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WO2022253306A1 - Anticorps ciblant le coronavirus et son utilisation - Google Patents

Anticorps ciblant le coronavirus et son utilisation Download PDF

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Publication number
WO2022253306A1
WO2022253306A1 PCT/CN2022/096800 CN2022096800W WO2022253306A1 WO 2022253306 A1 WO2022253306 A1 WO 2022253306A1 CN 2022096800 W CN2022096800 W CN 2022096800W WO 2022253306 A1 WO2022253306 A1 WO 2022253306A1
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Prior art keywords
seq
sequence
heavy chain
amino acid
hcdr3
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PCT/CN2022/096800
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English (en)
Chinese (zh)
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
梁世德
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 WO2022253306A1 publication Critical patent/WO2022253306A1/fr

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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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • 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
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • 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
    • C07K2317/50—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
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to antibodies targeting coronaviruses, including bispecific antibodies, single domain antibodies, heavy chain antibodies and applications thereof.
  • Coronavirus is a single-stranded positive-sense RNA virus that is not segmented. According to the serotype and genomic characteristics, the subfamily Coronaviridae is divided into four genera: ⁇ , ⁇ , ⁇ , and ⁇ . Protruding, shaped like a corolla and named after it.
  • the new coronavirus (SARS-CoV-2 or 2019-nCoV) discovered in 2019 belongs to the new coronavirus of the genus ⁇ , with an envelope, and the particles are round or oval, often pleomorphic, with a diameter of 60-140nm. Current research shows that SARS-CoV-2 is highly homologous to SARS-CoV.
  • the novel coronavirus pneumonia COVID-19 is mainly transmitted through the respiratory tract, and it may also be transmitted through contact.
  • the crowd is generally susceptible, and the elderly and those with underlying diseases are more seriously ill after infection, and children and infants also have the disease.
  • the incubation period of the new coronavirus is generally 1-14 days, most of which are 3-7 days.
  • the main clinical symptoms of infected people 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 is normal or decreased, or the number of lymphocytes is decreased, and some patients have increased liver enzymes, muscle enzymes and myoglobin.
  • Chest imaging showed multiple small patchy shadows and interstitial changes in the early stage, especially in the extrapulmonary zone; then it developed into multiple ground-glass shadows and infiltration shadows in both lungs, and in severe cases, lung consolidation and dyspnea gradually appeared.
  • Acute Respiratory Distress Syndrome (ARDS) shock, and various tissue damage and dysfunction of lung tissue, heart, and kidney occurred in patients. Most patients with mild infection have a good prognosis, while those with severe infection are often in critical condition and even die.
  • the invention provides antibodies with high affinity for the spike protein of SARS-CoV-2, including bispecific antibodies, single domain antibodies, heavy chain antibodies or antigen-binding fragments.
  • the invention provides a bispecific antibody or an antigen-binding fragment with high affinity for the spike protein of SARS-CoV-2.
  • Bispecific antibodies or antigen-binding fragments can bind to spike proteins, prevent virus particles from binding to cells, and mediate immune cell phagocytosis and clearance of virus particles.
  • Bispecific antibodies or antigen-binding fragments can be used to prevent, treat or improve COVID-19, and can also be used to diagnose COVID-19.
  • spike protein S protein or spike protein
  • ACE2 angiotensin-converting enzyme 2
  • SARS-CoV-2 enters the cell and uses the cell for its Synthesize new virus particles; the new virus particles are released outside the cell, and in the same way, the virus infects surrounding normal cells.
  • the bispecific antibody or antigen-binding fragment targeting the spike protein can block the binding of the spike protein to ACE2, thereby blocking the virus from entering the cell and exerting an antiviral effect.
  • the bispecific antibody or antigen-binding fragment of the present invention can also mediate immune cell phagocytosis and virus clearance.
  • Some embodiments provide a bispecific antibody targeting coronavirus, the bispecific antibody comprising a first binding moiety that binds a spike protein and a second binding moiety that binds a spike protein linked by a linker L1.
  • the first binding moiety that binds a spike protein comprises:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 1 or 2, or its variants with single or multiple site substitutions, deletions or insertions
  • HCDR2 which comprises such as SEQ ID NO : the amino acid sequence shown in 3 or 4, or its single or multiple site substitution, deletion or insertion variant
  • HCDR3 which comprises any one of SEQ ID NO:5-42 The amino acid sequences shown, or variants thereof with single or multiple site substitutions, deletions or insertions.
  • the first binding moiety comprises:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 1 or 2, or its variants with single or multiple site substitutions, deletions or insertions
  • HCDR2 which comprises such as SEQ ID NO : the amino acid sequence shown in 3 or 4, or a variant with single or multiple site substitutions, deletions or insertions
  • HCDR3 which comprises any one of SEQ ID NOs: 5-42 amino acid sequence, or its variants with single or multiple site substitutions, deletions or insertions.
  • the first binding moiety comprises:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 1 or 2
  • HCDR2 which comprises the amino acid sequence shown in SEQ ID NO: 3 or 4
  • HCDR3 which comprises Amino acid sequence as shown in any one of SEQ ID NO:5-42.
  • the first binding moiety comprises:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 1 or 2, or its variants with single or multiple site substitutions, deletions or insertions
  • HCDR2 which comprises such as SEQ ID NO : the amino acid sequence shown in 3 or 4, or its single or multiple site substitution, deletion or insertion variant
  • HCDR3 which comprises any one of SEQ ID NO:5-42 Amino acid sequence, or its single or multiple position substitution, deletion or insertion variant
  • LCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 43 or 44, or its single or multiple position A variant of point substitution, deletion or insertion
  • LCDR2 which comprises the amino acid sequence shown in SEQ ID NO: 45 or 46, or a variant with single or multiple site substitutions, deletions or insertions
  • LCDR3 which comprises the amino acid sequence shown in SEQ ID NO: 47 or 48, or its variants with single or multiple site substitutions, deletions or insertions.
  • the first binding moiety comprises:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 1 or 2, or its variants with single or multiple site substitutions, deletions or insertions
  • HCDR2 which comprises such as SEQ ID NO : the amino acid sequence shown in 3 or 4, or its single or multiple site substitution, deletion or insertion variant
  • HCDR3, which comprises any one of SEQ ID NO:5-42 Amino acid sequence, or its single or multiple position substitution, deletion or insertion variant
  • LCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 43 or 44, or its single or multiple position A variant of point substitution, deletion or insertion
  • LCDR2 which comprises the amino acid sequence shown in SEQ ID NO: 45 or 46, or a variant with single or multiple site substitutions, deletions or insertions
  • LCDR3, which comprises the amino acid sequence shown in SEQ ID NO: 47 or 48, or its variants with single or multiple site substitutions, deletions or insertions.
  • the substitution variants are conservative amino acid substitution variants.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO: 1 or 2, HCDR2 as shown in SEQ ID NO: 3 or 4, any of SEQ ID NOs: 5-42
  • HCDR1 shown in SEQ ID NO: 1 or 2
  • HCDR2 as shown in SEQ ID NO: 3 or 4
  • any of SEQ ID NOs: 5-42 One or two of HCDR3 shown in item, LCDR1 shown in SEQ ID NO: 43 or 44, LCDR2 shown in SEQ ID NO: 45 or 46, and LCDR3 shown in SEQ ID NO: 47 or 48 , three, four, five or all.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:5, HCDR3 as shown in SEQ ID NO LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:6, HCDR3 as shown in SEQ ID NO LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:7, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:8, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:9, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:10, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:11, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:12, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:13, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:14, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:15, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:16, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:17, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:18, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:19, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:20, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:21, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:22, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:23, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:24, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:25, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:26, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:27, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:28, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:29, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:30, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:31, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:32, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:33, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:34, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:35, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:36, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:37, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:38, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:39, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:40, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:3, HCDR3 as shown in SEQ ID NO:41, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :43, LCDR2 shown in SEQ ID NO:45 and LCDR3 shown in SEQ ID NO:47.
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:4, HCDR3 as shown in SEQ ID NO:42, HCDR3 as shown in SEQ ID NO: LCDR1 shown in :44, LCDR2 shown in SEQ ID NO:46 and LCDR3 shown in SEQ ID NO:48.
  • the first binding moiety comprises a heavy chain variable region and/or a light chain variable region.
  • the framework region of the heavy chain variable region of the first binding moiety comprises heavy chain FR1, heavy chain FR2, heavy chain FR3, and heavy chain FR4;
  • the heavy chain FR1 comprises SEQ ID NO: 49 or 50 The sequence shown, or a sequence having at least 90% identity to the sequence shown in SEQ ID NO: 49 or 50, or an amino acid having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 49 or 50 sequence;
  • the heavy chain FR2 comprises the sequence shown in SEQ ID NO: 51 or 52, or a sequence with at least 90% identity to the sequence shown in SEQ ID NO: 51 or 52, or a sequence shown in SEQ ID NO: 51 or 52 Amino acid sequences having one or more conservative amino acid substitutions compared to the sequence; and/or
  • the heavy chain FR3 comprises the sequence shown in SEQ ID NO: 53 or 54, or a sequence with at least 90% identity to the sequence shown in SEQ ID NO: 53 or 54, or a sequence shown in SEQ ID NO: 53 or 54 Amino acid sequences having one or more conservative amino acid substitutions compared to the sequence; and/or
  • the heavy chain FR4 comprises the sequence shown in SEQ ID NO:55, or a sequence having at least 90% identity with the sequence shown in SEQ ID NO:55, or has one or more of the sequences shown in SEQ ID NO:55 Amino acid sequence with multiple conservative amino acid substitutions.
  • the heavy chain FR1 comprises the sequence shown in SEQ ID NO: 49 or 50, or a sequence having at least 90% identity with the sequence shown in SEQ ID NO: 49 or 50, or a sequence with SEQ ID NO: 49 or 50 Compared with the sequence shown in: 49 or 50, there is an amino acid sequence with one or more conservative amino acid substitutions;
  • 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 with the sequence shown in SEQ ID NO: 51 or 52;
  • the heavy chain FR3 comprises SEQ ID NO: 53 or 54 or a sequence having at least 90% identity with 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:55, or a sequence with
  • the heavy chain FR1 comprises the sequence shown in SEQ ID NO:49
  • the heavy chain FR2 comprises the sequence shown in SEQ ID NO:51
  • the heavy chain FR3 comprises the sequence shown 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 shown in SEQ ID NO:50
  • the heavy chain FR2 comprises the sequence shown in SEQ ID NO:52
  • the heavy chain FR3 comprises the sequence shown in SEQ ID NO:54
  • the sequence shown, 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 chain FR2-HCDR2-heavy chain FR3-HCDR3-heavy chain FR4.
  • the heavy chain variable region of the first binding moiety comprises heavy chain FR1 as set forth in SEQ ID NO:49, HCDR1 as set forth in SEQ ID NO:1, HCDR1 as set forth in 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 NO:5-41 and as shown in SEQ ID Heavy chain FR4 shown in NO:55.
  • the heavy chain variable region of the first binding moiety comprises heavy chain FR1 as set forth in SEQ ID NO:50, HCDR1 as set forth in SEQ ID NO:2, HCDR1 as set forth 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 HCDR3 as shown in SEQ ID NO:55 Heavy chain FR4.
  • the first binding moiety heavy chain variable region comprises the sequence set forth in SEQ ID NO: 56 or 57, or a sequence at least 80% identical to the sequence set forth in SEQ ID NO: 56 or 57 , or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 56 or 57.
  • the first binding moiety light chain variable region comprises the sequence set forth in SEQ ID NO: 58 or 59, or a sequence having at least 80% identity 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 first binding portion heavy chain variable region comprises the sequence set forth in SEQ ID NO:56, and the first binding portion light chain variable region comprises the sequence set forth in SEQ ID NO:58.
  • the first binding portion heavy chain variable region comprises the sequence set forth in SEQ ID NO:57
  • the first binding portion light chain variable region comprises the sequence set forth in SEQ ID NO:59.
  • the first binding moiety 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 first binding moiety is an isotype of IgG, IgM, IgA, IgE, or IgD, or a fragment thereof.
  • the isotype is IgGl, IgG2, IgG3 or IgG4, or a fragment thereof.
  • the C-terminus of the heavy chain constant region in the first binding moiety is truncated.
  • the C-terminus of the heavy chain constant region in the first binding portion of type IgGl or IgG4 lacks amino acid residues G and K.
  • the first binding moiety is a chimeric antibody, a humanized antibody or a fully human antibody. In a certain aspect, the first binding moiety is a fully 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 parental Fc region.
  • the amino acid modification of the Fc region alters effector function activity relative to the activity of the parental Fc region.
  • the variant Fc region may have altered (i.e., increased or decreased) antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), phagocytosis, opsonization, or cell binding .
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-mediated cytotoxicity
  • phagocytosis opsonization
  • cell binding i.e., phagocytosis
  • amino acid modifications of the Fc region can alter the affinity of the variant Fc region for Fc ⁇ R (Fc ⁇ receptor) 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 heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 60 or 61, or a sequence at least 80% identical to the sequence set forth in SEQ ID NO: 60 or 61, or An amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 60 or 61; and/or
  • the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO:62, or a sequence having at least 80% identity with the sequence shown in SEQ ID NO:62, or a sequence similar to the sequence shown in SEQ ID NO:62 Amino acid sequences having one or more conservative amino acid substitutions are compared.
  • the heavy chain constant region comprises an amino acid sequence as shown in SEQ ID NO:60 or 61, and/or the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO:62 .
  • the heavy chain constant region comprises an amino acid sequence as shown in SEQ ID NO:60, and the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO:62.
  • the heavy chain constant region comprises an amino acid sequence as shown in SEQ ID NO:61, and the light chain constant region comprises an amino acid sequence as shown in SEQ ID NO:62.
  • the first binding moiety comprises a heavy chain and/or a light chain.
  • the heavy chain comprises an amino acid sequence as shown in SEQ ID NO: 71 or 72, or a sequence having at least 80% identity with the sequence shown in SEQ ID NO: 71 or 72, or a sequence with SEQ ID NO: 71 or 72.
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 73 or 74, or a sequence having at least 80% identity with the sequence shown in SEQ ID NO: 73 or 74, or a sequence with SEQ ID NO: 73 or 74
  • the sequences shown are compared to amino acid sequences having one or more conservative amino acid substitutions.
  • the heavy chain of the first binding moiety comprises an amino acid sequence as shown in SEQ ID NO: 71 or 72, and/or the light chain of the first binding moiety comprises an amino acid sequence such as SEQ ID NO : the sequence shown in 73 or 74.
  • the heavy chain of the first binding portion comprises an amino acid sequence as shown in SEQ ID NO:71, and the light chain of the first binding portion comprises an amino acid sequence as shown in SEQ ID NO:73 sequence. In some embodiments, the heavy chain of the first binding portion comprises an amino acid sequence as shown in SEQ ID NO:72, and the light chain of the first binding portion comprises an amino acid sequence as shown in SEQ ID NO:74 sequence.
  • the heavy chain comprises an amino acid sequence such as the sequence shown in amino acid 1 to amino acid 450 in SEQ ID NO:71 or amino acid 1 to amino acid 451 in SEQ ID NO:72, Or a sequence having at least 80% identity to the sequence shown in amino acid 1 to amino acid 450 in SEQ ID NO: 71 or amino acid 1 to amino acid 451 in SEQ ID NO: 72, or to SEQ ID NO An amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in amino acid 1 to amino acid 450 in :71 or amino acid 1 to amino acid 451 in SEQ ID NO:72; and/or
  • the light chain comprises an amino acid sequence as shown in SEQ ID NO: 73 or 74, or a sequence having at least 80% identity with the sequence shown in SEQ ID NO: 73 or 74, or a sequence with SEQ ID NO: 73 or 74
  • the sequences shown are compared to amino acid sequences having one or more conservative amino acid substitutions.
  • the heavy chain of the first binding portion comprises an amino acid sequence such as amino acid 1 to amino acid 450 in SEQ ID NO:71 or amino acid 1 to amino acid 451 in SEQ ID NO:72
  • the sequence shown, and/or the light chain of the first binding portion comprises an amino acid sequence as shown in SEQ ID NO:73 or 74.
  • the heavy chain of the first binding portion comprises an amino acid sequence as shown in amino acid 1 to amino acid 450 in SEQ ID NO:71, and the light chain of the first binding portion comprises amino acid The sequence is as shown in SEQ ID NO:73. In some embodiments, the heavy chain of the first binding portion comprises an amino acid sequence as shown in amino acid 1 to amino acid 451 in SEQ ID NO:72, and the light chain of the first binding portion comprises amino acid The sequence is as shown in SEQ ID NO:74.
  • the first binding moiety comprises 2 heavy chains with the same sequence and 2 light chains with the same sequence.
  • the linker L1 is a polypeptide comprising glycine and serine.
  • the sequence of the linker L1 is (G m S ) 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 L1 is (GGGGS) n , and the n is 1, 2, 3, 4 or 5 independently. In some embodiments, the linker L1 is GGGGS. In some embodiments, the linker L1 is (GGGGS) 2 , as shown in SEQ ID NO:65. In some embodiments, the linker L1 is (GGGGS) 3 . In some embodiments, the linker L1 is (GGGGS) 4 , as shown in SEQ ID NO:63. In some embodiments, the linker L1 is (GGGGS) 5 , as shown in SEQ ID NO:64.
  • the second binding moiety is a single domain antibody.
  • the single domain antibody is VHH.
  • the second binding moiety is a single domain antibody and comprises:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 66, or its variants with single or multiple site substitutions, deletions or insertions
  • HCDR2 which comprises the amino acid sequence shown in SEQ ID NO: 67
  • HCDR3 which comprises the amino acid sequence shown in SEQ ID NO: 68, or its single or variants with substitutions, deletions or insertions at multiple sites.
  • the second binding moiety is a single domain antibody and comprises:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 66, or its variants with single or multiple site substitutions, deletions or insertions
  • HCDR2 which comprises the amino acid sequence shown in SEQ ID NO: 67 The amino acid sequence shown, or its single or multiple site substitution, deletion or insertion variant
  • HCDR3 which comprises the amino acid sequence shown in SEQ ID NO: 68, or its single or multiple variants of substitutions, deletions or insertions.
  • the second binding moiety is a single domain antibody and comprises:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO:66
  • HCDR2 which comprises the amino acid sequence shown in SEQ ID NO:67
  • HCDR3 which comprises the amino acid sequence shown in SEQ ID NO : the amino acid sequence shown in 68.
  • the single domain antibody is VHH.
  • the single domain antibody comprises the sequence set forth in SEQ ID NO: 69, or a sequence at least 80% identical to the sequence set forth in SEQ ID NO: 69, or to SEQ ID NO: The sequence shown in 69 is compared to the amino acid sequence having one or more conservative amino acid substitutions.
  • the second binding moiety is a single domain antibody and comprises the amino acid sequence shown in SEQ ID NO:69.
  • the bispecific antibody comprises the above-mentioned first binding portion that binds to the spike protein and the second binding portion that binds to the spike protein, connected through the above-mentioned linker L1.
  • the bispecific antibody comprises the following characteristics:
  • the first binding moiety at least comprises HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:4, HCDR3 as shown in SEQ ID NO:42, HCDR3 as shown in SEQ ID NO:44
  • HCDR1 as shown in SEQ ID NO:2
  • HCDR2 as shown in SEQ ID NO:4
  • HCDR3 as shown in SEQ ID NO:42
  • HCDR3 as shown in SEQ ID NO:44
  • the second binding part is VHH, and the second binding part at least comprises HCDR1 shown in SEQ ID NO: 66, HCDR2 shown in SEQ ID NO: 67, and HCDR3 shown in SEQ ID NO: 68 one, two or three; and/or
  • the C-terminal of the first binding part is connected to the N-terminal of the second binding part through the linker L1, and the C-terminal of the first binding part is the C-terminal of the heavy chain or the light chain of the first binding part. the C-terminus of the chain; and/or
  • the amino acid sequence of the linker L1 is (GGGGS) n , and the n is 1, 2, 3, 4 or 5 independently.
  • the bispecific antibody comprises the following characteristics:
  • the first binding moiety comprises HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:4, HCDR3 as shown in SEQ ID NO:42, LCDR1 as shown in SEQ ID NO:44 , LCDR2 as shown in SEQ ID NO:46 and LCDR3 as shown in SEQ ID NO:48; and/or
  • the second binding moiety is VHH, the second binding moiety comprises HCDR1 as shown in SEQ ID NO:66, HCDR2 as shown in SEQ ID NO:67, and HCDR3 as shown in SEQ ID NO:68; and /or
  • the C-terminal of the first binding part is connected to the N-terminal of the second binding part through the linker L1, and the C-terminal of the first binding part is the C-terminal of the heavy chain or the light chain of the first binding part. the C-terminus of the chain; and/or
  • the sequence of the linker L1 is (GGGGS) n , and the n is 1, 2, 3, 4 or 5 independently.
  • the first binding moiety heavy chain variable region comprises the sequence set forth in SEQ ID NO:57, or a sequence at least 80% identical to the sequence set forth in SEQ ID NO:57, or An amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:57.
  • the first binding moiety light chain variable region comprises the sequence set forth in SEQ ID NO:59, or a sequence at least 80% identical to the sequence set forth in SEQ ID NO:59, or An amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:59.
  • the first binding portion heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:57
  • the first binding portion light chain variable region comprises the amino acid sequence shown in SEQ ID NO:59 sequence.
  • the first binding moiety 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 first binding moiety is of the IgG, IgM, IgA, IgE or IgD type, or a fragment thereof.
  • the first binding moiety is of the IgGl, IgG2, IgG3 or IgG4 type, or a fragment thereof.
  • the C-terminus of the heavy chain constant region in the first binding moiety is truncated.
  • the C-terminus of the heavy chain constant region in the first binding portion of type IgGl or IgG4 lacks amino acid residues G and K.
  • 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 parental Fc region.
  • the first binding moiety is scFv, Fab, Fab', F(ab) 2 or F(ab) 2 '.
  • the first binding moiety and/or the second binding moiety is a chimeric antibody, a humanized antibody, or a fully human antibody.
  • the heavy chain constant region of the first binding part comprises an amino acid sequence such as the sequence shown in amino acid 1 to amino acid 328 in SEQ ID NO: 60 or 61, or with SEQ ID NO: 60 or A sequence having at least 80% identity compared to the sequence shown in amino acid 1 to amino acid 328 in 61, or a sequence with amino acid 1 to amino acid 328 in SEQ ID NO: 60 or 61 Amino acid sequences with one or more conservative amino acid substitutions; and/or
  • the light chain constant region of the first binding moiety comprises an amino acid sequence as shown in SEQ ID NO:62, or a sequence having at least 80% identity compared with the sequence shown in SEQ ID NO:62, or a sequence with SEQ ID NO:62 Amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in :62.
  • the heavy chain constant region of the first binding portion comprises an amino acid sequence such as the sequence shown in amino acid 1 to amino acid 328 in SEQ ID NO:60, and the light chain of the first binding portion The constant region comprises an amino acid sequence as shown in SEQ ID NO:62.
  • the heavy chain constant region of the first binding portion comprises an amino acid sequence as shown in amino acid 1 to amino acid 328 in SEQ ID NO:61, and the light chain of the first binding portion The constant region comprises an amino acid sequence as shown in SEQ ID NO:62.
  • the heavy chain constant region of the first binding moiety comprises an amino acid sequence as set forth in SEQ ID NO: 60 or 61, or has at least 80% amino acid sequence compared to the sequence set forth in SEQ ID NO: 60 or 61 A sequence of identity, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 60 or 61; and/or
  • the light chain constant region of the first binding moiety comprises an amino acid sequence as shown in SEQ ID NO:62, or a sequence having at least 80% identity compared with the sequence shown in SEQ ID NO:62, or a sequence with SEQ ID NO:62 Amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in :62.
  • the heavy chain constant region of the first binding portion comprises the amino acid sequence shown in SEQ ID NO:60, and the light chain constant region of the first binding portion comprises the amino acid sequence shown in SEQ ID NO:62 sequence. In some embodiments, the heavy chain constant region of the first binding portion comprises the amino acid sequence shown in SEQ ID NO:61, and the light chain constant region of the first binding portion comprises the amino acid sequence shown in SEQ ID NO:62 sequence.
  • the second binding moiety is VHH. In some embodiments, the second binding moiety comprises a sequence as set forth in SEQ ID NO: 69, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 69, or to a sequence set forth in SEQ ID NO: 69 Amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in :69.
  • Some embodiments provide a bispecific antibody comprising a first binding moiety and a single domain antibody, and comprising the following features:
  • the first binding part includes a heavy chain and a light chain; the heavy chain of the first binding part comprises an amino acid sequence such as the sequence shown in amino acid 1 to amino acid 451 in SEQ ID NO: 72, or the sequence shown in SEQ ID NO:72 A sequence having at least 80% identity compared to the sequence shown in amino acid 1 to amino acid 451 in NO:72, or compared to the sequence shown in amino acid 1 to amino acid 451 in SEQ ID NO:72 Amino acid sequence with one or more conservative amino acid substitutions; and/or
  • the light chain of the first binding portion comprises an amino acid sequence as shown in SEQ ID NO:74, or a sequence having at least 80% identity compared with the sequence shown in SEQ ID NO:74, or with SEQ ID NO: An amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in 74; and/or
  • the C-terminus of the heavy chain of the first binding moiety is covalently linked to the single domain antibody via a linker L1 as shown in SEQ ID NO:63; and/or
  • the single domain antibody comprises a sequence as shown in SEQ ID NO:69, or a sequence having at least 80% identity compared to the sequence shown in SEQ ID NO:69, or compared to the sequence shown in SEQ ID NO:69 An amino acid sequence with one or more conservative amino acid substitutions.
  • the heavy chain of the first binding portion comprises an amino acid sequence as shown in amino acid 1 to amino acid 451 in SEQ ID NO:72, and the light chain of the first binding portion comprises amino acid The sequence is as shown in SEQ ID NO: 74; the C-terminal (ie CH3 end) of the heavy chain of the first binding part is covalently linked to the single domain antibody through the linker L1 shown in SEQ ID NO: 63, so Said single domain antibody comprises the sequence shown in SEQ ID NO:69.
  • Some embodiments provide a bispecific antibody comprising a first binding moiety and a single domain antibody, and comprising the following features:
  • the first binding part comprises a heavy chain and a light chain; the heavy chain of the first binding part comprises an amino acid sequence as shown in SEQ ID NO:72, or has an amino acid sequence compared with the sequence shown in SEQ ID NO:72 A sequence of at least 80% identity, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 72; and/or
  • the light chain of the first binding portion comprises an amino acid sequence as shown in SEQ ID NO:74, or a sequence having at least 80% identity compared with the sequence shown in SEQ ID NO:74, or with SEQ ID NO: An amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in 74; and/or
  • the C-terminus of the light chain of the first binding moiety is covalently linked to the single domain antibody via a linker L1 as shown in SEQ ID NO:64; and/or
  • the single domain antibody comprises a sequence as shown in SEQ ID NO:69, or a sequence having at least 80% identity compared to the sequence shown in SEQ ID NO:69, or compared to the sequence shown in SEQ ID NO:69 An amino acid sequence with one or more conservative amino acid substitutions.
  • the heavy chain of the first binding portion comprises an amino acid sequence as shown in SEQ ID NO:72, and the light chain of the first binding portion comprises an amino acid sequence as shown in SEQ ID NO:74 Sequence; the C-terminus (i.e. CL terminus) of the light chain of the first binding moiety is covalently linked to a single domain antibody comprising a single domain antibody comprising SEQ ID NO: The sequence shown in 69.
  • a bispecific antibody comprises a first polypeptide and a second polypeptide. In some embodiments, the bispecific antibody comprises two first polypeptides with the same sequence and two second polypeptides with the same sequence.
  • the first polypeptide comprises, or consists of, the heavy chain of the first binding moiety, the linker L1, the single domain antibody sequentially from the N-terminus to the C-terminus; the second polypeptide comprises Light chains, or consisting of them.
  • the first polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 77, or a sequence having at least 80% identity with the sequence shown in SEQ ID NO: 77, or with SEQ ID NO: 77.
  • the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 74, or a sequence having at least 80% identity compared with the sequence shown in SEQ ID NO: 74, or a sequence shown in SEQ ID NO: 74 An amino acid sequence having, or consisting of, one or more conservative amino acid substitutions compared to the sequence.
  • the first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:77
  • the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:74.
  • the first polypeptide comprises, or consists of, a heavy chain
  • the second polypeptide comprises, from the N-terminus to the C-terminus, the light chain of the first binding moiety, the linker L1, the single A domain antibody, or consisting of it.
  • the first polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 72, or a sequence with at least 80% identity compared with the sequence shown in SEQ ID NO: 72, or a sequence with SEQ ID NO: 72 NO:72 shows an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence, or consists of it; and/or
  • the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:78, or a sequence with at least 80% identity compared with the sequence shown in SEQ ID NO:78, or a sequence similar to the sequence shown in SEQ ID NO:78 An amino acid sequence having, or consisting of, one or more conservative amino acid substitutions.
  • the first polypeptide comprises an amino acid sequence as shown in SEQ ID NO:72
  • the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:78.
  • the diabody is an isolated diabody.
  • the bispecific antibody is a bispecific monoclonal antibody.
  • the isolated bispecific antibody is a monoclonal antibody.
  • the first binding portion of the bispecific antibody specifically binds a spike protein. In some embodiments, the second binding portion of the bispecific antibody specifically binds a spike protein.
  • the present invention also provides a single-domain antibody with high affinity for the spike protein of SARS-CoV-2.
  • Single-domain antibodies can bind to spike proteins, prevent virus particles from binding to cells, and mediate immune cell phagocytosis and clearance of virus particles.
  • Single domain antibodies can be used to prevent, treat or improve COVID-19, and can also be used to diagnose COVID-19.
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 66, or its variants with single or multiple site substitutions, deletions or insertions
  • HCDR2 which comprises the amino acid sequence shown in SEQ ID NO: 67
  • HCDR3 which comprises the amino acid sequence shown in SEQ ID NO: 68, or its single or variants with substitutions, deletions or insertions at multiple sites.
  • the single domain antibody comprises:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 66, or its variants with single or multiple site substitutions, deletions or insertions
  • HCDR2 which comprises the amino acid sequence shown in SEQ ID NO: 67 The amino acid sequence shown, or its single or multiple site substitution, deletion or insertion variant
  • HCDR3 which comprises the amino acid sequence shown in SEQ ID NO: 68, or its single or multiple variants of substitutions, deletions or insertions.
  • the single domain antibody comprises HCDR1 as set forth in SEQ ID NO:66, HCDR2 as set forth in SEQ ID NO:67, and HCDR3 as set forth in SEQ ID NO:68.
  • the single domain antibody is VHH. In some embodiments, the single domain antibody comprises the sequence set forth in SEQ ID NO: 69, or a sequence at least 80% identical to the sequence set forth in SEQ ID NO: 69, or to SEQ ID NO: The sequence shown in 69 is compared to the amino acid sequence having one or more conservative amino acid substitutions.
  • the single domain antibody comprises or consists of the sequence shown in SEQ ID NO: 69.
  • single domain antibodies are isolated antibodies. In some embodiments, a single domain antibody is an isolated monoclonal antibody.
  • the invention also provides a heavy chain antibody comprising a single domain antibody that can bind a spike protein, such as a single domain antibody as described herein.
  • Heavy chain antibodies prevent virus particles from binding to cells, and can mediate immune cell phagocytosis and clearance of virus particles. Heavy chain antibodies can be used to prevent, treat or improve COVID-19, and can also be used to diagnose COVID-19.
  • variable region of the heavy chain antibody comprising:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 66, or its variants with single or multiple site substitutions, deletions or insertions
  • HCDR2 which comprises the amino acid sequence shown in SEQ ID NO: 67
  • HCDR3 which comprises the amino acid sequence shown in SEQ ID NO: 68, or its single or variants with substitutions, deletions or insertions at multiple sites.
  • the heavy chain antibody comprises:
  • HCDR1 which comprises the amino acid sequence shown in SEQ ID NO: 66, or its variants with single or multiple site substitutions, deletions or insertions
  • HCDR2 which comprises the amino acid sequence shown in SEQ ID NO: 67 The amino acid sequence shown, or its single or multiple site substitution, deletion or insertion variant
  • HCDR3 which comprises the amino acid sequence shown in SEQ ID NO: 68, or its single or multiple variants of substitutions, deletions or insertions.
  • variable region of the heavy chain antibody comprises HCDR1 as set forth in SEQ ID NO:66, HCDR2 as set forth in SEQ ID NO:67, and HCDR3 as set forth in SEQ ID NO:68.
  • variable region of the heavy chain antibody comprises a sequence as set forth in SEQ ID NO: 69, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 69, or to An amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 69, or consisting of it.
  • variable region of the heavy chain antibody comprises, or consists of, the sequence shown in SEQ ID NO:69.
  • a heavy chain antibody comprises a variable region, a linker L2 and an Fc region.
  • the linker L2 is a polypeptide comprising glycine and serine.
  • the sequence of the linker L2 is (G m S ) n , wherein each m is independently 2, 3, 4 or 5, and n is independently 1, 2, 3, 4 or 5.
  • the sequence of the linker L2 is (GGGGS) n , and the n is 1, 2, 3, 4 or 5 independently.
  • the linker L2 is GGGGS.
  • the linker L2 is (GGGGS) 2 , as shown in SEQ ID NO:65.
  • the linker L2 is (GGGGS) 3 .
  • the linker L2 is (GGGGS) 4 , as shown in SEQ ID NO:63.
  • the linker L2 is (GGGGS) 5 , as shown in SEQ ID NO:64.
  • the Fc region comprises an amino acid sequence as set forth in SEQ ID NO: 70, or a sequence having at least 80% identity to the sequence set forth in SEQ ID NO: 70, or to a sequence set forth in SEQ ID NO: 70
  • the sequences shown are compared to amino acid sequences having one or more conservative amino acid substitutions.
  • the heavy chain antibody comprises an amino acid sequence as set forth in SEQ ID NO: 79, or a sequence at least 80% identical to the sequence set forth in SEQ ID NO: 79, or to SEQ ID NO: The sequence shown in 79 is compared to the amino acid sequence having one or more conservative amino acid substitutions.
  • the heavy chain antibody comprises an amino acid sequence as set forth in SEQ ID NO:79.
  • the antibody specifically binds a spike protein.
  • the antibody or antigen-binding fragment thereof is an isolated antibody or antigen-binding fragment.
  • the present invention also provides a method for preparing the antibody (including bispecific antibody, single domain antibody and heavy chain antibody), which comprises culturing host cells containing the nucleic acid encoding the antibody in a culture medium.
  • the method further comprises purifying the antibody. Purification can be carried out by conventional methods, such as centrifuging the cell suspension first, collecting the supernatant, and centrifuging again to further remove impurities. Methods such as ProteinA affinity column and ion exchange column can be used to purify antibody protein.
  • the present invention also provides nucleic acid encoding the antibody (including bispecific antibody, single domain antibody and heavy chain antibody).
  • the nucleic acid is an isolated nucleic acid.
  • the nucleic acid sequences are shown in Table 5 and Table 6.
  • the nucleic acid sequence encoding antibody heavy chain SEQ ID NO:72 is shown in SEQ ID NO:75.
  • the nucleic acid sequence encoding antibody light chain SEQ ID NO:74 is shown in SEQ ID NO:76.
  • the nucleic acid sequence encoding the first antibody polypeptide SEQ ID NO:77 is shown in SEQ ID NO:80 or 83.
  • nucleic acid sequence encoding the antibody second polypeptide SEQ ID NO:78 is shown in SEQ ID NO:81. In some embodiments, the nucleic acid sequence encoding heavy chain antibody SEQ ID NO:79 is shown in SEQ ID NO:82.
  • 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 a host cell comprising the nucleic acid or the vector.
  • the host cell is an isolated host cell.
  • the host cells are CHO cells, HEK293 cells, Cos1 cells, Cos7 cells, CV1 cells, and murine L cells.
  • the present invention also provides a pharmaceutical composition, which comprises the antibody and pharmaceutically acceptable auxiliary materials.
  • the invention also provides treatment methods and uses.
  • a method for preventing, treating or improving COVID-19 is provided, the method comprising administering an effective dose of the antibody (including bispecific antibody, single domain antibody and heavy chain antibody) to a patient.
  • the use of the antibodies (including bispecific antibodies, single domain antibodies and heavy chain antibodies) in preventing, treating or improving COVID-19 is provided.
  • the application of the antibodies (including bispecific antibodies, single domain antibodies and heavy chain antibodies) in the preparation of medicaments for preventing, treating or improving COVID-19 is provided.
  • the invention also provides diagnostic methods and uses.
  • a method for detecting the expression of SARS-CoV-2 in a sample is provided, the sample is contacted with the antibody (including bispecific antibody, single domain antibody and heavy chain antibody), so that the antibody (including bispecific antibody) Specific antibodies, single domain antibodies, and heavy chain antibodies) bind spike proteins and detect their binding, that is, the amount of spike proteins in the sample.
  • the use of the antibody (including bispecific antibody, single domain antibody and heavy chain antibody) in the preparation of a kit for diagnosing COVID-19 is provided.
  • diagnostic kits comprising the antibodies, including bispecific antibodies, single domain antibodies, and heavy chain antibodies, are provided.
  • the invention provides a bispecific antibody targeting coronavirus and its application.
  • the first binding part and the second binding part in the bispecific antibody cooperate to prevent SARS-CoV-2 virus particles from infecting cells, and mediate immune cell phagocytosis and clearance Virus particles can prevent, treat or improve COVID-19;
  • the bispecific antibody of the present invention can also be used to diagnose and detect whether a patient is infected with SARS-CoV-2.
  • Figure 1 is a graph showing the inhibition of the binding of SARS-CoV-2 to ACE2 by some anti-spike protein antibodies of the present invention in an ELISA experiment.
  • the abscissa represents the concentration, and the ordinate represents the OD value; among them, 1 represents antibody 1, 7 represents antibody 7, 8 represents antibody 8, 9 represents antibody 9, 12 represents antibody 12, 18 represents antibody 18, and 19 represents antibody 19 , 20 represents antibody 20, 21 represents antibody 21, and 22 represents antibody 22.
  • Figure 2 shows that the antibody blocks the binding of spike RBD to ACE2; wherein, no antibody was added to the ACE2 control group.
  • Figure 3 shows that the antibody blocks the virus from infecting mice; in the figure, the ordinate indicates the lung virus titer.
  • Fig. 4 shows the effect of the antibody on the body weight of the mice; in the figure, the ordinate indicates the percentage of the mouse body weight, and the abscissa indicates the days.
  • an entity refers to one or more such entities, for example "an antibody” should be understood as one or more antibodies, therefore, the term “a” (or “an” ), “one or more” and “at least one” may be used interchangeably herein.
  • compositions, methods, etc. include the listed elements, such as components or steps, but not exclude others.
  • Consisting essentially of means that the compositions and methods exclude other elements that substantially affect the characteristics of the combination, but do not exclude elements that do not substantially affect the composition or method.
  • Consisting of means excluding elements not specifically recited.
  • polypeptide is intended to encompass the singular as well as the plural “polypeptides” and refers to a molecule formed of amino acid monomers linked linearly by amide bonds (also known as peptide bonds).
  • polypeptide refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product.
  • the definition of “polypeptide” includes peptide, dipeptide, tripeptide, oligopeptide, "protein”, “amino acid chain” or any other term used to refer to a chain of two or more amino acids, and the term “polypeptide” may Used in place of, or interchangeably with, any of the above terms.
  • polypeptide is also intended to refer to the products 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-natural Amino acid modifications that occur.
  • a polypeptide may be derived from natural biological sources or produced by recombinant techniques, but it need not be translated from a specified nucleic acid sequence, it may be produced by any means including chemical synthesis.
  • amino acid refers to an organic compound containing both amino and carboxyl groups, such as an ⁇ -amino acid, which can be encoded by a nucleic acid directly or in the form of a precursor.
  • 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 fact that the same amino acid is encoded by different codons is 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).
  • a “conservative amino acid substitution” refers to the replacement of one amino acid residue with another amino acid residue containing a side chain (R group) of similar chemical properties (eg, charge or hydrophobicity). In general, conservative amino acid substitutions are unlikely to substantially alter the functional properties of a protein.
  • classes of amino acids that contain 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, VH, VHH" 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 between any two of these values (inclusive), or any value therein.
  • the number of amino acids in the "heavy chain constant region, light chain constant region, heavy chain or light chain, fusion protein first polypeptide or second polypeptide conservative amino acid substitution" 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 ( inclusive) or any value in it.
  • isolated used in the present invention with respect to cells, nucleic acids, polypeptides, antibodies, etc., for example, "isolated" DNA, RNA, polypeptides, antibodies refers to the isolated components of the cell's natural environment, such as DNA or RNA. One or more of the isolated molecules.
  • isolated as used herein also 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 fragments of nucleic acid that do not occur in nature, and do not exist in nature.
  • isolated is also used herein to refer to cells or polypeptides that are separated from other cellular proteins or tissues.
  • Isolated polypeptide is intended to include purified and recombinant polypeptides.
  • Isolated polypeptides, antibodies, etc. will usually be prepared by at least one purification step.
  • the purity of the isolated nucleic acid, polypeptide, antibody, etc. is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or some of these values The range between any two values of , including the endpoints, or any value therein.
  • polypeptides or polynucleotides refers to polypeptides or polynucleotides, meaning forms of polypeptides or polynucleotides that do not occur in nature, non-limiting examples may be produced by combination of polynucleotides or polynucleotides that do not normally exist or peptide.
  • Homology refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology or identity can be determined by comparing the alignable positions in each sequence. When a position in the sequences being compared is occupied by the same base or amino acid, then the molecules are homologous or identical at that position. The degree of homology between sequences is a function of the number of matching 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 (inclusive) between any two values in Numeric 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 between any two of these values (inclusive), or any value therein.
  • a nucleic acid or polynucleotide sequence (or polypeptide or antibody sequence) having a certain percentage (eg, 90%, 95%, 98%, or 99%) of "identity” or "sequence identity” with another sequence means When sequences are aligned, the percentage of bases (or amino acids) in the two sequences being compared are the same.
  • This alignment percent identity 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. It is preferred to use the default parameters for the alignment.
  • Biologically equivalent polynucleotides are polynucleotides that share the above indicated percentages of identity and encode a polypeptide having the same or similar biological activity.
  • a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), or when a polynucleotide In the case of RNA, thymine is replaced by uracil (U).
  • a "polynucleotide sequence” may be denoted by the letters of the polynucleotide molecule. This letter designation can be entered into a database in a computer with a central processing unit and used in bioinformatics applications such as for functional genomics and homology searches.
  • polynucleotide refers to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or their analog.
  • a polynucleotide can have any three-dimensional structure and can perform any function, known or unknown.
  • polynucleotides genes or gene fragments (e.g., 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.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • nucleotides can be made before or after assembly of the polynucleotide.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • Polynucleotides may be further modified after polymerization, for example by conjugation with labeling components.
  • the term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiment of a polynucleotide of the present disclosure includes 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 which is said to "encode” a polypeptide which, in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and/or translated to produce 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 whose molecule contains at least a portion of an immunoglobulin molecule that has the biological activity to bind an antigen.
  • Antibodies and antigen-binding fragments include, but are not limited to, complementarity determining regions (CDRs), heavy chain variable regions (VH), light chain variable regions (VL) of heavy or light chains or their ligand-binding portions described in the Examples.
  • CDRs complementarity determining regions
  • VH heavy chain variable regions
  • VL light chain variable regions
  • a heavy chain constant region (CH)
  • a light chain constant region CL
  • a framework region 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).
  • a 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 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 variable regions of the heavy (VH) and light (VL) chains 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 for flexibility, and serine or threonine for solubility, and can connect the N-terminus of VH to the C-terminus of VL, or vice versa. Although the protein has had its constant regions removed and a linker introduced, it retains the specificity of the original immunoglobulin. ScFv molecules are generally known in the art, for example as described in US Pat. No. 5,892,019.
  • single domain antibody or “sdAb” refers to a single antigen-binding polypeptide having three complementarity determining regions (CDRs). An sdAb alone is capable of binding the antigen but not paired with the corresponding CDR-containing polypeptide. In some cases, sdAbs were engineered from camelid HCAbs, and their heavy chain variable domains are referred to herein as "VHH".
  • Camelid sdAbs are one of the smallest known antigen-binding antibody fragments (see, e.g., Hamers-Casterman et al., Nature 363:446-8 (1993); Greenberg et al., Nature 374:168-73 (1995); Hassanzadeh- Ghassabeh et al., Nanomedicine (Lond), 8:1013-26 (2013)).
  • HcAb heavy chain antibody
  • VHH, CH2 and CH3 heavy chains
  • Camelids such as camels, llamas or alpacas are known to produce HcAbs.
  • the first binding moiety in the bispecific antibody is an antibody targeting spike protein, which is a whole antibody or an antigen-binding fragment. In some embodiments, the first binding moiety is an IgG type antibody. In some embodiments, the first binding moiety is an IgG type antibody, and the C-terminus of its heavy chain is truncated.
  • the second binding moiety in the bispecific antibody is an antibody targeting spike protein, which is a whole antibody or an antigen-binding fragment. In some embodiments, the second binding moiety in the bispecific antibody is a single domain antibody.
  • antibody includes a wide variety of polypeptides that can be distinguished biochemically.
  • classes of heavy chains include gamma, mu, alpha, delta or epsilon ( ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ), with some subclasses (eg ⁇ 1- ⁇ 4).
  • the nature of this chain determines the "class” of the antibody as IgG, IgM, IgA, IgG or IgE, respectively.
  • the immunoglobulin subclasses (isotypes), eg, IgGl, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and the functional specificities conferred are also known.
  • the immunoglobulin molecule is of the IgG class.
  • IgG typically comprises two identical light chain polypeptides with a molecular weight of about 23,000 Daltons and two identical heavy chain polypeptides with a molecular weight of about 53,000-70,000.
  • the four chains are linked by disulfide bonds in a "Y" configuration, with the light chain starting at the mouth of the "Y” and continuing through the variable region surrounding the heavy chain.
  • Antibodies, antigen-binding fragments, or derivatives disclosed in the present invention include but are not limited to polyclonal, monoclonal, multispecific, fully human, humanized, primatized, chimeric antibody/single chain antibody, epitope 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 Generated fragments and anti-idiotypic (anti-Id) antibodies.
  • epitope 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 Generated fragments and anti-idiotypic (anti-Id) antibodies.
  • immunoglobulins or antibody molecules disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulins or subclass.
  • type e.g., IgG, IgE, IgM, IgD, IgA, and IgY
  • class e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2
  • Light chains can be classified as kappa ( ⁇ ) or lambda ( ⁇ ). Each heavy chain can be associated with a kappa or lambda light chain.
  • ⁇ kappa
  • lambda ⁇
  • Each heavy chain can be associated with 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" portions of the two heavy chains are linked by covalent disulfide bonds or non-covalent bonding.
  • the amino acid sequence extends from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain.
  • the variable region of the immunoglobulin kappa light chain is V ⁇ ; the variable region of the 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 variable region of the light chain (VL) and the variable region of the heavy chain (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, etc. By convention, the numbering of constant regions increases as they become farther away 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-terminal ends of the heavy and light chains, respectively.
  • variable regions enable the antibody to selectively recognize and specifically bind epitopes on antigens.
  • 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 the Y. More specifically, the antigen binding site is defined by three CDRs (ie, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) in each of the VH and VL chains.
  • immunoglobulin molecules 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 short, A non-contiguous sequence of amino acids that specifically binds to an antigen. The remaining other amino acids in the antigen-binding domain, referred to as the "framework" regions, show less inter-molecular variability.
  • the framework regions mostly adopt a ⁇ -sheet conformation, and the CDRs form loop structures attached to them, or in some cases form part of the ⁇ -sheet structure. Thus, the framework regions position the CDRs in the correct orientation by forming a scaffold through non-covalent interchain interactions.
  • the antigen-binding domain with the CDRs in specific positions forms a surface complementary to the epitope on the antigen that facilitates the non-covalent binding of the antibody to its antigenic epitope.
  • a surface complementary to the epitope on the antigen that facilitates the non-covalent binding of the antibody to its antigenic epitope.
  • those of ordinary skill in the art can identify the amino acids comprising CDR and framework regions 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 regions
  • CDRs as defined by Kabat and Chothia include overlapping or subsets of amino acid residues when compared to each other. Nevertheless, it is within the scope of the invention to use either definition to refer to the CDRs of an antibody or variant thereof.
  • the exact residue numbers comprising a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can generally determine which specific residues are included in the CDRs based on the amino acid sequence of the variable region of the antibody.
  • Kabat et al. also defined a numbering system applicable to the variable region sequences of any antibody.
  • One of ordinary skill in the art can apply this "Kabat numbering" system to any variable region sequence independently of other experimental data other than the sequence itself.
  • “Kabat numbering” refers to 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 can also use the EU numbering system.
  • the antibodies or antigen-binding fragments disclosed herein may 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).
  • a "heavy chain constant region” includes an amino acid sequence derived from an immunoglobulin heavy chain.
  • a polypeptide comprising a heavy chain constant region 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 antibody or antigen-binding fragment disclosed in the present invention comprises a CH1 domain; comprises a CH1 domain, at least a part of the hinge region and a CH2 domain; comprises a CH1 domain and a CH3 domain; comprises a CH1 domain and at least a part of the hinge region and a CH3 domain; or comprising a CH1 domain, at least a portion of the hinge region, and a CH2 domain and a CH3 domain.
  • an antibody or antigen-binding fragment disclosed herein comprises a CH3 domain.
  • antibodies or antigen-binding fragments used in the present invention may lack part or all of the CH2 domain.
  • heavy chain constant regions may be modified such that the amino acid sequence of their naturally occurring immunoglobulin molecule is altered.
  • 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.
  • part of the heavy chain may comprise a chimeric hinge region derived partly from an IgG 1 molecule and partly from an IgG4 molecule.
  • a “light chain constant region” includes an amino acid sequence from an antibody light chain.
  • 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 form dimers through disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain.
  • a "VH domain” includes the amino-terminal variable domain of an immunoglobulin heavy chain
  • a "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 amino-terminal to the hinge region of an immunoglobulin heavy chain molecule.
  • the CH2 domain is not closely 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.
  • a "hinge region” includes part of the heavy chain region connecting the CH1 domain and the CH2 domain.
  • the hinge region comprises approximately 25 residues and is flexible, 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.
  • a thiol group of cysteine can form a disulfide bond or bridge with a second thiol group.
  • the CH1 and CL regions are linked by a disulfide bond, and the two heavy chains are linked by two disulfide bonds at positions 239 and 242 in the Kabat numbering system (positions 226 and 229 in the EU numbering system) connected everywhere.
  • a “chimeric antibody” refers to any antibody whose variable regions are obtained or derived from a first species and whose constant regions (which may be complete, partial or modified) are derived from a second species.
  • the variable regions are of non-human origin (eg, mouse or primate) and the constant regions are of human origin.
  • Specific binding or “specific for” generally refers to the complementary binding of an antibody or antigen-binding fragment to a specific antigen through its antigen-binding domain and epitope to form a relatively stable complex.
  • 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, antibody “A” may be said to have a higher specificity for that antigen than antibody “B” if it has a greater relative affinity for the same antigen than antibody "B”.
  • Specific binding can be described by an equilibrium dissociation constant (KD), with a smaller KD implying a tighter binding.
  • KD equilibrium dissociation constant
  • An antibody that "specifically binds" to a spike protein includes an equilibrium dissociation constant KD 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.
  • Monospecific antibodies can specifically bind one antigen or one epitope, while bispecific antibodies can specifically bind two different antigens or two different epitopes.
  • Treatment means therapeutic treatment and prophylactic or preventive measures, the purpose of which is to prevent, slow down, 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 Relief of symptoms, reduction of disease extent, stabilization of disease state (i.e. not worsening), delay or slowing of disease progression, amelioration, remission, alleviation or disappearance of disease state (whether partial or total), prolongation and Expected survival without treatment, etc.
  • Patients in need of treatment include those who already have a condition or disorder, are susceptible to having a condition or disorder, or are in need of prevention of the condition or disorder, and can or are expected to benefit from the administration of an antibody or pharmaceutical composition 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.
  • ECMO refers to extracorporeal membrane oxygenation (Extracorporeal Membrane Oxygenation, ECMO), which is a medical emergency technology equipment, mainly used to provide continuous extracorporeal respiration and circulation for patients with severe cardiopulmonary failure to maintain their lives.
  • ICU refers to the intensive care unit (Intensive Care Unit), where treatment, nursing, and rehabilitation can be carried out simultaneously, providing isolation places and equipment for severe or comatose patients, providing the best nursing care, comprehensive treatment, combination of medical care and nursing care, and surgery Postoperative early rehabilitation, joint care and sports therapy and other services.
  • Intensive Care Unit intensive care unit
  • Treatment, nursing, and rehabilitation can be carried out simultaneously, providing isolation places and equipment for severe or comatose patients, providing the best nursing care, comprehensive treatment, combination of medical care and nursing care, and surgery Postoperative early rehabilitation, joint care and sports therapy and other services.
  • IMV intermittent mandatory ventilation
  • intermittent mandatory ventilation implements periodic volume or pressure ventilation according to a preset time interval, that is, 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. According to most ventilators can provide pressure support during spontaneous breathing.
  • HFNC High-flow nasal cannula oxygen therapy
  • High-flow nasal cannula oxygen therapy which is an oxygen therapy method that directly delivers a certain oxygen concentration of air and oxygen mixed with high-flow gas to patients through a nasal catheter without sealing.
  • a form of non-invasive respiratory support that rapidly improves oxygenation. At present, it can be applied to patients with acute hypoxic respiratory failure, patients after surgery, patients with respiratory failure without endotracheal intubation, immunosuppressed patients, patients with cardiac insufficiency, etc.
  • NMV Non-invasive Ventilation, which refers to non-invasive mechanical ventilation except tracheal intubation and tracheotomy.
  • EC 50 means half maximum effect concentration (concentration for 50% of maximal effect, EC 50 ) refers to the concentration that can cause 50% of the maximum effect.
  • IC50 means 50% inhibitory concentration, ie the concentration of drug or inhibitor required to inhibit a given biological process by half.
  • the "parental Fc region" in the present invention 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 of SEQ ID NO:61 or SEQ ID NO:66.
  • the invention provides antibodies with high affinity for spike protein, including bispecific antibodies, single domain antibodies and heavy chain antibodies.
  • Bispecific antibodies, single domain antibodies, and heavy chain antibodies exhibit potent binding activity and are useful for therapeutic and diagnostic applications.
  • these antibodies can prevent the fusion of SARS-CoV-2 virus particles and cell membranes, and mediate immune cell phagocytosis and clearance of virus particles.
  • bispecific antibodies in which the C-terminal (ie CH3 terminal) of the heavy chain of the first binding moiety is covalently linked to the single domain antibody through the linker L1.
  • the bispecific antibody comprises two first polypeptides with the same sequence and two second polypeptides with the same sequence; the amino acid sequence of the first polypeptide is shown in SEQ ID NO: 77, the The amino acid sequence of the second polypeptide is shown in SEQ ID NO:74.
  • bispecific antibodies in which the C-terminus (ie, CL terminus) of the light chain of the first binding moiety is covalently linked to the single domain antibody through a linker L1.
  • the bispecific antibody comprises two first polypeptides with the same sequence and two second polypeptides with the same sequence; the amino acid sequence of the first polypeptide is shown in SEQ ID NO: 72, and the The amino acid sequence of the second polypeptide is shown in SEQ ID NO:78.
  • Some embodiments provide a single domain antibody, the amino acid sequence of the single domain antibody is shown in SEQ ID NO:69.
  • Some embodiments provide a heavy chain antibody, the heavy chain antibody contains two heavy chains with the same sequence, the heavy chain sequence of which is shown in SEQ ID NO:79.
  • the diabody, single domain antibody or heavy chain antibody may also be linked with an amino acid sequence or one or more modifying groups.
  • the diabodies, single domain antibodies or heavy chain antibodies disclosed herein may contain a flexible linker sequence, or may be modified to add functional groups (eg PEG, drug, toxin or tag).
  • the bispecific antibody, single domain antibody or heavy chain antibody disclosed in the present invention also includes modified derivatives, that is, modified by covalent attachment of any type of molecule to the antibody, wherein the covalent attachment does not prevent the antibody from binding to the epitope .
  • modified derivatives that is, modified by covalent attachment of any type of molecule to the antibody, wherein the covalent attachment does not prevent the antibody from binding to the epitope .
  • antibodies may be glycosylated, acetylated, pegylated, phosphorylated, amidated, derivatized by known protecting/blocking groups, proteolytically cleaved, linked to cellular ligands, or other proteins etc. Any of the numerous chemical modifications can be performed by existing techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc.
  • a bispecific antibody, single domain antibody or heavy chain antibody can be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG.
  • Bispecific antibodies, single domain antibodies or heavy chain antibodies can be conjugated or fused to therapeutic agents which can include detectable labels such as radiolabels, immunomodulators, hormones, enzymes, oligonucleotides, photosensitive therapeutics , diagnostic agents, cytotoxic agents, ultrasound enhancing agents, non-radioactive labels and compositions thereof, and other such agents known in the art.
  • therapeutic agents can include detectable labels such as radiolabels, immunomodulators, hormones, enzymes, oligonucleotides, photosensitive therapeutics , diagnostic agents, cytotoxic agents, ultrasound enhancing agents, non-radioactive labels and compositions thereof, and other such agents known in the art.
  • a diabody, single domain antibody or heavy chain antibody can be detectably labeled by coupling it to a chemiluminescent compound.
  • the presence of the chemiluminescently labeled antibody is then determined by detecting the luminescence that occurs during the chemical reaction.
  • chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters.
  • the present invention also discloses polynucleotides or nucleic acid molecules encoding the bispecific antibody, single domain antibody and heavy chain antibody of the present invention.
  • the polynucleotide 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, bispecific, single domain antibody or heavy chain antibody.
  • Methods of making antibodies are well known in the art and described herein.
  • both the variable and constant regions of the antibodies 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 to a particular antigen can be produced by administering the antigen to transgenic animals that have been modified to produce fully human antibodies in response to antigen challenge.
  • Exemplary techniques that can be used to prepare such antibodies are found in US Patent Nos. 6,458,592; 6,420,140, the entire contents of which are incorporated herein by reference.
  • antibodies are produced that do not elicit an adverse immune response in the animal (eg, human) to be treated.
  • the antibodies disclosed herein are modified using art-recognized techniques to reduce their immunogenicity.
  • 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, which retain or substantially retain the antigen-binding properties of the parent antibody but are less immunogenic in humans.
  • framework residues in the human framework regions will be replaced by corresponding residues from the CDR donor antibody, such as residues that improve antigen binding.
  • framework substitutions can be identified by methods known in the art, such as by modeling the interaction of CDRs and framework residues to identify framework residues important for antigen binding and by sequence alignment to identify abnormal framework residues at specific positions. (Refer to US Patent 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 Patents 5,225,539, 5,530,101 and 5,585,089), repair or surface rearrangement (EP 592,106; EP 519,596) , and chain rearrangements (US Patent 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/9852976 A1 and WO/0034317 A2).
  • the heavy and light chain variable region sequences from a starting antibody are analyzed and a human T cell epitope "map" from each variable region is generated, showing the epitopes relative to the complementarity determining regions (CDRs) and the positions of other key residues within the sequence.
  • CDRs complementarity determining regions
  • Individual T-cell epitopes from T-cell epitope maps are analyzed to identify alternative amino acid substitutions with lower risk of altering antibody activity.
  • 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.
  • Genes for the complete heavy and light chains containing the modified variable and human constant regions are then cloned into expression vectors, and the plasmids are subsequently transformed into cell lines to produce complete antibodies.
  • Antibodies are then compared using appropriate biochemical and biological assays to identify the best antibody.
  • the binding specificity of the antibodies disclosed in the present invention can be detected by in vitro experiments, such as co-immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • in vitro experiments such as co-immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • scFv Single-chain fusion peptides are generated by amino acid bridging of the heavy and light chain fragments of the Fv region to form single-chain units.
  • the technique of assembling functional Fv fragments in E. coli can also be used (Skerra et al., Science 242:1038-1041 (1988)).
  • scFv single chain Fv
  • antibodies examples include those described in US Pat. Nos. 4,946,778 and 5,258,498.
  • chimeric, humanized or fully human antibodies may be used.
  • Chimeric antibodies are molecules in which different parts of the antibody are derived from different animal species, such as antibodies that have the variable regions of a murine monoclonal antibody and the constant regions of a human immunoglobulin.
  • Methods of producing chimeric antibodies are known in the art, see US Patent Nos. 5,807,715, 4,816,567, and 4,816,397, the entire contents of which are incorporated herein by reference.
  • Naturally occurring VHH domains directed against particular antigens or targets can be obtained from (native or immune) libraries of Camelidae VHH sequences. Such libraries and techniques are described, for example, in WO 99/37681, WO 01/90190, WO 03/025020 and WO 03/035694.
  • improved synthetic or semi-synthetic libraries derived from (native or immune) VHH libraries can be used, for example VHH libraries obtained from (native or immune) VHH libraries by techniques such as random mutagenesis and/or CDR shuffling, e.g. WO 00 /43507 described.
  • Antibodies can be prepared by a variety of methods known in the art, including phage display methods using antibody libraries derived from immunoglobulin sequences. See also U.S. Patents 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 content of the patent is incorporated herein by reference.
  • DNA encoding the desired monoclonal antibody can be isolated and sequenced using conventional methods (e.g., using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). sequencing. Isolated and subcloned hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into 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 cells that do not produce other immunoglobulins in cells.
  • prokaryotic or eukaryotic host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce other immunoglobulins in cells.
  • Isolated DNA (which may be synthetic as described herein) can also be used to prepare the constant and variable region sequences of antibodies as described in US Pat. No. 5,658,570, the entire contents of which are incorporated herein by reference. This method extracts RNA from selected cells and converts it into cDNA, which is then amplified by PCR using Ig-specific primers. Suitable probes for this purpose are also mentioned in US Patent No. 5,658,570.
  • one or more CDRs of an antibody of the invention can be inserted into a framework region, eg, into a human framework region, to construct a humanized non-fully human antibody.
  • the framework regions may be naturally occurring or consensus framework regions, preferably human framework regions (see Chothia et al., J. Mol. Biol. 278:457-479 (1998) for a list of human framework regions).
  • Some polynucleotides may encode an antibody that specifically binds at least one epitope of an antigen of interest produced by a combination of framework regions and CDRs.
  • One or more amino acid substitutions may be made within the framework regions, and the 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 in this way, thereby producing antibody molecules lacking one or more interchain disulfide bonds.
  • Other modifications to polynucleotides within the skill of the art are also encompassed in the present invention.
  • the DNA encoding the antibody can be designed and synthesized according to the amino acid sequence of the antibody described herein according to conventional methods, placed into an expression vector, and then transfected into a host cell, and the transfected host cell is cultured in a culture medium produce antibodies.
  • 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 achieved by early and late promoters of SV40, long terminal repeats from retroviruses such as RSV, HTLV1, HIVI, and early promoters of cytomegalovirus (CMV), and other cellular promoters promoters such as the actin promoter.
  • 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, pCS2 or pCHO1.0 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 selection markers, common selection markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance and other selection genes, so as to facilitate transfection Screening of successful cell isolation.
  • selection markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance and other selection genes, so as to facilitate transfection Screening of successful cell isolation.
  • the constructed plasmid is transfected into host cells without the above-mentioned genes, and cultured in a selective medium, the successfully transfected cells grow in large numbers and produce the desired target protein.
  • mutations can be introduced into the nucleotide sequence encoding the antibodies of the present invention using standard techniques known to those skilled in the art, including but not limited to site-directed mutagenesis and PCR-mediated mutations resulting in amino acid substitutions.
  • Variants include derivatives
  • mutations 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 may be conservative amino acid substitutions.
  • the invention also provides treatment methods and uses.
  • a method for preventing, treating or improving COVID-19 comprising administering an effective dose of an antibody (including a bispecific antibody, a single domain antibody or a heavy chain antibody) to a patient.
  • the application of the antibody (including bispecific antibody, single domain antibody or heavy chain antibody) in preventing, treating or improving COVID-19 is provided.
  • the use of the antibody (including bispecific antibody, single domain antibody or heavy chain antibody) in the preparation of a medicament for preventing, treating or improving COVID-19 is provided.
  • the patient is a patient suspected of being infected with the SARS-CoV-2 virus.
  • the patient is a patient who has been in contact with a SARS-CoV-2 virus carrier. In some embodiments, the patient is a patient confirmed to be infected with the 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 antibody (including bispecific, single domain, or heavy chain antibody) used, the patient's age and weight, general health, sex, and diet , as well as the timing of administration, frequency of excretion, drug combination, and severity of the particular condition being treated. These factors are in the judgment of the medical caregiver, who is 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 employed, the severity of the disease and the effect desired.
  • the dosage used can be determined by principles of pharmacology and pharmacokinetics well known in the art.
  • Methods of administration of antibodies include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, epidural, and oral injections.
  • the pharmaceutical compositions may be administered by any convenient route, such as by infusion or bolus injection, absorbed through the epithelium or mucous membranes (eg, oral mucosa, rectal and intestinal mucosa, etc.), and may be co-administered with other biologically active agents.
  • compositions containing an antibody of the invention can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (e.g., by powder, ointment, drops, etc.) or transdermal patch), orally, or by oral or nasal spray.
  • parenteral refers to modes of administration including intravenous, intramuscular, intranasal, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
  • the mode of administration can be systemic administration or local administration.
  • compositions of the invention comprise nucleic acids or polynucleotides encoding proteins, which can be administered in vivo to promote expression of the encoded proteins by constructing them as part of a suitable nucleic acid expression vector,
  • the above-mentioned part of the vector is then administered to make it an intracellular part, for example by using a retroviral vector (see US Patent No. 4,980,286), or by direct injection, or by using particle bombardment (for example, a gene gun; Biolistic, Dupont) , or coated with liposomes or cell surface receptors or transfection reagents, or administered via linkage to homeobox peptides known to enter the nucleus (see e.g.
  • nucleic acid can be introduced into the cell by homologous recombination and integrated into the host cell DNA for expression.
  • the antibodies of the present invention are administered to patients at a dose of 0.01 mg/kg to 100 mg/kg of patient body weight, or 0.1 mg/kg to 20 mg/kg of patient weight. Each administration may be separated by at least 1 day to 3 days; or at least one week.
  • the dose and frequency of administration of the antibodies of the invention can be reduced by enhancing antibody uptake and tissue penetration (eg, into the brain) through modifications such as lipidation.
  • bispecific antibodies single domain antibodies or heavy chain antibodies of the present invention or polynucleotides encoding them, for example encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing said compounds , receptor-mediated endocytosis (see eg Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of nucleic acids as part of retroviruses or other vectors, etc.
  • antibodies of the invention may be combined with other therapeutic or prophylactic regimens, including administration of one or more antibodies of the invention and one or more other therapeutic agents or methods used together or in combination.
  • antibodies of the invention may be administered simultaneously or separately with other therapeutic agents.
  • an antibody of the invention may be administered before or after another other therapeutic agent is administered.
  • the therapeutic agent used in combination with the antibody of the invention is one of the following: HIV drugs, antimalarial drugs, RNA polymerase inhibitors, antiviral drugs, and monoclonal antibodies.
  • HIV medications include lopinavir/ritonavir, ASC09/ritonavir, and darunavir; lopinavir/ritonavir and ribavirin alone are not recommended.
  • the antimalarial drug includes chloroquine phosphate.
  • antiviral drugs include arbidol, favipiravir, and alpha-interferon.
  • the monoclonal antibody comprises BDB-001.
  • 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 tocilizumab (tochilizumab, e.g.
  • the patient treated by this method is confirmed to be infected with novel coronavirus and has 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 chemoattractant protein -1 (MCP1), macrophage inflammatory protein 1 ⁇ (MIP1A)) increased.
  • TNF- ⁇ tumor necrosis factor alpha
  • IFN- ⁇ IFN- ⁇
  • IL-1 ⁇ interferon-inducible protein-10
  • MCP1A monocyte chemoattractant protein -1
  • MIP1A macrophage inflammatory protein 1 ⁇
  • the subject treated by the present methods has elevated TNF-alpha.
  • one or more cytokines are at least 50% above normal levels. In some embodiments, the one or more cytokines are at least 2-fold, 3-fold, or 4-fold the normal level.
  • the patient has fever, hypotension, hypoxia, and/or acute respiratory distress syndrome (ARDS) prior to treatment by the method.
  • ARDS acute respiratory distress syndrome
  • the patient has lungs filled with inflammatory fluid (so-called "white lung") prior to treatment by the method.
  • the patient has Cytokine Release Syndrome (CRS) caused by cytokine storm before treatment with the method.
  • CRS Cytokine Release Syndrome
  • antibodies of the invention are used in conjunction with ICU therapy. In some embodiments, antibodies of the invention are combined with ECMO and/or IMV therapy in vitro. In some embodiments, an antibody of the invention is combined with oxygen therapy. In some embodiments, antibodies of the invention are combined with NIV/HFNC therapy. In some embodiments, after treatment, one or more cytokines in the patient is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% lower than before treatment . In some embodiments, the method heals the patient.
  • the antibodies of the invention can also be used for detection and diagnosis.
  • a sample can be obtained from a patient. Following optional pretreatment of the sample, the sample can be incubated with an antibody of the invention under conditions that allow the antibody to interact with spike proteins that may be present in the sample. Antibodies can be used to detect the presence of spike proteins in a sample using methods such as ELISA.
  • the presence (eg, amount or concentration) of a spike protein in a sample can be used to diagnose a relevant disease, as an indication that a patient is suitable for antibody therapy, or as an indication that a patient has (or has not) responded to treatment for a disorder.
  • a prognostic approach one, two or more tests can be performed at a particular stage when starting treatment for the disease to indicate the progress of the treatment.
  • compositions comprise effective doses of antibodies and pharmaceutically acceptable adjuvants.
  • the term "pharmaceutically acceptable” refers to a substance approved by a governmental regulatory agency or listed in other recognized pharmacopoeias for use in animals, particularly in humans.
  • pharmaceutically acceptable excipients generally refer to any type of non-toxic solid, semi-solid or liquid fillers, diluents, encapsulating materials or formulation aids, etc.
  • 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 a 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, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, Propylene, ethylene glycol, water, ethanol, etc.
  • the pharmaceutical composition if desired, can also contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates.
  • Antibacterial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, and tonicity adjusting agents such as sodium chloride or dextrose are also contemplated.
  • These pharmaceutical compositions may 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 can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • compositions will contain a clinically effective dose of the antibody or antigen-binding fragment, preferably in purified form, together with an appropriate amount of carrier to provide a form suitable for administration to the patient.
  • the formulation should be suitable for the mode of administration.
  • the preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • the composition is formulated into a pharmaceutical composition suitable for intravenous injection to human body according to conventional procedures.
  • Compositions for intravenous administration are generally 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 individually or in admixture in unit dosage form, such as in the form of a dry lyophilized powder or an anhydrous concentrate in a hermetically sealed container (such as an ampoule, vial, or sachet).
  • the composition can be dispensed with an infusion bottle or bag containing sterile pharmaceutical grade water, physiological saline or glucose injection.
  • an ampoule or vial of sterile water for injection or physiological saline or glucose injection can be used so that the active ingredients can be mixed before administration.
  • the compounds of the present invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those derived from, for example, salts formed with anions of hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc. - Salts of cations such as ethylaminoethanol, histidine, procaine and the like.
  • Antibodies can be prepared by the following method or other known methods: sequence optimization is performed according to the CHO codon preference characteristics of the host cell, and the DNA sequence is obtained from the amino acid sequence.
  • sequence optimization is performed according to the CHO codon preference characteristics of the host cell, and the DNA sequence is obtained from the amino acid sequence.
  • the optimized and synthesized sequences were cloned into vectors, and then a large number of plasmids were extracted to construct stable expression cell lines: the linearized expression vector was mixed with CHO cells and then added to a 0.4cm electroporation cup for electroporation; after electroporation was completed, press 1,200 cells per well are plated in 96-well cell culture plates, and after about 2-3 weeks, select the mother clones with high expression levels to carry out cell expansion culture and expression level detection from 96-well to 24-well to 6-well to shake flasks, and shake flasks are selected Clones with high expression levels were subcloned, subclones were expanded and cultured and expressed to identify the
  • variable region compositions of exemplary antibodies 1-23 and 2F8 are shown in Table 7, VH and CH (as shown in SEQ ID NO:61) form the heavy chain of the antibody, and VL and CL (as shown in SEQ ID NO:62) form The light chain of the antibody; wherein, the antibody 2F8 (monospecific antibody) contains two identical heavy chains (as shown in SEQ ID NO:72) and two identical light chains (as shown in SEQ ID NO:74)
  • the nucleotide sequence corresponding to its heavy chain is shown in SEQ ID NO: 75, and the nucleotide sequence corresponding to its light chain is shown in SEQ ID NO: 76; the amino acid sequence of the single domain antibody VHH18 is shown in SEQ ID NO: 69;
  • Chain antibody VHH18-Fc contains two heavy chains with the same sequence (as shown in SEQ ID NO: 79), the heavy chain is composed of single domain antibody, linker L2 and Fc, and the nucleic acid sequence corresponding to the heavy chain is shown in
  • the sequence of the purified antibody was confirmed by sequencing as described above.
  • Elisa detection was carried out for the above antibodies, and the detection method was as follows: a 96-well plate (Corning, 9018) was coated with spike-RBD-mFC (sino biologicals), sealed with tape and stored; the plate was washed in washing buffer PBST (containing 0.05% Tween 20 in PBS) for 3 washes, then add blocking solution (200 ⁇ L of 10 mg/ml BSA per well, the solvent is wash buffer); after incubation (1 hour (h), 37°C), wash the plate with wash buffer Wash 3 times, then add 100 ⁇ L of serially diluted samples to each well; after incubation (1.5h, 37°C), wash the plate with washing buffer, and then add anti-human ⁇ light chain antibody-peroxidase conjugate ( Diluted to 1:2000 in blocking solution, 100 ⁇ L/well); the plate was washed with washing buffer, and the test samples were incubated (1 h, 37 h) before adding 100 ⁇ L TMB (Tetramethylbenzidine, Biop
  • the EC 50 was calculated by the absorbance value, and the EC 50 values of various monospecific antibodies binding to the SARS-CoV-2 spike protein are shown in Table 8.
  • ACE2 angiotensin-converting enzyme 2
  • the detection method is as follows: a 96-well plate (Corning, 9018) is coated with spike-RBD-mFC (sino biologicals), sealed with tape and stored at 4°C overnight; the plate is washed in a washing buffer (PBS containing 0.05% Tween 20) Wash 3 times in washing buffer, then add blocking solution (200 ⁇ L of 10 mg/ml BSA per well, the solvent is washing buffer); after incubation (2h, 37°C), wash the plate 3 times in washing buffer, add different concentrations of Antibody samples were then added with biotinylated angiotensin-converting enzyme 2 (50ng/ml); after incubation (1h, 37°C), the plate was washed 3 times with wash buffer, and then 100 ⁇ L streptavidin was added per well Peroxidase conjugate (diluted 1:10,000 in blocking solution), after incubation (1h
  • SPR Surface Plasmon Resonance
  • antibodies 2F8 and 2F8-VH-VHH18 can block the binding of ACE2 to spike RBD, and antibody VHH18-Fc can partially block the binding of ACE2 to RBD.
  • BiaCore T200 (GE Healthcare) (Biomolecular Interaction Analysis) was detected at 25°C: Protein A chip was used for detection, and the antibody was diluted with 1 ⁇ HBS EP+ (0.1M HEPES, 1.5M NaCl, 0.03M EDTA, supplemented with 0.005 %surfactant P20), captured through the experimental flow path (Fc2, Fc4) at a flow rate of 10 ⁇ l/min; then the flow rate was adjusted to 30 ⁇ l/min, and the dilutions of different concentrations of Spike S1 RBD and mutants (0nM, 3.125nM, 6.25nM, 12.5nM, 25nM and 50nM, diluted with 1 ⁇ HBS EP+), flow through the surface of the experimental flow path (Fc2, Fc4) and reference flow path (Fc1, Fc3) at the same time, for binding and dissociation, Finally, pH 1.5 Glycine buffer was added to regenerate the chip and enter the next cycle.
  • 1 ⁇ HBS EP+ 0.1M HEPES
  • the kinetic constant spike trimer (Acrobiosystems, SPN-C52H8) was calculated using the 1:1 Langmuir binding model on BiaCore Data Analysis software (ka is the binding rate, kd is the dissociation rate, and kD is the binding-dissociation equilibrium constant).
  • antibodies 2F8, 2F8-VL-VHH18, 2F8-VH-VHH18 and VHH18-Fc bound well to Spike S1 RBD and its mutants.
  • antibodies VHH18-Fc and 2F8-VH-VHH18 can bind spike RBD protein and mutant protein; the results show that the EC 50 value of antibody 2F8-VH-VHH18 binding to spike RBD is about 10 ng/ml.
  • the main principle is: use ACE2 + 293F cells as susceptible cells, and incubate different concentrations of antibodies with the SARS-CoV-2-Fluc pseudovirus system; when the antibody is combined with the pseudovirus, it will block the virus from entering ACE2 + 293 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 Ability to inhibit virus infection in vitro.
  • the article number of the wild-type pseudovirus strain WT is GM-0220PV07 (Jiman Bio)
  • the article number of the mutant strain 1 (E484K) is GM-0220PV35 (Jiman Bio)
  • the article number of the mutant strain 2 (W436R) is GM-0220PV26 (Jiman Biological)
  • GM-0220PV33 of Mutant 3 (B.1.1.7/VUI-202012/01del 145Y) (Jiman Biological)
  • Mutant 4 (B.1.1.7/VUI-202012/01del 144Y/ 145Y) is GM-0220PV34 (Jiman Biology)
  • the article number of mutant strain 5 (B.1.351/501Y.V2, Beta) is GM-0220PV32-96T (Jiman Biology)
  • the article number of mutant 6 (D614G) GM-0220PV14 (Jiman Biology)
  • mutant strain 7 (D614G, D936
  • the detection method of pseudovirus inhibitory ability is as follows: the antibody is diluted to 4 ⁇ g/ml, then 4-fold gradient dilution, transferred to a 96-well detection plate according to the volume of 50 ⁇ l per well, and used; The DMEM medium of FBS was diluted, and the diluted pseudovirus solution was transferred to the above-mentioned 96-well plate containing the antibody according to 25 ⁇ l per well, and after mixing, it was allowed to stand at room temperature for 1 h; ACE2 + 293 cells were treated with 0.25% Trypsin-EDTA ( Gibco, 25200-072) was digested and counted, and the cell density was adjusted to 4 ⁇ 10 5 cells/ml, and the cells were added to the above-mentioned 96-well detection plate according to the volume of 50 ⁇ l per well, and cultured in a 37°C incubator for 48 hours; 50 ⁇ l was added to each well Bio-Lite luciferase assay system (Novizan, DD1201-0
  • HEK293 cells (ACS-4500TM, ATCC) were cultured in DMEM complete medium containing 10% FBS, and the ACE2 expression plasmid (Yiqiao Shenzhou , HG10108-M) transfection, followed by hygromycin (200 ⁇ g/ml) pressure selection and flow sorting (using 10 ⁇ g/ml anti-ACE2 and PE-coupled Anti-Human IgG-Fc), the cells Continue to amplify and select single clones with PE positive rate>90% for the next step of amplifying, and screen out HEK293 cells expressing ACE2, that is, ACE2 + 293 cells.
  • E484K+K417N+N501Y (mutant strain 12) 6 W436R (mutant strain 11) 60 E484K (mutant strain 1) 20 K417N (mutant strain 14) 25 D614G (mutant strain 6) 16 N501Y, D614G (mutant strain 15) 28 del144,145 (mutant strain 4) 7 N354D, D364Y (mutant strain 16) 45 D839Y (mutant strain 8) 6 D614G, D936Y (mutant strain 7) 6 V483A (mutant strain 9) 36 B.1.1.529 (mutant strain 13) 30
  • test antibody was serially diluted (initial concentration was 60nM, 3-fold serial dilution), and the antibody dilution was mixed with 200 PFU SARS-CoV-2 wild-type new coronavirus particles (virus strain number: 2019-nCoV/ IQTC01/human/2020/Guangzhou, GenBank is MT123290.1) or SARS-CoV-2delta (from the Guangdong Provincial Center for Disease Control and Prevention) were mixed in equal volumes, and a virus-free control group and a virus-free cell control group were set up at the same time; Set up 3 duplicate wells for each experimental group, and let stand at 37°C for 1 hour; discard the supernatant of Vero E6 cells (ATCC CRL-1587) in the 96-well plate, and take 50 ⁇ l of the virus-antibody mixture after incubation Transfer to a Vero E6 cell plate, place in a cell culture incubator at 37°C, and incubate for 1 hour; discard the supernatant in
  • mice transfected with hACE2 (adenovirus (Ad5-hACE2)) (Hunan Slack Jingda Experimental Animal Co., Ltd.) were used for virus infection test, divided into 5 groups, 12 in each group
  • G1 group Mice infected with 105 new coronaviruses (virus strain number: 2019-nCoV/IQTC01/human/2020/Guangzh ou, GenBank: MT123290.1) were administered PBS as a control
  • G2 group mice Mice were intraperitoneally injected with 1mg antibody 2F8-VH-VHH18, and 105 new coronaviruses were infected by intranasal drip after 24 hours (h);
  • G3 group mice were infected with 105 new coronaviruses by intranasal drip, and 1mg antibody 2F8 was injected intraperitoneally after 18 hours -VH-VHH18;
  • G4 group Mice were given 1 mg of antibody
  • mice in each group On the third day after infection with the new coronavirus, the lung tissues of 4 mice in each group were homogenized, and the live virus titer in the lungs of the mice was detected by the FRNT method. The body weight of mice in each group was detected every day after virus infection and antibody injection for a total of 14 days.
  • G2 group, G3 group and G4 group can all block the infection of lung tissue by the new coronavirus.
  • the neutralization level of 2F8-VH-VHH18 to SARS-CoV-2 wild strain, Alpha, Beta, Gamma, Delta and Omicron mutant strain was determined by microdilution neutralization method.

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Abstract

L'invention concerne un anticorps bispécifique, un anticorps à domaine unique et un anticorps à chaîne lourde qui se lient au SARS-CoV-2 ou à une protéine de spicule du SARS-CoV, et leur utilisation.
PCT/CN2022/096800 2021-06-04 2022-06-02 Anticorps ciblant le coronavirus et son utilisation WO2022253306A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105820245A (zh) * 2015-01-06 2016-08-03 珠海亿胜生物制药有限公司 抗vegf抗体
WO2020169755A2 (fr) * 2019-02-20 2020-08-27 Harbour Antibodies Bv Anticorps
CN111620946A (zh) * 2020-05-09 2020-09-04 江苏省疾病预防控制中心(江苏省公共卫生研究院) 分离的新型冠状病毒单克隆抗体或其抗原结合部分
CN112225806A (zh) * 2020-11-13 2021-01-15 李亚峰 一种人源抗新型冠状病毒（SARS-CoV-2）的中和活性单克隆抗体
CN112552399A (zh) * 2021-02-24 2021-03-26 恒翼生物医药科技(上海)有限公司 抗sars-cov-2中和抗体
CN113045647A (zh) * 2021-03-22 2021-06-29 南京传奇生物科技有限公司 新冠病毒SARS-CoV-2的中和性抗体及其应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105820245A (zh) * 2015-01-06 2016-08-03 珠海亿胜生物制药有限公司 抗vegf抗体
WO2020169755A2 (fr) * 2019-02-20 2020-08-27 Harbour Antibodies Bv Anticorps
CN111620946A (zh) * 2020-05-09 2020-09-04 江苏省疾病预防控制中心(江苏省公共卫生研究院) 分离的新型冠状病毒单克隆抗体或其抗原结合部分
CN112225806A (zh) * 2020-11-13 2021-01-15 李亚峰 一种人源抗新型冠状病毒（SARS-CoV-2）的中和活性单克隆抗体
CN112552399A (zh) * 2021-02-24 2021-03-26 恒翼生物医药科技(上海)有限公司 抗sars-cov-2中和抗体
CN113045647A (zh) * 2021-03-22 2021-06-29 南京传奇生物科技有限公司 新冠病毒SARS-CoV-2的中和性抗体及其应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DE GASPARO RAOUL; PEDOTTI MATTIA; SIMONELLI LUCA; NICKL PETR; MUECKSCH FRAUKE; CASSANITI IRENE; PERCIVALLE ELENA; LORENZI JULIO C.: "Bispecific IgG neutralizes SARS-CoV-2 variants and prevents escape in mice", NATURE, NATURE PUBLISHING GROUP UK, LONDON, vol. 593, no. 7859, 25 March 2021 (2021-03-25), London, pages 424 - 428, XP037456144, ISSN: 0028-0836, DOI: 10.1038/s41586-021-03461-y *
LIM SHION A., GRAMESPACHER JOSEF A., PANCE KATARINA, RETTKO NICHOLAS J., SOLOMON PAIGE, JIN JING, LUI IRENE, ELLEDGE SUSANNA K., L: "Bispecific VH/Fab antibodies targeting neutralizing and non-neutralizing Spike epitopes demonstrate enhanced potency against SARS-CoV-2", MABS, LANDES BIOSCIENCE, US, vol. 13, no. 1, 1 January 2021 (2021-01-01), US , XP093010387, ISSN: 1942-0862, DOI: 10.1080/19420862.2021.1893426 *
WU RUIJUN, LI ZHI-FEI, ZHANG XIN, PU RUN, AO YI, SUN YAN-RONG: "Development and Prospect of Antibody Drugs for SARS－CoV－2", CHINA BIOTECHNOLOGY, vol. 40, no. 5, 15 May 2020 (2020-05-15), pages 1 - 6, XP055878556, DOI: 10.13523/j.cb.2003076 *

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