内注射于人体的药物组合物。用于静脉内给药的组合物通常是在无菌等渗水性缓冲液中的溶液。药物组合物还可包含增溶剂和局部麻醉剂如利多卡因，从而缓解注射部位的疼痛。一般而言，有效成分以单位剂量形式单独供给或混在一起供给，如以干燥的冻干粉末或无水浓缩物的形式装在可指示活性剂份量的密封容器(如安瓿瓶或小袋)中。在通过输注施用组合物的情况下，可以用含有无菌药用级水或盐水的输液瓶来分装组合物。在通过注射施用组合物的情况下，可以使用注射用的无菌水或盐水的安瓿瓶，使得可以在施用之前混合有效成分。
本发明的化合物可以配制成中性的或盐的形式。药学上可接受的盐包括衍生自如与盐酸、磷酸、乙酸、草酸、酒石酸等的阴离子形成的盐，以及衍生自如与钠、钾、铵、钙、氢氧化铁、异丙胺、三乙胺、2-乙氨基乙醇、组氨酸、普鲁卡因等的阳离子形成的盐。
“约”指相关技术领域技术人员容易知道的相应数值的常规误差范围。在一些实施方式中，本文中提到“约”指所描述的数值以及其±10％、±5％或±1％的范围。
“ECMO”即指体外膜肺氧合(Extracorporeal Membrane Oxygenation，ECMO)，其是一种医疗急救技术设备，主要用于对重症心肺功能衰竭患者提供持续的体外呼吸与循环，以维持患者生命。
“ICU”是指重症加强护理病房(Intensive Care Unit)，治疗、护理、康复均可同步进行，为重症或昏迷患者提供隔离场所和设备，提供最佳护理、综合治疗、医养结合，以及术后早期康复、关节护理运动治疗等服务。
“IMV”即指间歇性指令通气(intermittent mandatory ventilation)，其是根据预先设置的时间间隔即时间触发，来实施周期性的容量或压力通气。这期间允许患者在指令通气期间以任何设定的基础压力水平进行自主呼吸。在自主呼吸时，患者可以在持续气流支持下自主呼吸，或者机器将按需阀门打开以允许自主呼吸。据大多数呼吸 机都可以在自主呼吸时提供压力支持。
“HFNC”即经鼻高流量氧疗(High-flow nasal cannula oxygen therapy)，其是通过无需密封的鼻塞导管直接将一定氧浓度的空氧混合高流量气体输送给患者的一种氧疗方式，作为一种无创呼吸支持的形式，其能迅速地改善氧合。目前可以应用于急性低氧性呼吸衰竭患者、外科手术后患者、呼吸衰竭未行气管插管患者、免疫抑制患者、心功能不全患者等。
“NIV”即指无创通气(Non-invasine Ventilation)，是指除气管插管、气管切开以外的无创伤的机械通气。
“EC 50”即半最大效应浓度(concentration for 50％of maximal effect,EC50)是指能引起50％最大效应的浓度。
“IC 50”表示50％抑制浓度，即对指定的生物过程抑制一半时所需的药物或者抑制剂的浓度。
本发明中“亲本Fc区”可以为天然存在的Fc区，编码Fc区的基因可来自人、鼠、兔、骆驼、猴子，优选为人和小鼠；例如，亲本Fc区为SEQ ID NO:60、SEQ ID NO:61或SEQ ID NO:66中Fc区。
具体实施方式
以下通过具体的实施例进一步说明本发明的技术方案，具体实施例不代表对本发明保护范围的限制。其他人根据本发明理念所做出的一些非本质的修改和调整仍属于本发明的保护范围。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1抗spike蛋白抗体和融合蛋白的制备
抗体和融合蛋白可以通过以下方法或其他已知方法制备：依照宿主细胞CHO密码子偏好性特点进行序列优化，由氨基酸序列得到DNA序列。将优化并合成的序列分别克隆入载体，然后分别抽提大量质粒，进行稳定表达细胞株的构建：线性化的表达载体与CHO细胞混匀后加入0.4cm电转杯进行电转；电转完成后，按1200个细胞每孔铺到96孔细胞培养板，约2-3周后选择表达量高的母克隆进行96孔到24孔到6孔到摇瓶的细胞扩大培养和表达量检测，选择摇瓶表达量高的克隆进行亚克隆，亚克隆扩大培养和表达鉴定同母克隆，根据表达量水平和细胞株稳定性选择单克隆稳定细胞株，悬浮培养12天左右收获上清，进行proA亲和捕获，阴离子及阳离子层析获得纯度大于95％的抗体或融合蛋白。
抗体和融合蛋白的相关序列见表1-4，VH与CH(如SEQ ID NO:61所示)组成抗 体的重链，VL与CL(如SEQ ID NO:62所示)组成抗体的轻链。其中，抗体2F8含有两条序列相同的重链(如SEQ ID NO:66所示)和两条序列相同的轻链(如SEQ ID NO:68所示)；抗体anti-19含有两条序列相同的重链(如SEQ ID NO:65所示)和两条序列相同的轻链(如SEQ ID NO:67所示)；融合蛋白20包含两条序列相同的多肽A(如SEQ ID NO:69所示)和两条序列相同的多肽B(如SEQ ID NO:67所示)；多肽A包含重链片段、连接子和6-HB干扰多肽，多肽B包含轻链；表2和表3中重链的Fc区采用下单划线标出，表3中多肽A的连接子和6-HB干扰多肽分别采用单下划线和双下划线标出。
纯化后的抗体和融合蛋白经测序证实序列如上所述。
表1抗spike蛋白抗体中VH和VL的组成
Figure PCTCN2022091204-appb-000005
表2抗体各部分的氨基酸序列
Figure PCTCN2022091204-appb-000006
Figure PCTCN2022091204-appb-000007
表3抗体全长和融合蛋白的示例氨基酸序列
Figure PCTCN2022091204-appb-000008
Figure PCTCN2022091204-appb-000009
表4抗体2F8的核酸序列
Figure PCTCN2022091204-appb-000010
Figure PCTCN2022091204-appb-000011
实施例2抗体与SARS-CoV-2 spike蛋白结合活性的检测
针对上述抗体进行Elisa检测，检测方法为：将96孔板(Corning,9018)用spike-RBD-mFC(sino biologicals)包被，并用胶带密封并储存；将板在清洗缓冲液PBST(PBS,0.05％Tween 20)中清洗3次，之后加入封闭溶液(每孔200μL的10mg/ml BSA，溶剂为清洗缓冲液)；孵育(1h，37℃)后，将板用清洗缓冲液洗涤3次，然后每孔添加梯度稀释好的样品100μL；孵育(1.5h，37℃)后，将板用清洗缓冲液洗涤，然后加入抗人κ轻链抗体-过氧化物酶偶联物(在封闭溶液中稀释至1:2000，100μL/孔)；将板用清洗缓冲液洗涤，在添加100μL TMB(Tetramethylbenzidine，Biopanda TMB-S-001)底物/孔之前，将测试样品孵育(1h，37℃)；显色10分钟后，加入100μL/孔0.1M H 2SO 4终止反应，然后在450nm的吸光度下测量96孔板。
通过吸光值计算EC 50，各种抗体与SARS-CoV-2 spike蛋白结合的EC 50值见表5。
表5
Figure PCTCN2022091204-appb-000012
实施例3抗体和融合蛋白与spike蛋白亲和力的测定
BiaCore(生物分子相互作用分析)：将抗体以10μg/mL的浓度包被到protein A的芯片上，使用BiaCore在25℃下测量结合动力学，平衡探针；重组spike S1 RBD蛋白(Acrobiosystems，SPD-C52H3)或三聚体spike trimer(Acrobiosystems，SPN-C52H8)以10-400nM浓度通过包被着抗体的芯片；仅通过流穿缓冲液(含有0.05％Tween-20的PBS缓冲液)作为对照减去背景和非特异性结合信号；在BiaCore Data Analysis软件上使用1:1 Langmuir结合模型计算动力学常数(ka为结合速率，kd为解离速率，kD为结合解离平衡常数)。
1)如表6所示，抗体2F8与Spike S1 RBD和三聚体spike trimer的结合力良好。
表6抗体2F8与Spike S1 RBD和三聚体spike trimer的亲和力常数
抗原 K a(1/Ms) K d(1/s) K D(M)
spike S1 RBD 4.7E+07 1.23E-03 2.61E-11
spike trimer 4.44E+07 2.45E-06 5.51E-14
2)如表7所示，抗体anit-19和融合蛋白20与三聚体spike trimer的结合力良好。
表7抗体anti-19和融合蛋白20与三聚体spike trimer的亲和力常数
样品 K a(1/Ms) K d(1/s) K D(M)
anti-19 4.751E+04 4.363E-03 9.183E-10
融合蛋白20 6.595E+04 5.099E-05 7.732E-10
实施例4抗spike蛋白抗体结合活性的检测
针对抗体2F8进行ELISA检测，检测方法为：将spike RBD蛋白(Acrobiosytems， SPD-C52H3)稀释至2μg/ml，每孔100μl置于96孔板(Corning，9018)中，4℃包被过夜；将96孔板在清洗缓冲液PBST(含0.05％Tween-20的PBS缓冲液)中清洗3次，之后加入封闭溶液(每孔200μL 3mg/ml BSA，溶剂为清洗缓冲液)，37℃孵育2h；然后，将96孔板用清洗缓冲液洗涤3次，每孔添加梯度稀释的抗体溶液100μL，37℃孵育1.5h后；将96孔板用清洗缓冲液洗涤5次，加入100μL的抗人κ轻链抗体-过氧化物酶偶联物(在封闭溶液中稀释至1:2000)，37℃孵育1h；将96孔板用清洗缓冲液洗涤8次，再添加100μL TMB(Tetramethylbenzidine，Biopanda TMB-S-001)底物进行显色；显色10-15min后，加入50μL 0.1M H 2SO 4终止反应，450nm的吸光度下测量光吸收值。
结果显示，抗体2F8与spike RBD蛋白结合的EC 50值约为1nM。
实施例5抗体阻断ACE2与spikeRBD的结合
采用SPR(表面等离子共振)技术进行检测：先将100nM biotinylated spike RBD蛋白(Acrobiosystems，SPD-C82E9)结合在streptavidin探针上，结合了biotinylated spike RBD的探针与100nM抗体溶液进行孵育，将捕获了抗体的探针与100nM ACE2(近岸生物，C419)蛋白进行孵育，以便检测已经结合了抗体的spike RBD蛋白是否还可以与溶液中的ACE2进行结合。
如图1显示，抗体2F8阻断了ACE2与spike RBD的结合。
实施例6抗体抑制假病毒侵染ACE2 +293F细胞
本实验是从体外评估抗体抑制spike-pseudotyped假病毒(吉满生物)侵染表达ACE2细胞(即ACE2 +293F细胞)的能力。采用ACE2 +293F细胞检测抗体抑制带有荧光素酶基因的SARS-CoV-2假病毒侵染细胞的能力。其主要原理是：采用ACE2 +293F细胞作为易感染细胞，将不同浓度的抗体与SARS-CoV-2-Fluc假病毒系统孵育；当抗体与假病毒孵育结合后，将阻断病毒侵染进入ACE2 +293F细胞；假病毒无法有效侵染细胞，其基因组上的luciferase报告基因便无法在细胞内表达且产生荧光信号；由于荧光信号的信号值与加入的抗体浓度成负相关，从而可以检测抗体的体外抑制病毒侵染的能力。其中，突变株D614G，D936Y货号为GM-0220PV19-96T(吉满生物)，突变株D839Y的货号为GM-0220PV6-480T(吉满生物)，突变株V483A的货号为GM-0220PV17-480T(吉满生物)，突变株D614G，A831V的货号为GM-0220PV24-480T(吉满生物)，突变株B.1.351/501Y.V2的货号为GM-0220PV32-96T(吉满生物)。
假病毒抑制能力检测方法为：抗体稀释至6μg/ml，后4倍梯度稀释，按照每孔50μl体积转移至96孔检测板中，待用；将不同突变株的假病毒原液分别用含有10％FBS的DMEM培养基进行稀释，将稀释后的假病毒溶液按照25μl每孔转移至上述含有抗体的96孔板中，混匀后室温静置1h；将ACE2 +293F细胞用0.25％Trypsin-EDTA (Gibco，25200-072)消化后计数，将细胞密度调至4×10 5cells/ml，按照每孔50μl体积将细胞加入到上述96孔检测板中，37℃培养箱培养48h；每孔加入50μl Bio-Lite luciferase assay system(诺维赞，DD1201-03)检测试剂，静置3分钟后进行读数，并根据读数计算抑制率：抑制率＝[1-(样品组-空白对照组)/(阴性对照组-空白对照组)]×100％；其中，阴性对照组添加假病毒溶液且不添加抗体，空白对照组不添加假病毒溶液。
ACE2 +293F细胞的构建方法为：将HEK293F细胞用含10％FBS的DMEM完全培养基培养，采用lipofectamine 2000 transfection reagent(Thermo Fisher，11668019)进行ACE2表达质粒(义翘神州，HG10108-M)的转染，之后通过潮霉素(200μg/ml)的加压筛选和流式分选(采用10μg/ml anti-ACE2和PE耦联的Anti-Human IgG-Fc)，细胞继续扩增挑选出PE阳性率>90％的单克隆进行下一步扩增，筛选出表达ACE2的HEK293F细胞，即ACE2 +293F细胞。
如表8所示，抗体2F8可有效地抑制假病毒侵染细胞。
表8抗体2F8对假病毒的抑制IC 50(μg/ml)
病毒 D614G，D936Y D839Y V483A D614G，A831V B.1.351/501Y.V2
IC 50 0.003 0.002 0.002 0.002 0.003
实施例7抗体抑制真病毒
1)在BSL-3实验室将待测抗体进行梯度稀释(初始浓度为60nM，3倍梯度稀释)，将抗体稀释液与200PFU SARS-CoV-2新冠病毒粒子(病毒株编号：2019-nCoV/IQTC01/human/2020/Guangzhu)等体积混合，同时设立无抗体的病毒对照组和无病毒的细胞对照组；每实验组设置3个复孔，37℃静置1h；吸弃96孔板中的非洲绿猴肾细胞系Vero E6细胞(ATCC CRL-1587)的上清液，取50μl孵育后的病毒抗体混合物转移至Vero E6细胞板中，放置于细胞培养箱中37℃，孵育1小时；吸弃Vero E6细胞板中上清液，加入100μl 37℃预热的DMEM培养基(含1.6％CMC(羧甲基纤维素))，放置于37℃细胞培养箱培养24h；24h后取出细胞板，加入200μl的4％PFA(多聚甲醛溶液)，4℃过夜孵育固定灭活病毒及细胞；第二天，吸弃上清后更换新鲜4％PFA，将96孔细胞板带入BSL-2实验室进行后续实验；使用FRNT50(plaque/focus reduction neutralization test(P/FRNT))实验进行检测：破膜、封闭后分别使用SARS-CoV-2 N rabbit polyclonal antibody(北京义翘神州，货号为40143-T62)和HRP goat-anti rabbit IgG(Jackson，货号为111-035-003)作为一抗和二抗，依次进行孵育；洗涤干净后使用True blue进行显色；Immuno ELISAPOT上机读取结果，Graphpad软件进行数据分析。
结果显示，抗体2F8可有效地抑制了真病毒侵染细胞，其IC 50为0.027nM。
2)采用细胞病变(CPE,cytopathic effect)减少法检测融合蛋白的预防效应。试验主要方法为：将抗体或融合蛋白加入等体积含100PFU COVID-19病毒液(Genebank accession no.MT123290.1)，混匀后置37℃孵育1h；混合物加入单层培养的Vero E6细胞中，37℃孵育1h；去除病毒液，用PBS洗涤1次，每孔加入200μL培养基。空白对照组和正常细胞组每孔加入等体积培养液，37℃培养4日，在显微镜下观察并记录细胞病变程度。细胞出现病变程度按以下6级标准记录：-无病变出现；±为细胞病变少于10％；+为细胞病变约25％；++为细胞病变约50％；+++为约75％细胞出现病变；++++为75％以上细胞病变。
结果显示，抗体anti-19和融合蛋白20有效地阻断病毒的侵染能力，并呈现出浓度依赖效应，两者的IC 50分别是5.68nM和4.3nM。
3)将含100PFU COVID-19病毒液加入96孔板中单层培养的Vero E6细胞中，混匀后置37℃孵育2小时；去除病毒液，用PBS洗涤1次，每孔加入200μl融合蛋白20稀释液(采用培养基进行梯度稀释)，置于37℃继续培养96小时；显微镜下观察并记录细胞病变程度。细胞出现病变程度按以下6级标准记录：-无病变出现；±为细胞病变少于10％；+为细胞病变约25％；++为细胞病变约50％；+++为约75％细胞出现病变：++++为75％以上细胞病变。
结果显示，融合蛋白20与侵染了SARS-CoV-2的Vero E6细胞孵育后，融合蛋白20有效地抑制病毒在细胞内进行复制，并呈现出浓度依赖效应，其IC 50是12nM。

Claims (17)

  1. 抗体或抗原结合片段，所述抗体或抗原结合片段特异性结合SARS-CoV-2或SARS-CoV的spike蛋白，所述抗体或抗原结合片段至少包含如SEQ ID NO:1或2所示的HCDR1、如SEQ ID NO:3或4所示的HCDR2、如SEQ ID NO:5-42中任一项所示的HCDR3、如SEQ ID NO:43或44所示的LCDR1、如SEQ ID NO:45或46所示的LCDR2和如SEQ ID NO:47或48所示的LCDR3中的一个或多个。
  2. 如权利要求1所述的抗体或抗原结合片段，所述抗体或抗原结合片段至少包含如SEQ ID NO:1或2所示的HCDR1、如SEQ ID NO:3或4所示的HCDR2、如SEQ ID NO:5-42中任一项所示的HCDR3、如SEQ ID NO:43或44所示的LCDR1、如SEQ ID NO:45或46所示的LCDR2和如SEQ ID NO:47或48所示的LCDR3。
  3. 如权利要求1所述的抗体或抗原结合片段，所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:6所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3；或
    所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:9所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3；或
    所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:15所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3；或
    所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:16所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3；或
    所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:18所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3；或
    所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:19所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3；或
    所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID  NO:3所示的HCDR2、如SEQ ID NO:20所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3；或
    所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:21所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3；或
    所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:22所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3；或
    所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:23所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3；或
    所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:24所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3；或
    所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:25所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3；或
    所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:28所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3；或
    所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:29所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3；或
    所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:30所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3；或
    所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:32所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3；或
    所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:33所示的HCDR3、如SEQ ID NO:43所示的 LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3；或
    所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:34所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3；或
    所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:35所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3；或
    所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:36所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3；或
    所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:37所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3；或
    所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:39所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3；或
    所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:40所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3；或
    所述抗体或抗原结合片段至少包含如SEQ ID NO:1所示的HCDR1、如SEQ ID NO:3所示的HCDR2、如SEQ ID NO:41所示的HCDR3、如SEQ ID NO:43所示的LCDR1、如SEQ ID NO:45所示的LCDR2和如SEQ ID NO:47所示的LCDR3；或
    所述抗体或抗原结合片段至少包含如SEQ ID NO:2所示的HCDR1、如SEQ ID NO:4所示的HCDR2、如SEQ ID NO:42所示的HCDR3、如SEQ ID NO:44所示的LCDR1、如SEQ ID NO:46所示的LCDR2和如SEQ ID NO:48所示的LCDR3。
  4. 如权利要求1-3任一项所述的抗体或抗原结合片段，所述抗体或抗原结合片段的重链可变区的框架区包含重链FR1、重链FR2、重链FR3和重链FR4，所述抗体或抗原结合片段的重链FR1包含SEQ ID NO:49或50所示的序列，或与SEQ ID NO:49或50所示序列具有至少90％同一性的序列，或与SEQ ID NO:49或50所示序列相比具有一或多个保守氨基酸取代的氨基酸序列；和/或
    所述抗体或抗原结合片段的重链FR2包含SEQ ID NO:51或52所示的序列，或与 SEQ ID NO:51或52所示序列具有至少90％同一性的序列，或与SEQ ID NO:51或52所示序列相比具有一或多个保守氨基酸取代的氨基酸序列；和/或
    所述抗体或抗原结合片段的重链FR3包含SEQ ID NO:53或54所示的序列，或与SEQ ID NO:53或54所示序列具有至少90％同一性的序列，或与SEQ ID NO:53或54所示序列相比具有一或多个保守氨基酸取代的氨基酸序列；和/或
    所述抗体或抗原结合片段的重链FR4包含SEQ ID NO:55所示的序列，或与SEQ ID NO:55所示序列具有至少90％同一性的序列，或与SEQ ID NO:55所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
  5. 如权利要求1-4任一项所述的抗体或抗原结合片段，所述抗体或抗原结合片段的重链可变区包含SEQ ID NO:56或57所示的序列，或与SEQ ID NO:56或57所示序列具有至少80％同一性的序列，或与SEQ ID NO:56或57所示序列相比具有一或多个保守氨基酸取代的氨基酸序列；和/或
    所述抗体或抗原结合片段的轻链可变区包含SEQ ID NO:58或59所示的序列，或与SEQ ID NO:58或59所示序列具有至少80％同一性的序列，或与SEQ ID NO:58或59所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
  6. 抗体或抗原结合片段，所述抗体或抗原结合片段特异性结合SARS-CoV-2或SARS-CoV的spike蛋白抗原，所述抗体或抗原结合片段包含氨基酸序列如SEQ ID NO:56所示的重链可变区和氨基酸序列如SEQ ID NO:58所示的轻链可变区；或
    所述抗体或抗原结合片段包含氨基酸序列如SEQ ID NO:57所示的重链可变区和氨基酸序列如SEQ ID NO:59所示的轻链可变区。
  7. 如权利要求1-6任一项所述的抗体或抗原结合片段，所述抗体或抗原结合片段的重链恒定区包含氨基酸序列如SEQ ID NO:60或61所示的序列，或与SEQ ID NO:60或61所示序列具有至少80％同一性的序列，或与SEQ ID NO:60或61所示序列相比具有一或多个保守氨基酸取代的氨基酸序列；和/或
    所述抗体或抗原结合片段的轻链恒定区包含氨基酸序列如SEQ ID NO:62所示的序列，或与SEQ ID NO:62所示序列具有至少80％同一性的序列，或与SEQ ID NO:62所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
  8. 一种与SARS-CoV-2或SARS-CoV的spike蛋白特异性结合的抗体，所述抗体 的重链包含氨基酸序列如SEQ ID NO:65或66所示的序列，或与SEQ ID NO:65或66所示序列具有至少80％同一性的序列，或与SEQ ID NO:65或66所示序列相比具有一或多个保守氨基酸取代的氨基酸序列；和/或
    所述抗体的轻链包含氨基酸序列如SEQ ID NO:67或68所示的序列，或与SEQ ID NO:67或68所示序列具有至少80％同一性的序列，或与SEQ ID NO:67或68所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
  9. 一种与SARS-CoV-2或SARS-CoV的spike蛋白特异性结合的融合蛋白，所述融合蛋白包含如权利要求1-8任一项所述的抗体或抗原结合片段以及6-HB干扰多肽。
  10. 如权利要求9所述的融合蛋白，所述抗体或抗原结合片段的至少一个C-末端或N-末端通过连接子与6-HB干扰多肽进行连接。
  11. 如权利要求9所述的融合蛋白，所述抗体或抗原结合片段的重链或轻链C-末端通过连接子与6-HB干扰多肽进行连接。
  12. 如权利要求9-11任一项所述的融合蛋白，所述6-HB干扰多肽包含如SEQ ID NO:63所示的序列，或与SEQ ID NO:63所示序列具有至少90％同一性的序列，或与SEQ ID NO:63所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
  13. 如权利要求9-12任一项所述的融合蛋白，所述连接子为包含甘氨酸和丝氨酸的多肽；或者，所述连接子的序列为(G mS) n，其中每个m独立为2、3、4或5，n独立为1、2、3、4或5；或者，所述连接子的序列为(GGGGS) n，其中n独立为1、2、3、4或5。
  14. 一种与SARS-CoV-2或SARS-CoV的spike蛋白特异性结合的融合蛋白，所述融合蛋白包含氨基酸序列如SEQ ID NO:69所示的序列，或与SEQ ID NO:69所示序列具有至少80％同一性的序列，或与SEQ ID NO:69所示序列相比具有一或多个保守氨基酸取代的氨基酸序列；所述融合蛋白还包含氨基酸序列如SEQ ID NO:67所示的序列，或与SEQ ID NO:67所示序列具有至少80％同一性的序列，或与SEQ ID NO:67所示序列相比具有一或多个保守氨基酸取代的氨基酸序列。
  15. 一种编码权利要求1-7任一项所述的抗体或抗原结合片段，或权利要求8所述的抗体，或权利要求9-14任一项所述的融合蛋白的核酸。
  16. 一种药物组合物，所述药物组合物包含如权利要求1-7任一项所述的抗体或抗原结合片段，或权利要求8所述的抗体，或权利要求9-14任一项所述的融合蛋白。
  17. 权利要求1-7任一项所述的抗体或抗原结合片段或权利要求8所述的抗体或权利要求9-14任一项所述的融合蛋白或权利要求16所述的药物组合物在预防、治疗或改善SARS或COVID-19中的应用；或权利要求1-7任一项所述的抗体或抗原结合片段或权利要求8所述的抗体或权利要求9-14任一项所述的融合蛋白或权利要求16所述的药物组合物在制备用于预防、治疗或改善SARS或COVID-19的药物中的应用。
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