WO2021249547A1 - Anti-coronavirus antibodies and uses thereof - Google Patents

Anti-coronavirus antibodies and uses thereof Download PDF

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Publication number
WO2021249547A1
WO2021249547A1 PCT/CN2021/099779 CN2021099779W WO2021249547A1 WO 2021249547 A1 WO2021249547 A1 WO 2021249547A1 CN 2021099779 W CN2021099779 W CN 2021099779W WO 2021249547 A1 WO2021249547 A1 WO 2021249547A1
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seq
amino acid
set forth
cdrs
nos
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PCT/CN2021/099779
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French (fr)
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Yi Yang
Lei Chen
Jingshu XIE
Yuelei SHEN
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Biocytogen Pharmaceuticals (Beijing) Co., Ltd.
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • 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]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/524—CH2 domain
    • 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
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • This disclosure relates to anti-coronavirus antibodies or antigen-binding fragments and uses thereof.
  • Coronaviruses are a group of related RNA viruses that cause diseases in mammals and birds. In humans, these viruses cause respiratory tract infections that can range from mild to lethal. Mild illnesses include some cases of the common cold (which is also caused by other viruses, predominantly rhinoviruses) , while more lethal varieties can cause SARS, MERS, and COVID-19.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • This disclosure relates to anti-coronavirus S protein antibodies, antigen-binding fragment thereof, and the uses thereof.
  • an antibody or antigen-binding fragment thereof that binds to a coronavirus spike protein, comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR2 amino acid sequence, and the VL
  • the selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are one of the following:
  • the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 55, 56, 57, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 58, 59, 60, respectively;
  • the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 61, 62, 63, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 64, 65, 66, respectively;
  • the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 67, 68, 69, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 70, 71, 72, respectively;
  • the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 73, 74, 75, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 76, 77, 78, respectively;
  • the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 79, 80, 81, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 82, 83, 84, respectively;
  • the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 85, 86, 87, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 88, 89, 90, respectively;
  • the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 91, 92, 93, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 94, 95, 96, respectively;
  • the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 97, 98, 99, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 100, 101, 102, respectively;
  • the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 103, 104, 105, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 106, 107, 108, respectively;
  • the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 109, 110, 111, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 112, 113, 114, respectively;
  • the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 115, 116, 117, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 118, 119, 120, respectively.
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3 respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively according to Kabat numbering scheme.
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively according to Kabat numbering scheme.
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively according to Kabat numbering scheme.
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively according to Kabat numbering scheme.
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 25, 26, and 27, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively according to Kabat numbering scheme.
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively according to Kabat numbering scheme.
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 37, 38, and 39, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 40, 41, and 42, respectively according to Kabat numbering scheme.
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 43, 44, and 45, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 46, 47, and 48, respectively according to Kabat numbering scheme.
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 52, 53, and 54, respectively according to Kabat numbering scheme.
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 55, 56, and 57, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 58, 59, and 60, respectively according to Kabat numbering scheme.
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 61, 62, and 63, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 64, 65, and 66, respectively according to Chothia numbering scheme.
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 67, 68, and 69, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 70, 71, and 72, respectively according to Chothia numbering scheme.
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 73, 74, and 75, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 76, 77, and 78, respectively according to Chothia numbering scheme.
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 79, 80, and 81, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 82, 83, and 84, respectively according to Chothia numbering scheme.
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 85, 86, and 87, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 88, 89, and 90, respectively according to Chothia numbering scheme.
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 91, 92, and 93, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 94, 95, and 96, respectively according to Chothia numbering scheme.
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 97, 98, and 99, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 100, 101, and 102, respectively according to Chothia numbering scheme.
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 103, 104, and 105, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 106, 107, and 108, respectively according to Chothia numbering scheme.
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 109, 110, and 111, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 112, 113, and 114, respectively according to Chothia numbering scheme.
  • the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 115, 116, and 117, respectively
  • the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 118, 119, and 120, respectively according to Chothia numbering scheme.
  • the antibody or antigen-binding fragment thereof specifically binds to a human coronavirus spike protein.
  • the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof (e.g., a human IgG1 antibody) .
  • the antibody or antigen-binding fragment thereof comprises a mouse constant domain. In some embodiments, the antibody or antigen-binding fragment thereof comprises a human constant domain. In some embodiments, the human constant domain comprises LALA mutations.
  • nucleic acid comprising a polynucleotide encoding a polypeptide comprising:
  • an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively; and in some embodiments, the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 122 binds to a coronavirus spike protein;
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • VL light chain variable region
  • VH heavy chain variable region
  • VL light chain variable region
  • an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively; and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 123 binds to the coronavirus spike protein;
  • an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively; and in some embodiments, the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 126 binds to the coronavirus spike protein;
  • VH heavy chain variable region
  • VL light chain variable region
  • an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively, and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 125 binds to the coronavirus spike protein;
  • an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively, and in some embodiments, the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 128 binds to the coronavirus spike protein;
  • VH heavy chain variable region
  • VL light chain variable region
  • an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively, and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 127 binds to the coronavirus spike protein;
  • an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 25, 26, and 27, respectively, and in some embodiments, the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 130 binds to the coronavirus spike protein;
  • VH heavy chain variable region
  • VL light chain variable region
  • an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively, and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 129 binds to the coronavirus spike protein;
  • VH heavy chain variable region
  • VL light chain variable region
  • an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively, and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 131 binds to the coronavirus spike protein;
  • an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 37, 38, and 39, respectively, and in some embodiments, the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 134 binds to the coronavirus spike protein;
  • VH heavy chain variable region
  • VL light chain variable region
  • an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 40, 41, and 42, respectively, and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 133 binds to the coronavirus spike protein;
  • an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 43, 44, and 45, respectively, and in some embodiments, the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 136 binds to the coronavirus spike protein;
  • VH heavy chain variable region
  • VL light chain variable region
  • an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 46, 47, and 48, respectively, and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 135 binds to the coronavirus spike protein;
  • an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively, and in some embodiments, the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 138 binds to the coronavirus spike protein;
  • VH heavy chain variable region
  • VL light chain variable region
  • an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 52, 53, and 54, respectively, and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 137 binds to the coronavirus spike protein;
  • an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 58, 59, and 60, respectively, and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 139 binds to the coronavirus spike protein.
  • the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively.
  • the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively.
  • the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively.
  • the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively.
  • the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively.
  • the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively.
  • the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively.
  • the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively.
  • the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 25, 26, and 27, respectively.
  • the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively.
  • the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively.
  • the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively.
  • the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 37, 38, and 39, respectively.
  • the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 40, 41, and 42, respectively.
  • the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 43, 44, and 45, respectively.
  • the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 46, 47, and 48, respectively.
  • the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively.
  • the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 52, 53, and 54, respectively.
  • the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 55, 56, and 57, respectively.
  • the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 58, 59, and 60, respectively.
  • the VH when paired with a VL specifically binds to the coronavirus spike protein, or the VL when paired with a VH specifically binds to the coronavirus spike protein.
  • the immunoglobulin heavy chain or the fragment thereof is a human immunoglobulin heavy chain or a fragment thereof
  • the immunoglobulin light chain or the fragment thereof is a human immunoglobulin light chain or a fragment thereof.
  • the nucleic acid is cDNA.
  • a vector comprising one or more of the nucleic acids as described herein.
  • a vector comprising two of the nucleic acids as described herein.
  • the vector encodes the VH region and the VL region that together bind to the coronavirus spike protein.
  • each vector comprises one of the nucleic acids as described herein.
  • together the pair of vectors encodes the VH region and the VL region that together bind to the coronavirus spike protein.
  • a cell comprising the vector or the pair of vectors as described herein.
  • the cell is a CHO cell.
  • a cell comprising one or more of the nucleic acids as described herein.
  • a cell comprising two of the nucleic acids as described herein.
  • the two nucleic acids together encode the VH region and the VL region that together bind to the coronavirus spike protein.
  • an antibody or antigen-binding fragment thereof that binds to a coronavirus spike protein comprising a heavy chain variable region (VH) comprising an amino acid sequence that is at least 80%identical to a selected VH sequence, and a light chain variable region (VL) comprising an amino acid sequence that is at least 80%identical to a selected VL sequence.
  • VH heavy chain variable region
  • VL light chain variable region
  • the selected VH sequence and the selected VL sequence are one of the following:
  • the selected VH sequence is SEQ ID NO: 121, and the selected VL sequence is SEQ ID NO: 122;
  • the selected VH sequence is SEQ ID NO: 123, and the selected VL sequence is SEQ ID NO: 124;
  • the selected VH sequence is SEQ ID NO: 125, and the selected VL sequence is SEQ ID NO: 126;
  • the selected VH sequence is SEQ ID NO: 127, and the selected VL sequence is SEQ ID NO: 128;
  • the selected VH sequence is SEQ ID NO: 129, and the selected VL sequence is SEQ ID NO: 130;
  • the selected VH sequence is SEQ ID NO: 131, and the selected VL sequence is SEQ ID NO: 132;
  • the selected VH sequence is SEQ ID NO: 133
  • the selected VL sequence is SEQ ID NO: 134;
  • the selected VH sequence is SEQ ID NO: 135, and the selected VL sequence is SEQ ID NO: 136;
  • the selected VH sequence is SEQ ID NO: 137, and the selected VL sequence is SEQ ID NO: 138;
  • the selected VH sequence is SEQ ID NO: 139
  • the selected VL sequence is SEQ ID NO: 140.
  • the VH comprises the sequence of SEQ ID NO: 121 and the VL comprises the sequence of SEQ ID NO: 122.
  • the VH comprises the sequence of SEQ ID NO: 123 and the VL comprises the sequence of SEQ ID NO: 124.
  • the VH comprises the sequence of SEQ ID NO: 125 and the VL comprises the sequence of SEQ ID NO: 126.
  • the VH comprises the sequence of SEQ ID NO: 127 and the VL comprises the sequence of SEQ ID NO: 128.
  • the VH comprises the sequence of SEQ ID NO: 129 and the VL comprises the sequence of SEQ ID NO: 130.
  • the VH comprises the sequence of SEQ ID NO: 131 and the VL comprises the sequence of SEQ ID NO: 132.
  • the VH comprises the sequence of SEQ ID NO: 133 and the VL comprises the sequence of SEQ ID NO: 134.
  • the VH comprises the sequence of SEQ ID NO: 135 and the VL comprises the sequence of SEQ ID NO: 136.
  • the VH comprises the sequence of SEQ ID NO: 137 and the VL comprises the sequence of SEQ ID NO: 138.
  • the VH comprises the sequence of SEQ ID NO: 139 and the VL comprises the sequence of SEQ ID NO: 140.
  • the antibody or antigen-binding fragment thereof specifically binds to a human coronavirus spike protein.
  • the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof (e.g., a human IgG1 antibody) .
  • the antibody or antigen-binding fragment thereof comprises a mouse constant domain. In some embodiments, the antibody or antigen-binding fragment thereof comprises a human constant domain. In some embodiments, the human constant domain comprises LALA mutations.
  • an antibody or antigen-binding fragment thereof comprising the VH CDRs 1, 2, 3, and the VL CDRs 1, 2, 3 of the antibody or antigen-binding fragment thereof as described herein.
  • the antibody or antigen-binding fragment thereof specifically binds to an S1 subunit of the coronavirus spike protein.
  • the antibody or antigen-binding fragment thereof specifically binds to a receptor binding domain (RBD) of the S1 subunit of the coronavirus spike protein.
  • RBD receptor binding domain
  • the amino acid sequence of the RBD is at least 80%identical to amino acids 319-541 of SEQ ID NO: 141.
  • an antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof as described herein.
  • a method of producing an antibody or an antigen-binding fragment thereof comprising (a) culturing the cell as described herein under conditions sufficient for the cell to produce the antibody or the antigen-binding fragment thereof; and (b) collecting the antibody or the antigen-binding fragment thereof produced by the cell.
  • provided herein is a method of treating a subject having a coronavirus-related disease, the method comprising administering a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof as described herein to the subject.
  • a method of neutralizing a coronavirus comprising contacting the coronavirus with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described herein.
  • a method of blocking internalization of a coronavirus by a cell comprising contacting the coronavirus with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof as described herein.
  • a method of identifying a subject as having a coronavirus disease comprisingdetecting a sample collected from the subject as having the coronavirus by the antibody or antigen-binding fragment thereof as described herein, thereby identifying the subject as having a coronavirus infection.
  • the sample is a blood sample, a saliva sample, a stool sample, or a liquid sample from the respiratory tract of the subject.
  • the coronavirus is SARS-CoV-2. In some embodiments, the coronavirus is MERS-CoV. In some embodiments, the coronavirus is SARS-CoV.
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described herein, and a pharmaceutically acceptable carrier.
  • a method of treating a subject having a coronavirus-related disease or neutralizing coronavirus in the subject comprising administering to the subject a therapeutically effective amount of a first antibody or antigen-binding fragment thereof, and a therapeutically effective amount of a second antibody or antigen-binding fragment thereof.
  • the first antibody or antigen-binding fragment thereof is the antibody or antigen-binding fragment thereof as described herein.
  • the second antibody or antigen-binding fragment thereof is an anti-S protein antibody or antigen-binding fragment thereof.
  • the method further comprises administering a third antibody or antigen-binding fragment thereof to the subject.
  • the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof target different epitopes of an S protein.
  • the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof are selected from FIG. 16.
  • the first antibody or antigen-binding fragment thereof comprises VH CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 37, 38, 39, respectively, and VL CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 40, 41, 42, respectively; and the second antibody or antigen-binding fragment thereof comprises VH CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 31, 32, 33, respectively, and VL CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 34, 35, 36, respectively.
  • the first antibody or antigen-binding fragment thereof comprises VH CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 13, 14, 15, respectively, and VL CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 16, 17, 18, respectively; and the second antibody or antigen-binding fragment thereof comprises VH CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 49, 50, 51, respectively, and VL CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 52, 53, 54, respectively.
  • a pharmaceutical composition comprising two or more antibodies or antigen-binding fragment thereof.
  • one of the two or more antibodies or antigen-binding fragment thereof is the antibody or antigen-binding fragment thereof as described herein.
  • the pharmaceutical composition further comprises a third antibody or antigen-binding fragment thereof.
  • each antibody or antigen-binding fragment thereof targets different epitopes of an S protein.
  • two antibodies or antigen-binding fragments thereof are selected from FIG. 16.
  • a first antibody or antigen-binding fragment thereof comprises VH CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 37, 38, 39, respectively, and VL CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 40, 41, 42, respectively; and a second antibody or antigen-binding fragment thereof comprises VH CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 31, 32, 33, respectively, and VL CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 34, 35, 36, respectively.
  • a first antibody or antigen-binding fragment thereof comprises VH CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 13, 14, 15, respectively, and VL CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 16, 17, 18, respectively; and a second antibody or antigen-binding fragment thereof comprises VH CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 49, 50, 51, respectively, and VL CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 52, 53, 54, respectively.
  • an anti-coronavirus spike protein antibody or antigen binding fragment thereof comprising: a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3; and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3.
  • VH heavy chain variable region
  • VL light chain variable region
  • the VH CDR1 sequence is SX 1 X 2 X 3 X 4 X 5 X 6 (SEQ ID NO: 142) .
  • X 1 is N, F, G, Y, or null
  • X 2 is G, or null
  • X 3 is Y, or null
  • X 4 is Y, A, or null
  • X 5 is M, L, or W
  • X 6 is S, N, T, A, or H.
  • the VH CDR2 sequence is X 7 IX 8 X 9 X 10 GX 11 X 12 X 13 X 14 YX 15 X 16 SX 17 X 18 X 19 (SEQ ID NO: 143) .
  • the VH CDR3 sequence is X 20 X 21 X 22 X 23 X 24 X 25 X 26 X 27 X 28 X 29 X 30 X 31 DX 32 (SEQ ID NO: 144) .
  • X 20 is D, E, or Q;
  • X 21 is R, L, V, T, or A;
  • X 22 is G, or null;
  • X 23 is Y, or null;
  • X 24 is S, V, L, or null;
  • X 25 is S, G, D, or null;
  • X 26 is D, S, P, K, N, G, or null;
  • X 27 is Y, N, V, L, W, or null;
  • X 28 is N, S, L, T, or null;
  • X 29 is Y, S, F, G, or D;
  • X 30 is G, N, F, or S;
  • X 31 is M, or F;
  • X 32
  • X 1 is N
  • X 2 is null
  • X 3 is null
  • X 4 is Y
  • X 5 is M
  • X 6 is S.
  • X 7 is V
  • X 8 is Y
  • X 9 is Y
  • X 10 is S
  • X 11 is G
  • X 12 is S
  • X 13 is T
  • X 14 is Y
  • X 15 is A
  • X 16 is D
  • X 17 is V
  • X 18 is K
  • X 19 is G.
  • X 20 is D
  • X 21 is R
  • X 22 is null
  • X 23 is null
  • X 24 is null
  • X 25 is null
  • X 27 is Y
  • X 28 is null
  • X 29 is Y
  • X 30 is G
  • X 31 is M
  • X 32 is V.
  • the VL CDR1 sequence is X 33 AX 34 QX 35 IX 36 X 37 X 38 LX 39 (SEQ ID NO: 145) .
  • X 33 is Q, or R;
  • X 34 is S, or R;
  • X 35 is D, or G;
  • X 36 is S, N, or T;
  • X 37 is N, I, S, or K;
  • X 38 is Y, or F;
  • X 39 is N, or A.
  • the VL CDR2 sequence is X 40 ASX 41 LX 42 X 43 (SEQ ID NO: 146) .
  • X 40 is D, or A; X 41 is N, T, or S; X 42 is E, Q, or L; X 43 is T, or S.
  • the VL CDR3 sequence is X 44 X 45 X 46 X 47 X 48 X 49 X 50 X 51 X 52 T (SEQ ID NO: 147) .
  • X 33 is Q
  • X 34 is S
  • X 35 is D
  • X 36 is S
  • X 37 is N
  • X 38 is Y
  • X 39 is N
  • X 40 is D
  • X 41 is N
  • X 42 is E
  • X 43 is T
  • X 44 is Q
  • X 45 is Q
  • X 46 is Y
  • X 47 is D
  • X 48 is N
  • X 49 is L
  • X 50 is P
  • X 51 is null
  • X 52 is null.
  • antibody refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementary determining region (CDR) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope.
  • CDR complementary determining region
  • Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bi-specific antibodies) , single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies.
  • an antibody can contain an Fc region of a human antibody.
  • the term antibody also includes derivatives, e.g., bi-specific antibodies, single-chain antibodies, diabodies, linear antibodies, and multi-specific antibodies formed from antibody fragments.
  • the term “antigen-binding fragment” refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen.
  • the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of a heavy chain or a variable domain of light chain) .
  • variable domains include, e.g., Fab, Fab’, F (ab’) 2 , and Fv fragments.
  • human antibody refers to an antibody that is encoded by a nucleic acid (e.g., rearranged human immunoglobulin heavy or light chain locus sequence) derived from a human.
  • a human antibody is collected from a human or produced in a human cell culture (e.g., human hybridoma cells) .
  • a human antibody is produced in a non-human cell (e.g., a mouse or hamster cell line) .
  • a human antibody is produced in a bacterial or yeast cell.
  • a human antibody is derived from a transgenic non-human animal (e.g., a mouse) containing an unrearranged or rearranged human immunoglobulin locus (e.g., heavy or light chain human immunoglobulin locus) .
  • a transgenic non-human animal e.g., a mouse
  • an unrearranged or rearranged human immunoglobulin locus e.g., heavy or light chain human immunoglobulin locus
  • chimeric antibody refers to an antibody that contains a sequence present in at least two different species (e.g., antibodies from two different mammalian species such as a human and a mouse antibody) .
  • a non-limiting example of a chimeric antibody is an antibody containing the variable domain sequences (e.g., all or part of a light chain and/or heavy chain variable domain sequence) of a human antibody and the constant domains of a non-human antibody. Additional examples of chimeric antibodies are described herein and are known in the art.
  • humanized antibody refers to a non-human antibody which contains minimal sequence derived from a non-human (e.g., mouse) immunoglobulin and contains sequences derived from a human immunoglobulin.
  • humanized antibodies are human antibodies (recipient antibody) in which hypervariable (e.g., CDR) region residues of the recipient antibody are replaced by hypervariable (e.g., CDR) region residues from a non-human antibody (e.g., a donor antibody) , e.g., a mouse, rat, or rabbit antibody, having the desired specificity, affinity, and capacity.
  • the Fv framework residues of the human immunoglobulin are replaced by corresponding non-human (e.g., mouse) immunoglobulin residues.
  • humanized antibodies may contain residues which are not found in the recipient antibody or in the donor antibody. These modifications can be made to further refine antibody performance.
  • the humanized antibody contains substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops (CDRs) correspond to those of a non-human (e.g., mouse) immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin.
  • CDRs hypervariable loops
  • the humanized antibody can also contain at least a portion of an immunoglobulin constant region (Fc) , typically, that of a human immunoglobulin.
  • the chimeric antibody has a human heavy chain variable domain and a human light chain variable domain, and mouse constant domains.
  • the humanization can involve replace mouse constant domains with human constant domains, thereby making a full human antibody.
  • Humanized antibodies can be produced using molecular biology methods known in the art. Non-limiting examples of methods for generating humanized antibodies are described herein.
  • single-chain antibody refers to a single polypeptide that contains at least two immunoglobulin variable domains (e.g., a variable domain of a mammalian immunoglobulin heavy chain or light chain) that is capable of specifically binding to an antigen.
  • immunoglobulin variable domains e.g., a variable domain of a mammalian immunoglobulin heavy chain or light chain
  • single-chain antibodies are described herein.
  • multimeric antibody refers to an antibody that contains four or more (e.g., six, eight, or ten) immunoglobulin variable domains.
  • the multimeric antibody is able to crosslink one target molecule (e.g., S protein) to at least one second target molecule (e.g., CD3) on the surface of a mammalian cell (e.g., a human T-cell) .
  • the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present invention is provided.
  • Veterinary and non-veterinary applications are contemplated in the present disclosure.
  • Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old) .
  • patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates.
  • non-human primates e.g., monkey, chimpanzee, gorilla, and the like
  • rodents e.g., rats, mice, gerbils, hamsters, ferrets, rabbits
  • lagomorphs e.g., swine (e.g., pig, miniature pig)
  • equine canine, feline, bovine, and other domestic, farm, and zoo animals.
  • the phrases “specifically binding” and “specifically binds” mean that the antibody interacts with its target molecule preferably to other molecules, because the interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) on the target molecule; in other words, the reagent is recognizing and binding to molecules that include a specific structure rather than to all molecules in general.
  • An antibody that specifically binds to the target molecule may be referred to as a target-specific antibody.
  • an antibody that specifically binds to a S protein molecule may be referred to as a S protein-specific antibody or an anti-S protein antibody.
  • polypeptide, ” “peptide, ” and “protein” are used interchangeably to refer to polymers of amino acids of any length of at least two amino acids.
  • nucleic acid molecule As used herein, the terms “polynucleotide, ” “nucleic acid molecule, ” and “nucleic acid sequence” are used interchangeably herein to refer to polymers of nucleotides of any length of at least two nucleotides, and include, without limitation, DNA, RNA, DNA/RNA hybrids, and modifications thereof.
  • FIG. 1 is a set of flow cytometry results showing the blocking effects of anti-SARS-CoV-2 antibodies in hybridoma supernatant.
  • PC1 is positive control.
  • NC1 and NC2 are negative control 1 and 2, respectively.
  • FIG. 2 shows antigen-binding curves of 09-4E5-IgG1-LALA at 0.78125nM, 1.5625nM, 3.125 nM, 6.25 nM, 12.5 nM, 25 nM and 50 nM, 100nM.
  • FIG. 3 shows fitting curves of 03-9A8-IgG1-LALA and 09-7B8-IgG1-LALA.
  • FIG. 4 is a matrix table showing binding ratios between analyte 1 and analyte 2.
  • the antibody numbers 1-10 are purified human anti-SARS-CoV-2 antibodies (1) 01-2H10-IgG1-LALA, (2) 03-10D12-IgG1-LALA, (3) 03-10F9-IgG1-LALA, (4) 03-1F9-IgG1-LALA, (5) 05-8G6-IgG1-LALA, (6) 05-9G11-IgG1-LALA, (7) 09-2F7-IgG1-LALA, (8) 09-4E5-IgG1-LALA, (9) 09-7B8-IgG1-LALA, and (10) 03-9A8-IgG1-LALA, respectively.
  • FIG. 5 shows epitope correlation of the human anti-SARS-CoV-2 antibodies 01-2H10-IgG1-LALA (01-2H10) , 03-10F9-IgG1-LALA (03-10F9) , 03-10D12-IgG1-LALA (03-10D12) , 03-1F9-IgG1-LALA (03-1F9) , 03-9A8-IgG1-LALA (03-9A8) , 09-2F7-IgG1-LALA (09-2F7) , 09-4E5-IgG1-LALA (09-4E5) , 09-7B8-IgG1-LALA (09-7B8) , 05-8G6-IgG1-LALA (05-8G6) , and 05-9G11-IgG1-LALA (05-9G11) .
  • FIG. 6 shows epitope clusters of the human anti-SARS-CoV-2 antibodies 01-2H10-IgG1-LALA (2H10) , 03-10F9-IgG1-LALA (10F9) , 03-10D12-IgG1-LALA (10D12) , 03-1F9-IgG1-LALA (1F9) , 03-9A8-IgG1-LALA (9A8) , 09-2F7-IgG1-LALA (2F7) , 09-4E5-IgG1-LALA (4E5) , 09-7B8-IgG1-LALA (7B8) , 05-8G6-IgG1-LALA (8G6) , and 05-9G11-IgG1-LALA (9G11) .
  • FIGS. 7A-7B show a set of binding curves of captured 2019-nCoV S protein RBD by ACE2 alone, or a human anti-SARS-CoV-2 antibody followed by ACE2.
  • FIGS. 8A-8E show a set of binding curves of 03-9A8-IgG1-LALA (Ab. 01) , 03-10D12-IgG1-LALA (Ab. 02) , 05-9G11-IgG1-LALA (Ab. 03) , 09-4E5-IgG1-LALA (Ab. 04) , and 09-7B8-IgG-LALA (Ab. 05) alone, or in combination.
  • FIG. 9 shows IC 50 of 03-9A8-IgG1-LALA (Ab. 01, or 9A8) , 03-10D12-IgG1-LALA (Ab. 02, or 03-10D12) , 05-9G11-IgG1-LALA (Ab. 03, or 05-9G11) , 09-4E5-IgG1-LALA (Ab. 04, or 09-4E5) , and 09-7B8-IgG-LALA (Ab. 05, or 09-7B8) alone, or in combination.
  • FIG. 10 shows COVID-10 pseudovirus neutralizing activity curves of 03-10D12-IgG1-LALA (Ab1) , 03-9A8-IgG1-LALA (Ab2) , 09-4E5-IgG1-LALA (Ab3) , 09-7B8-IgG-LALA (Ab4) , 05-9G11-IgG1-LALA (Ab5) , and Ab1 + Ab5 combination.
  • FIG. 11A shows CDR1 consensus sequence of the heavy chain variable region (VH) of anti-SARS-CoV-2 antibodies.
  • FIG. 11B shows CDR2 consensus sequence of the VH of anti-SARS-CoV-2 antibodies.
  • FIG. 11C shows CDR3 consensus sequence of the VH of anti-SARS-CoV-2 antibodies.
  • FIG. 12A shows CDR1 consensus sequence of the light chain variable region (VL) of anti-SARS-CoV-2 antibodies.
  • FIG. 12B shows CDR2 consensus sequence of the VL of anti-SARS-CoV-2 antibodies.
  • FIG. 12C shows CDR3 consensus sequence of the VL of anti-SARS-CoV-2 antibodies.
  • FIG. 13 lists CDR sequences of several anti-SARS-CoV-2 antibodies as defined by Kabat numbering.
  • FIG. 14 lists CDR sequences of several anti-SARS-CoV-2 antibodies as defined by Chothia numbering.
  • FIG. 15 lists sequences that are described in the present disclosure.
  • FIG. 16 lists different pairs of antibodies or antigen binding fragments thereof.
  • Coronavirus uses its spike glycoprotein (S) , a main target for neutralization antibody, to bind its receptor, and mediate membrane fusion and virus entry.
  • S spike glycoprotein
  • Each monomer of trimeric S protein is about 180 kDa, and contains two subunits, S1 and S2, mediating attachment and membrane fusion, respectively.
  • the SARS-CoV-2 virion consists of a helical capsid formed by nucleocapsid (N) proteins bound to the RNA genome, which is enclosed by membrane (M) proteins, envelope (E) proteins and trimeric spike (S) proteins that render them their “corona-like” appearance (See Zhou P, Yang XL, Wang XG, et al., A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature.
  • the S protein receptor binding domain (RBD) in the S1 subunit binds to the angiotensin converting enzyme (Angiotensin I Converting Enzyme 2; ACE2) on the cell membranes of type 2 pneumocytes and intestinal epithelial cells.
  • ACE2 angiotensin converting enzyme
  • TMPRSS2 host cell transmembrane serine protease 2
  • the present disclosure provides antibodies, antigen-binding fragments thereof that specifically bind to a coronavirus (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV) S protein.
  • coronavirus e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV
  • These antibodies or antigen-binding fragments thereof are high titer neutralizing antibodies or antigen-binding fragments thereof.
  • Therapeutic antibodies can neutralize viral infections via two mechanisms of action, e.g., Fc-independent functions that block capsid/host receptor interaction, and induce virus aggregation, and/or Fc-dependent functions that cause Fc-FcR interaction to activate immune cells leading to killing of virus.
  • the disclosure also provides methods of treating COVID-19 using the antibodies or antigen-binding fragments thereof as described herein, and methods of diagnosing COVID-19 using the antibodies or antigen-binding fragments thereof as described herein.
  • the disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to coronavirus spike proteins.
  • the antibodies and antigen-binding fragments described herein are capable of binding to coronavirus spike proteins.
  • these antibodies can block human ACE2 binding to the coronavirus spike proteins.
  • these antibodies can neutralize the coronavirus.
  • the disclosure provides e.g., anti-coronavirus spike protein antibodies 01-2H10, 03-1F9, 03-9A8, 03-10F9, 05-8G6, 05-9G11, 03-10D12, 09-2F7, 09-7B8, and 09-4E5, and any antibodies derived therefrom (e.g., the chimeric antibodies, the humanized antibodies, andthe full human antibodies) .
  • the CDR sequences for 01-2H10, and 01-2H10 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 1, 2, 3, and CDRs of the light chain variable domain, SEQ ID NOs: 4, 5, 6 as defined by Kabat numbering.
  • the CDRs can also be defined by Chothia system. Under the Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 61, 62, 63, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 64, 65, 66.
  • the CDR sequences for 03-1F9, and 03-1F9 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 7, 8, 9, and CDRs of the light chain variable domain, SEQ ID NOs: 10, 11, 12, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 67, 68, 69, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 70, 71, 72.
  • the CDR sequences for 03-9A8, and 03-9A8 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 13, 14, 15, and CDRs of the light chain variable domain, SEQ ID NOs: 16, 17, 18, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 73, 74, 75, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 76, 77, 78.
  • the CDR sequences for 03-10F9, and 03-10F9 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 19, 20, 21, and CDRs of the light chain variable domain, SEQ ID NOs: 22, 23, 24, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 79, 80, 81, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 82, 83, 84.
  • the CDR sequences for 05-8G6, and 05-8G6 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 25, 26, 27, and CDRs of the light chain variable domain, SEQ ID NOs: 28, 29, 30, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 85, 86, 87, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 88, 89, 90.
  • the CDR sequences for 05-9G11, and 05-9G11 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 31, 32, 33, and CDRs of the light chain variable domain, SEQ ID NOs: 34, 35, 36, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 91, 92, 93, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 94, 95, 96.
  • the CDR sequences for 03-10D12, and 03-10D12 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 37, 38, 39, and CDRs of the light chain variable domain, SEQ ID NOs: 40, 41, 42, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 97, 98, 99, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 100, 101, 102.
  • the CDR sequences for 09-2F7, and 09-2F7 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 43, 44, 45, and CDRs of the light chain variable domain, SEQ ID NOs: 46, 47, 48, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 103, 104, 105, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 106, 107, 108.
  • the CDR sequences for 09-7B8, and 09-7B8 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 49, 50, 51, and CDRs of the light chain variable domain, SEQ ID NOs: 52, 53, 54, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 109, 110, 111, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 112, 113, 114.
  • the CDR sequences for 09-4E5, and 09-4E5 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 55, 56, 57, and CDRs of the light chain variable domain, SEQ ID NOs: 58, 59, 60, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 115, 116, 117, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 118, 119, 120.
  • the amino acid sequence for the heavy chain variable region of 01-2H10 antibody is set forth in SEQ ID NO: 121.
  • the amino acid sequence for the light chain variable region of 01-2H10 antibody is set forth in SEQ ID NO: 122.
  • the amino acid sequence for the heavy chain variable region of 03-1F9 antibody is set forth in SEQ ID NO: 123.
  • the amino acid sequence for the light chain variable region of 03-1F9 antibody is set forth in SEQ ID NO: 124.
  • the amino acid sequence for the heavy chain variable region of 03-9A8 antibody is set forth in SEQ ID NO: 125.
  • the amino acid sequence for the light chain variable region of 03-9A8 antibody is set forth in SEQ ID NO: 126.
  • the amino acid sequence for the heavy chain variable region of 03-10F9 antibody is set forth in SEQ ID NO: 127.
  • the amino acid sequence for the light chain variable region of 03-10F9 antibody is set forth in SEQ ID NO: 128.
  • the amino acid sequence for the heavy chain variable region of 05-8G6 antibody is set forth in SEQ ID NO: 129.
  • the amino acid sequence for the light chain variable region of 05-8G6 antibody is set forth in SEQ ID NO: 130.
  • the amino acid sequence for the heavy chain variable region of 05-9G11 antibody is set forth in SEQ ID NO: 131.
  • the amino acid sequence for the light chain variable region of 05-9G11 antibody is set forth in SEQ ID NO: 132.
  • the amino acid sequence for the heavy chain variable region of 03-10D12 antibody is set forth in SEQ ID NO: 133.
  • the amino acid sequence for the light chain variable region of 03-10D12 antibody is set forth in SEQ ID NO: 134.
  • the amino acid sequence for the heavy chain variable region of 09-2F7 antibody is set forth in SEQ ID NO: 135.
  • the amino acid sequence for the light chain variable region of 09-2F7 antibody is set forth in SEQ ID NO: 136.
  • the amino acid sequence for the heavy chain variable region of 09-7B8 antibody is set forth in SEQ ID NO: 137.
  • the amino acid sequence for the light chain variable region of 09-7B8 antibody is set forth in SEQ ID NO: 138.
  • the amino acid sequence for the heavy chain variable region of 09-4E5 antibody is set forth in SEQ ID NO: 139.
  • the amino acid sequence for the light chain variable region of 09-4E5 antibody is set forth in SEQ ID NO: 140.
  • the heavy chain and the light chain of an antibody can have one or more mutations.
  • the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 121, 123, 125, 127, 129, 131, 133, 135, 137, or 139.
  • the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 122, 124, 126, 128, 130, 132, 134, 136, 138, or 140.
  • the heavy chain variable region sequence can be paired with the corresponding light chain variable region sequence, and together they bind to the coronavirus spike protein described herein.
  • the heavy chain variable region and the light chain variable region are completely obtained from a human sequence (e.g., human immunoglobulin heavy chain locus sequence and/or human immunoglobulin kappa chain locus sequence) .
  • the antibodies or antigen-binding fragments thereof described herein can also contain one, two, or three heavy chain variable region CDRs selected from the group of SEQ ID NOs: 1-3, SEQ ID NOs: 7-9, SEQ ID NOs: 13-15, SEQ ID NOs: 19- 21, SEQ ID NOs: 25-27, SEQ ID NOs: 31-33, SEQ ID NOs: 37-39, SEQ ID NOs: 43-45, SEQ ID NOs: 49-51, and SEQ ID NOs: 55-57; and/or one, two, or three light chain variable region CDRs selected from the group of SEQ ID NOs: 4-6, SEQ ID NOs: 10-12, SEQ ID NOs: 16-18, SEQ ID NOs: 22-24, SEQ ID NOs: 28-30, SEQ ID NOs: 34-36, SEQ ID NOs: 40-42, SEQ ID NOs: 46-48, SEQ ID NOs: 52-54, and SEQ ID NOs: 58-60
  • the antibodies can have a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR3 amino acid sequence.
  • VH heavy chain variable region
  • CDRs complementarity determining regions
  • the antibodies can have a light chain variable region (VL) comprising CDRs 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL CDR3 amino acid sequence.
  • the selected VH CDRs 1, 2, 3 amino acid sequences and the selected VL CDRs, 1, 2, 3 amino acid sequences are shown in FIG. 13 (Kabat CDR) and FIG. 14 (Chothia CDR) .
  • the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 1 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 2 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 3 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 7 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 8 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 9 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 13 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 14 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 15 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 19 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 20 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 21 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 25 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 26 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 27 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 31 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 32 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 33 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 37 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 38 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 39 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 43 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 44 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 45 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 49 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 50 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 51 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 55 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 56 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 57 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 4 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 5 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 6 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 10 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 11 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 12 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 16 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 17 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 18 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 22 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 23 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 24 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 28 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 29 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 30 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 34 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 35 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 36 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 40 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 41 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 42 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 46 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 47 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 48 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 52 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 53 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 54 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 58 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 59 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 60 with zero, one or two amino acid insertions, deletions, or substitutions.
  • the insertions, deletions, and substitutions can be within the CDR sequence, or at one or both terminal ends of the CDR sequence.
  • the CDR is determined based on Kabat numbering scheme. In some embodiments, the CDR is determined based on Chothia numbering scheme.
  • the disclosure also provides antibodies or antigen-binding fragments thereof that bind to coronavirus spike proteins.
  • the antibodies or antigen-binding fragments thereof contain a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH sequence, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL sequence.
  • VH heavy chain variable region
  • VL light chain variable region
  • the selected VH sequence is SEQ ID NO: 121
  • the selected VL sequence is SEQ ID NO: 122.
  • the selected VH sequence is SEQ ID NO: 123 and the selected VL sequence is SEQ ID NO: 124. In some embodiments, the selected VH sequence is SEQ ID NO: 125 and the selected VL sequence is SEQ ID NO: 126. In some embodiments, the selected VH sequence is SEQ ID NO: 127 and the selected VL sequence is SEQ ID NO: 128. In some embodiments, the selected VH sequence is SEQ ID NO: 129 and the selected VL sequence is SEQ ID NO: 130. In some embodiments, the selected VH sequence is SEQ ID NO: 131 and the selected VL sequence is SEQ ID NO: 132.
  • the selected VH sequence is SEQ ID NO: 133 and the selected VL sequence is SEQ ID NO: 134. In some embodiments, the selected VH sequence is SEQ ID NO: 135 and the selected VL sequence is SEQ ID NO: 136. In some embodiments, the selected VH sequence is SEQ ID NO: 137 and the selected VL sequence is SEQ ID NO: 138. In some embodiments, the selected VH sequence is SEQ ID NO: 139 and the selected VL sequence is SEQ ID NO: 140.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) .
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the disclosure also provides nucleic acid comprising a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin light chain.
  • the immunoglobulin heavy chain or immunoglobulin light chain comprises CDRs as shown in FIG. 13 or FIG. 14, or have sequences as shown in FIG. 15.
  • the polypeptides are paired with corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region)
  • the paired polypeptides bind to the coronavirus spike protein described herein.
  • the anti-coronavirus spike protein antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments and multi-specific (e.g., bi-specific) antibodies or antibody fragments.
  • Additional antibodies provided herein are polyclonal, monoclonal, multi-specific (multimeric, e.g., bi-specific) , human antibodies, chimeric antibodies (e.g., human-mouse chimera) , single-chain antibodies, intracellularly-made antibodies (i.e., intrabodies) , and antigen-binding fragments thereof.
  • the antibodies or antigen-binding fragments thereof can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) , class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) , or subclass.
  • the antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof.
  • Fragments of antibodies are suitable for use in the methods provided so long as they retain the desired affinity and specificity of the full-length antibody.
  • a fragment of an antibody that binds to the coronavirus spike protein described herein will retain an ability to bind to the coronavirus spike protein.
  • An Fv fragment is an antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight association, which can be covalent in nature, for example in scFv. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs or a subset thereof confer antigen binding specificity to the antibody.
  • Single-chain Fv or (scFv) antibody fragments comprise the VH and VL domains (or regions) of antibody, wherein these domains are present in a single polypeptide chain.
  • the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.
  • the present disclosure also provides an antibody or antigen-binding fragment thereof that cross-competes with any antibody or antigen-binding fragment as described herein.
  • the cross-competing assay is known in the art, and is described in the present disclosure (e.g., by testing the binding of two antibodies to Recombinant 2019-nCoV S protein) .
  • the present disclosure also provides an antibody or antigen-binding fragment thereof that binds to the same epitope or region as any antibody or antigen-binding fragment as described herein.
  • the epitope binning assay is known in the art, and is described e.g., in Estep et al., "High throughput solution-based measurement of antibody-antigen affinity and epitope binning. " MAbs. Vol. 5. No. 2. Taylor &Francis, 2013, which is incorporated herein reference in its entirety.
  • antibodies also called immunoglobulins
  • antibodies are made up of two classes of polypeptide chains, light chains and heavy chains.
  • a non-limiting examples of antibody of the present disclosure can be an intact, four immunoglobulin chain antibody comprising two heavy chains and two light chains.
  • the heavy chain of the antibody can be of any isotype including IgM, IgG, IgE, IgA, or IgD or sub-isotype including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc.
  • the light chain can be a kappa light chain or a lambda light chain.
  • An antibody can comprise two identical copies of a light chain and two identical copies of a heavy chain.
  • the heavy chains which each contain one variable domain (or variable region, V H ) and multiple constant domains (or constant regions) , bind to one another via disulfide bonding within their constant domains to form the “stem” of the antibody.
  • the light chains which each contain one variable domain (or variable region, V L ) and one constant domain (or constant region) , each bind to one heavy chain via disulfide binding.
  • the variable region of each light chain is aligned with the variable region of the heavy chain to which it is bound.
  • the variable regions of both the light chains and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FR) .
  • CDRs complementary determining regions
  • the four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting the beta-sheet structure, and in some cases forming part of, the beta-sheet structure.
  • the CDRs in each chain are held in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding region.
  • the CDRs are important for recognizing an epitope of an antigen.
  • an “epitope” is the smallest portion of a target molecule capable of being specifically bound by the antigen binding domain of an antibody.
  • the minimal size of an epitope may be about three, four, five, six, or seven amino acids, but these amino acids need not be in a consecutive linear sequence of the antigen’s primary structure, as the epitope may depend on an antigen’s three-dimensional configuration based on the antigen’s secondary and tertiary structure.
  • the antibody is an intact immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA) .
  • the IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, differ in their constant region, particularly in their hinges and upper CH2 domains. The sequences and differences of the IgG subclasses are known in the art, and are described, e.g., in Vidarsson, et al, "IgG subclasses and allotypes: from structure to effector functions.
  • the antibody can also be an immunoglobulin molecule that is derived from any species (e.g., human, rodent, mouse, camelid) .
  • Antibodies disclosed herein also include, but are not limited to, polyclonal, monoclonal, monospecific, polyspecific antibodies, and chimeric antibodies that include an immunoglobulin binding domain fused to another polypeptide.
  • the term “antigen binding domain” or “antigen binding fragment” is a portion of an antibody that retains specific binding activity of the intact antibody, i.e., any portion of an antibody that is capable of specific binding to an epitope on the intact antibody’s target molecule. It includes, e.g., Fab, Fab', F (ab') 2, and variants of these fragments.
  • an antibody or an antigen binding fragment thereof can be, e.g., a scFv, a Fv, a Fd, a dAb, a bispecific antibody, a bispecific scFv, a diabody, a linear antibody, a single-chain antibody molecule, a multi-specific antibody formed from antibody fragments, and any polypeptide that includes a binding domain which is, or is homologous to, an antibody binding domain.
  • Non-limiting examples of antigen binding domains include, e.g., the heavy chain and/or light chain CDRs of an intact antibody, the heavy and/or light chain variable regions of an intact antibody, full length heavy or light chains of an intact antibody, or an individual CDR from either the heavy chain or the light chain of an intact antibody.
  • the Fab fragment contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CH1) of the heavy chain.
  • F (ab') 2 antibody fragments comprise a pair of Fab fragments which are generally covalently linked near their carboxy termini by hinge cysteines between them. Other chemical couplings of antibody fragments are also known in the art.
  • Diabodies are small antibody fragments with two antigen-binding sites, which fragments comprise a VH connected to a VL in the same polypeptide chain (VH and VL) .
  • VH and VL polypeptide chain
  • Linear antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions.
  • Linear antibodies can be bispecific or monospecific.
  • Antibodies and antibody fragments of the present disclosure can be modified in the Fc region to provide desired effector functions or serum half-life.
  • Multimerization of antibodies may be accomplished through natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, some percentage of purified antibody preparations (e.g., purified IgG 1 molecules) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody multimers.
  • purified antibody preparations e.g., purified IgG 1 molecules
  • antibody homodimers may be formed through chemical linkage techniques known in the art.
  • heterobifunctional crosslinking agents including, but not limited to SMCC (succinimidyl 4- (maleimidomethyl) cyclohexane-1-carboxylate) and SATA (N-succinimidyl S-acethylthio-acetate) can be used to form antibody multimers.
  • SMCC succinimidyl 4- (maleimidomethyl) cyclohexane-1-carboxylate
  • SATA N-succinimidyl S-acethylthio-acetate
  • An exemplary protocol for the formation of antibody homodimers is described in Ghetie et al., (Proc. Natl. Acad. Sci. U.S.A. 94: 7509-7514, 1997) .
  • Antibody homodimers can be converted to Fab’ 2 homodimers through digestion with pepsin. Another way to form antibody homodi
  • the multi-specific antibody is a bi-specific antibody.
  • Bi-specific antibodies can be made by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers that are recovered from recombinant cell culture.
  • the interface can contain at least a part of the CH3 domain of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan) .
  • Compensatory “cavities” of identical or similar size to the large side chain (s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine) .
  • This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
  • This method is described, e.g., in WO 96/27011, which is incorporated by reference in its entirety.
  • Bi-specific antibodies include cross-linked or “heteroconjugate” antibodies.
  • one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin.
  • Heteroconjugate antibodies can also be made using any convenient cross-linking methods. Suitable cross-linking agents and cross-linking techniques are well known in the art and are disclosed in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.
  • bi-specific antibodies can be prepared using chemical linkage.
  • Brennan et al. (Science 229: 81, 1985) describes a procedure where intact antibodies are proteolytically cleaved to generate F (ab’) 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation.
  • the Fab’ fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • TNB thionitrobenzoate
  • One of the Fab’ TNB derivatives is then reconverted to the Fab’ thiol by reduction with mercaptoethylamine, and is mixed with an equimolar amount of another Fab’ TNB derivative to form the bi-specific antibody.
  • any of the antibodies or antigen-binding fragments described herein may be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or in solution) .
  • stabilizing molecules include: a polymer (e.g., a polyethylene glycol) or a protein (e.g., serum albumin, such as human serum albumin) .
  • the conjugation of a stabilizing molecule can increase the half-life or extend the biological activity of an antibody or an antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in a human) .
  • the antibodies or antigen-binding fragments described herein can be conjugated to a therapeutic agent.
  • the antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof can covalently or non-covalently bind to a therapeutic agent.
  • the therapeutic agent is a cytotoxic or cytostatic agent.
  • the scFV has one heavy chain variable domain, and one light chain variable domain. In some embodiments, the scFV has two heavy chain variable domains, and two light chain variable domains.
  • the antibodies or antigen-binding fragments thereof described herein can block the binding between the coronavirus (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV) S protein and ACE2.
  • the antibody by binding to coronavirus S protein, the antibody can neutralize coronavirus.
  • the antibody can promote virus aggregation.
  • the antibody can induce Fc-dependent antiviral functions.
  • the antibody can inhibit cleavage of the S protein by host cell TMPRSS2.
  • the antibody can block viral entry into host cell.
  • the disclosure provides antibodies or antigen-binding fragments thereof that neutralize the coronavirus (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV) such that the neutralized coronavirus is at least or about 5%, at least or about 10%, at least or about 15%, at least or about 20%, at least or about 25%, at least or about 30%, at least or about 35%, at least or about 40%, at least or about 45%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, or at least or about 95%of the total amount of the coronavirus.
  • the coronavirus e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV
  • the neutralized coronavirus is at least or about 5%, at least or about 10%, at least or about 15%, at least
  • the disclosure provides antibodies or antigen-binding fragments thereof that promote the coronavirus (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV) aggregation by at least or about 1 fold, at least or about 2 folds, at least or about 3 folds, at least or about 4 folds, at least or about 5 folds, at least or about 6 folds, at least or about 7 folds, at least or about 8 folds, at least or about 9 folds, at least or about 10 folds, at least or about 20 folds, at least or about 30 folds, at least or about 40 folds, at least or about 50 folds, or at least or about 100 folds as compared when no antibodies or antigen-binding fragments thereof as described herein are present.
  • coronavirus e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV
  • the disclosure provides antibodies or antigen-binding fragments thereof comprising a human Fc domain, which induce Fc-dependent antiviral functions by at least or about at least or about 1 fold, at least or about 2 folds, at least or about 3 folds, at least or about 4 folds, at least or about 5 folds, at least or about 6 folds, at least or about 7 folds, at least or about 8 folds, at least or about 9 folds, at least or about 10 folds, at least or about 20 folds, at least or about 30 folds, at least or about 40 folds, at least or about 50 folds, or at least or about 100 folds as compared when no antibodies or antigen-binding fragments thereof as described herein are present.
  • the disclosure provides antibodies or antigen-binding fragments thereof comprising a human Fc domain, which induce host immune response by at least or about at least or about 1 fold, at least or about 2 folds, at least or about 3 folds, at least or about 4 folds, at least or about 5 folds, at least or about 6 folds, at least or about 7 folds, at least or about 8 folds, at least or about 9 folds, at least or about 10 folds, at least or about 20 folds, at least or about 30 folds, at least or about 40 folds, at least or about 50 folds, or at least or about 100 folds as compared when no antibodies or antigen-binding fragments thereof as described herein are present.
  • the disclosure provides antibodies or antigen-binding fragments thereof that block viral entry (or internalization) into host cell such that the internalization rate is less than or about 50%, less than or about 45%, less than or about 40%, less than or about 35%, less than or about 30%, less than or about 25%, less than or about 20%, less than or about 15%, less than or about 10%, less than or about 10%, or less than or about 5%of the internalization rate when no antibodies or antigen-binding fragments thereof as described herein are present.
  • the antibodies or antigen-binding fragments thereof as described herein can increase immune response, activity or number of immune cells (e.g., T cells, CD8+ T cells, CD4+ T cells, macrophages, antigen presenting cells) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 folds, 3 folds, 5 folds, 10 folds, or 20 folds.
  • immune cells e.g., T cells, CD8+ T cells, CD4+ T cells, macrophages, antigen presenting cells
  • the antibody specifically binds to the S protein (e.g., SARS-CoV-2 S protein, SARS-CoV S protein, or MERS-CoV S protein) with a dissociation rate (koff) of less than 0.1 s -1 , less than 0.01 s -1 , less than 0.001 s -1 , less than 0.0001 s -1 , or less than 0.00001 s -1 .
  • S protein e.g., SARS-CoV-2 S protein, SARS-CoV S protein, or MERS-CoV S protein
  • the dissociation rate (koff) is greater than 0.01 s -1 , greater than 0.001 s -1 , greater than 0.0001 s -1 , greater than 0.00001 s -1 , or greater than 0.000001 s -1 .
  • kinetic association rates (kon) is greater than 1 x 10 2 /Ms, greater than 1 x 10 3 /Ms, greater than 1 x 10 4 /Ms, greater than 1 x 10 5 /Ms, or greater than 1 x 10 6 /Ms. In some embodiments, kinetic association rates (kon) is less than 1 x 10 5 /Ms, less than 1 x 10 6 /Ms, or less than 1 x 10 7 /Ms.
  • KD is less than 1 x 10 -6 M, less than 1 x 10 -7 M, less than 1 x 10 -8 M, less than 1 x 10 -9 M, less than 1 x 10 -10 M, less than 1 x 10 -11 M, or less than 1 x 10 -12 M.
  • the KD is less than 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM or 0.05 nM.
  • KD is greater than 1 x 10 -7 M, greater than 1 x 10 -8 M, greater than 1 x 10 -9 M, greater than 1 x 10 -10 M, greater than 1 x 10 -11 M, greater than 1 x 10 -12 M, or greater than 1 x 10 -13 M.
  • the antibody binds to SARS-CoV-2 S protein, SARS-CoV S protein, MERS-CoV S protein, or other coronavirus S proteins. In some embodiments, the antibody does not bind to other coronavirus S proteins.
  • thermal stabilities are determined.
  • the antibodies or antigen binding fragments as described herein can have a Tm greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C.
  • Tm is less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C.
  • the antibodies or antigen binding fragments can enhance APC (e.g., DC cell) function, for example, inducing surface expression of costimulatory and MHC molecules, inducing production of proinflammatory cytokines, and/or enhancing T cell triggering function.
  • APC e.g., DC cell
  • the antibodies or antigen binding fragments can induce complement-dependent cytotoxicity (CMC) and/or antibody dependent cellular cytoxicity (ADCC) , and kill the infected cell.
  • CMC complement-dependent cytotoxicity
  • ADCC antibody dependent cellular cytoxicity
  • the antibodies or antigen binding fragments have a functional Fc region.
  • effector function of a functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC) .
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • effector function of a functional Fc region is phagocytosis.
  • effector function of a functional Fc region is ADCC and phagocytosis.
  • the antibodies or antigen binding fragments can induce complement mediated cytotoxicity (CMC) .
  • CMC complement mediated cytotoxicity
  • the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4.
  • the antibody is a human IgG1 antibody.
  • the antibodies or antigen binding fragments do not have a functional Fc region.
  • the antibodies or antigen binding fragments are Fab, Fab’, F (ab’) 2, and Fv fragments.
  • the Fc region has LALA mutations (L234A and L235A mutations in EU numbering) , or LALA-PG mutations (L234A, L235A, P329G mutations in EU numbering) .
  • An isolated fragment of S protein (e.g., RBD domain) can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation.
  • Polyclonal antibodies can be raised in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of an antigenic peptide or protein.
  • the antigenic peptide or protein is injected with at least one adjuvant.
  • the antigenic peptide or protein can be conjugated to an agent that is immunogenic in the species to be immunized. Animals can be injected with the antigenic peptide or protein more than one time (e.g., twice, three times, or four times) .
  • the full-length polypeptide or protein can be used or, alternatively, antigenic peptide fragments thereof can be used as immunogens.
  • the antigenic peptide of a protein comprises at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence of S protein and encompasses an epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with the protein.
  • the immunogen comprises or consists of RBD domain of the S protein (e.g., amino acid 319-541 of SEQ ID NO: 141) .
  • the immunogen polypeptide is linked to a Fc (e.g., a mouse Fc) .
  • An immunogen typically is used to prepare antibodies by immunizing a suitable subject (e.g., human or transgenic animal expressing at least one human immunoglobulin locus) .
  • the subject is a homozygous humanized heavy chain immunoglobulin locus and homozygous humanized light chain immunoglobulin locus (hVH/hVL mice) (Biocytogen RenMab TM Mouse) .
  • the humanized mice are described e.g., in PCT/CN2020/075698, which is incorporated herein by reference in its entirety.
  • An appropriate immunogenic preparation can contain, for example, a recombinantly-expressed or a chemically-synthesized polypeptide.
  • the preparation can further include an adjuvant, such as Freund’s complete or incomplete adjuvant, or a similar immunostimulatory agent.
  • Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide, or an antigenic peptide thereof (e.g., part of S protein) as an immunogen.
  • the antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme-linked immunosorbent assay (ELISA) using the immobilized polypeptide or peptide.
  • ELISA enzyme-linked immunosorbent assay
  • the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A of protein G chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al., (Nature 256: 495-497, 1975) , the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4: 72, 1983) , the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985) , or trioma techniques.
  • standard techniques such as the hybridoma technique originally described by Kohler et al., (Nature 256: 495-497, 1975) , the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4: 72, 1983) , the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R.
  • Hybridoma cells producing a monoclonal antibody are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide or epitope of interest, e.g., using a standard ELISA assay.
  • Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding a human, humanized, or chimeric antibody, or antigen-binding fragment thereof described herein, or by peptide synthesis.
  • Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acids sequences that make-up the antigen-binding site of the antibody or an antigen-binding domain.
  • some antibodies or antigen-binding fragments will have increased affinity for the target protein. Any combination of deletions, insertions, and/or combinations can be made to arrive at an antibody or antigen-binding fragment thereof that has increased binding affinity for the target.
  • the amino acid changes introduced into the antibody or antigen-binding fragment can also alter or introduce new post-translational modifications into the antibody or antigen-binding fragment, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence such that a different sugar is attached by enzymes present in a cell) , or introducing new glycosylation sites.
  • Antibodies disclosed herein can be derived from any species of animal, including mammals.
  • Non-limiting examples of native antibodies include antibodies derived from humans, primates, e.g., monkeys and apes, cows, pigs, horses, sheep, camelids (e.g., camels and llamas) , chicken, goats, and rodents (e.g., rats, mice, hamsters and rabbits) , including transgenic rodents genetically engineered to produce human antibodies.
  • Human and humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequence as those derived from) human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) , for example in the CDRs.
  • Identity or homology with respect to an original sequence is usually the percentage of amino acid residues present within the candidate sequence that are identical with a sequence present within the human, humanized, or chimeric antibody or fragment, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • a cysteine residue can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated may have any increased half-life in vitro and/or in vivo.
  • Homodimeric antibodies with increased half-life in vitro and/or in vivo can also be prepared using heterobifunctional cross-linkers as described, for example, in Wolff et al., ( "Monoclonal antibody homodimers: enhanced antitumor activity in nude mice. " Cancer research 53.11 (1993) : 2560-2565) .
  • an antibody can be engineered which has dual Fc regions.
  • a covalent modification can be made to the antibody or antigen-binding fragment thereof.
  • These covalent modifications can be made by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage.
  • Other types of covalent modifications of the antibody or antibody fragment are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with an organic derivatization agent that is capable of reacting with selected side chains or the N-or C-terminal residues.
  • antibody variants having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region.
  • the amount of fucose in such antibody composition may be from 1%to 80%, from 1%to 65%, from 5%to 65%or from 20%to 40%.
  • the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example.
  • Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues; or position 314 in Kabat numbering) ; however, Asn297 may also be located about ⁇ 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function.
  • the Fc region of the antibody can be further engineered to replace the Asparagine at position 297 with Alanine (N297A) .
  • the Fc region of the antibodies was further engineered to replace the serine at position 228 (EU numbering) of IgG4 with proline (S228P) .
  • S228P serine at position 228
  • a detailed description regarding S228 mutation is described, e.g., in Silva et al., "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation. " Journal of Biological Chemistry 290.9 (2015) : 5462-5469, which is incorporated by reference in its entirety.
  • the present disclosure also provides recombinant vectors (e.g., an expression vectors) that include an isolated polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein) , host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleotide) , and the production of recombinant antibody polypeptides or fragments thereof by recombinant techniques.
  • recombinant vectors e.g., an expression vectors
  • an isolated polynucleotide disclosed herein e.g., a polynucleotide that encodes a polypeptide disclosed herein
  • host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleo
  • a “vector” is any construct capable of delivering one or more polynucleotide (s) of interest to a host cell when the vector is introduced to the host cell.
  • An “expression vector” is capable of delivering and expressing the one or more polynucleotide (s) of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced.
  • the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and/or a poly-A tail, either within the vector or in the genome of the host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector.
  • regulatory elements such as a promoter, enhancer, and/or a poly-A tail
  • a vector can be introduced into the host cell by methods known in the art, e.g., electroporation, chemical transfection (e.g., DEAE-dextran) , transformation, transfection, and infection and/or transduction (e.g., with recombinant virus) .
  • vectors include viral vectors (which can be used to generate recombinant virus) , naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.
  • a polynucleotide disclosed herein e.g., a polynucleotide that encodes a polypeptide disclosed herein
  • a viral expression system e.g., vaccinia or other pox virus, retrovirus, or adenovirus
  • vaccinia or other pox virus, retrovirus, or adenovirus may involve the use of a non-pathogenic (defective) , replication competent virus, or may use a replication defective virus.
  • viral propagation generally will occur only in complementing virus packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86: 317-321; Flexner et al., 1989, Ann. N.Y.
  • the DNA insert comprising an antibody-encoding or polypeptide-encoding polynucleotide disclosed herein can be operatively linked to an appropriate promoter (e.g., a heterologous promoter) , such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters are known to the skilled artisan.
  • the promoter is a cytomegalovirus (CMV) promoter.
  • CMV cytomegalovirus
  • the expression constructs can further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs may include a translation initiating at the beginning and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated.
  • the expression vectors can include at least one selectable marker.
  • markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria.
  • Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces, and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells; and plant cells. Appropriate culture mediums and conditions for the host cells described herein are known in the art.
  • Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
  • Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.
  • Non-limiting bacterial promoters suitable for use include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter.
  • Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV) , and metallothionein promoters, such as the mouse metallothionein-I promoter.
  • yeast Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods.
  • Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986) , which is incorporated herein by reference in its entirety.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type.
  • enhancers include the SV40 enhancer, which is located on the late side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • secretion signals may be incorporated into the expressed polypeptide.
  • the signals may be endogenous to the polypeptide or they may be heterologous signals.
  • the polypeptide (e.g., antibody) can be expressed in a modified form, such as a fusion protein (e.g., a GST-fusion) or with a histidine-tag, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to the polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.
  • the antibodies or antigen-binding fragments thereof of the present disclosure can be used for various therapeutic purposes.
  • the disclosure provides methods for treating a coronavirus-related disease in a subject, methods of neutralizing a coronavirus, methods of blocking a coronavirus/ACE2 interaction, methods of promoting coronavirus aggregation, methods of inducing Fc-dependent antiviral functions, methods of blocking internalization of a coronavirus by a cell, methods of identifying a subject having a coronavirus-related disease.
  • the treatment can halt, slow, retard, or inhibit progression of a coronavirus-related disease.
  • the treatment can result in the reduction of in the number, severity, and/or duration of one or more symptoms of the coronavirus-related disease in a subject.
  • the disclosure features methods that include administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein to a subject in need thereof (e.g., a subject having, or identified or diagnosed as having, a coronavirus-related disease) .
  • the coronavirus-related disease is COVID-19 (Coronavirus disease 2019) , Severe acute respiratory syndrome (SARS) , or Middle East respiratory syndrome (MERS) .
  • COVID-19 Coronavirus disease 2019
  • SARS Severe acute respiratory syndrome
  • MERS Middle East respiratory syndrome
  • the coronavirus that causing the coronavirus-related disease is SARS-CoV, SARS-CoV-2, MERS-CoV, or other types of coronavirus having one or more S proteins.
  • the amino acid sequence of the S protein of the coronavirus described herein comprises a sequence that is at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, or at least or about 98%identical to the receptor-biding domain sequence of the SARS-CoV-2 S protein.
  • compositions and methods disclosed herein can be used for treatment of patients at risk for a coronavirus-related disease.
  • Patients with coronavirus-related disease can be identified with various methods known in the art.
  • an “effective amount” is meant an amount or dosage sufficient to effect beneficial or desired results including halting, slowing, retarding, or inhibiting progression of a disease, e.g., a coronavirus-related disease.
  • An effective amount will vary depending upon, e.g., an age and a body weight of a subject to which the antibody, antigen binding fragment, antibody-encoding polynucleotide, vector comprising the polynucleotide, and/or compositions thereof is to be administered, a severity of symptoms and a route of administration, and thus administration can be determined on an individual basis.
  • an effective amount can be administered in one or more administrations.
  • an effective amount of an antibody or an antigen binding fragment is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and/or delay progression of a coronavirus-related disease in a patient.
  • an effective amount of an antibody or antigen binding fragment may vary, depending on, inter alia, patient history as well as other factors such as the type (and/or dosage) of antibody used.
  • Effective amounts and schedules for administering the antibodies, antibody-encoding polynucleotides, and/or compositions disclosed herein may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal that will receive the antibodies, antibody-encoding polynucleotides, and/or compositions disclosed herein, the route of administration, the particular type of antibodies, antibody-encoding polynucleotides, antigen binding fragments, and/or compositions disclosed herein used and other drugs being administered to the mammal.
  • a typical daily dosage of an effective amount of an antibody is 0.01 mg/kg to 100 mg/kg (mg per kg of patient weight) .
  • the dosage can be less than 100 mg/kg, 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, or 0.1 mg/kg.
  • the dosage can be greater than 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, 0.1 mg/kg, 0.05 mg/kg, or 0.01 mg/kg.
  • the dosage is about 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.9 mg/kg, 0.8 mg/kg, 0.7 mg/kg, 0.6 mg/kg, 0.5 mg/kg, 0.4 mg/kg, 0.3 mg/kg, 0.2 mg/kg, or 0.1 mg/kg.
  • the at least one antibody, antigen-binding fragment thereof, or pharmaceutical composition e.g., any of the antibodies, antigen-binding fragments, or pharmaceutical compositions described herein
  • at least one additional therapeutic agent can be administered to the subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day) .
  • at least two different antibodies and/or antigen-binding fragments are administered in the same composition (e.g., a liquid composition) .
  • At least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition) .
  • the at least one antibody or antigen-binding fragment and the at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition containing at least one antibody or antigen-binding fragment and a solid oral composition containing at least one additional therapeutic agent) .
  • the at least one additional therapeutic agent is administered as a pill, tablet, or capsule.
  • the at least one additional therapeutic agent is administered in a sustained-release oral formulation.
  • the one or more additional therapeutic agents can be administered to the subject prior to, or after administering the at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) .
  • the one or more additional therapeutic agents and the at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition are administered to the subject such that there is an overlap in the bioactive period of the one or more additional therapeutic agents and the at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) in the subject.
  • the subject can be administered the at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) over an extended period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years) .
  • a skilled medical professional may determine the length of the treatment period using any of the methods described herein for diagnosing or following the effectiveness of treatment (e.g., the observation of at least one symptom of the coronavirus-related disease) .
  • a skilled medical professional can also change the identity and number (e.g., increase or decrease) of antibodies or antigen-binding antibody fragments (and/or one or more additional therapeutic agents) administered to the subject and can also adjust (e.g., increase or decrease) the dosage or frequency of administration of at least one antibody or antigen-binding antibody fragment (and/or one or more additional therapeutic agents) to the subject based on an assessment of the effectiveness of the treatment (e.g., using any of the methods described herein and known in the art) .
  • the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions can be combined to have an improved therapeutic effect.
  • the subject can be administered the at least one, two, three, four, or five antibody, antigen-binding fragment thereof selected from Table 1.
  • these antibodies or antigen-binding fragments thereof target different epitopes of an S protein.
  • these antibodies or antigen-binding fragments thereof do not cross-compete with each other.
  • at least two antibodies or antigen-binding fragments thereof are selected. Different pairs of antibodies or antigen-binding fragments thereof can be selected from FIG. 16. These pairs of antibodies or antigen-binding fragments include any antibodies or antigen-binding fragments derived from the antibodies as shown in FIG.
  • the subject is administered with 03-10D12-IgG1-LALA and 05-9G11-IgG1-LALA. In some embodiments, the subject is administered with 03-9A8-IgG1-LALA and 09-7B8-IgG-LALA..
  • the antibodies or antigen-binding fragments thereof can be used for detecting coronavirus (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV) in a subject (e.g., a human) or diagnosing a coronavirus related disease.
  • coronavirus e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV
  • Methods known in the art can be designed, e.g., ELISA, to produce a diagnostic test kit.
  • one or more antibodies or antigen-binding fragments comprising any of the heavy chain single variable domains as described herein can be used.
  • compositions that contain at least one (e.g., one, two, three, or four) of the antibodies or antigen-binding fragments described herein. Two or more (e.g., two, three, or four) of any of the antibodies or antigen-binding fragments described herein can be present in a pharmaceutical composition in any combination.
  • the pharmaceutical compositions may be formulated in any manner known in the art.
  • the pharmaceutical compositions can comprise one, two, three, four, five or more than five antibodies, antigen-binding fragments thereof as described herein (e.g., selected from Table 1) .
  • these antibodies or antigen-binding fragments thereof target different epitopes of an S protein.
  • these antibodies or antigen-binding fragments thereof do not cross-compete with each other.
  • at least two antibodies or antigen-binding fragments thereof are selected. Different pairs of antibodies or antigen-binding fragments thereof can be selected from FIG. 16. These pairs of antibodies or antigen-binding fragments include any antibodies or antigen-binding fragments derived from the antibodies as shown in FIG.
  • the pharmaceutical composition comprises 03-10D12-IgG1-LALA and 05-9G11-IgG1-LALA. In some embodiments, the pharmaceutical composition comprises 03-9A8-IgG1-LALA and 09-7B8-IgG-LALA..
  • compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) .
  • the compositions can include a sterile diluent (e.g., sterile water or saline) , a fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvents, antibacterial or antifungal agents, such as benzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid, buffers, such as acetates, citrates, or phosphates, and isotonic agents, such as sugars (e.g., dextrose) , polyalcohols (e.g., mannitol or
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. Preparations of the compositions can be formulated and enclosed in ampules, disposable syringes, or multiple dose vials. Where required (as in, for example, injectable formulations) , proper fluidity can be maintained by, for example, the use of a coating, such as lecithin, or a surfactant. Absorption of the antibody or antigen-binding fragment thereof can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin) .
  • an agent that delays absorption e.g., aluminum monostearate and gelatin
  • controlled release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid) .
  • biodegradable, biocompatible polymers e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid
  • compositions containing one or more of any of the antibodies or antigen-binding fragments described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage) .
  • parenteral e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal
  • dosage unit form i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage
  • compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under Good Manufacturing Practice (GMP) conditions.
  • Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration) .
  • Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries. The formulation depends on the route of administration chosen.
  • antibodies can be formulated in aqueous solutions, preferably in physiologically-compatible buffers to reduce discomfort at the site of injection.
  • the solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • antibodies can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • compositions as described herein can be administered through respiratory tract by various means, for example, nasal administration, nasal instillation, insufflation (e.g., nasal sprays) , inhalation (through nose or mouth) , intrapulmonary administration, intratracheal administration, or any combinations thereof.
  • nasal instillation refers to a procedure that delivers a therapeutic agent directly into the nose and onto the nasal membranes, wherein a portion of the therapeutic agent can pass through tracheas and is delivered into the lung.
  • Toxicity and therapeutic efficacy of compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys) .
  • Agents that exhibit high therapeutic indices are preferred. Where an agent exhibits an undesirable side effect, care should be taken to minimize potential damage (i.e., reduce unwanted side effects) .
  • Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.
  • a therapeutically effective amount of the one or more (e.g., one, two, three, or four) antibodies or antigen-binding fragments thereof (e.g., any of the antibodies or antibody fragments described herein) will be an amount that treats the disease in a subject (e.g., inhibits coronavirus) in a subject (e.g., a human subject identified as having COVID-19) , or a subject identified as being at risk of developing the disease (e.g., a subject who has previously infected by coronavirus but now has been cured) , decreases the severity, frequency, and/or duration of one or more symptoms of a disease in a subject (e.g., a human) .
  • any of the antibodies or antigen-binding fragments described herein can be determined by a health care professional or veterinary professional using methods known in the art, as well as by the observation of one or more symptoms of disease in a subject (e.g., a human) . Certain factors may influence the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and the presence of other diseases) .
  • Exemplary doses include milligram or microgram amounts of any of the antibodies or antigen-binding fragments described herein per kilogram of the subject’s weight (e.g., about 1 ⁇ g/kg to about 500 mg/kg; about 100 ⁇ g/kg to about 500 mg/kg; about 100 ⁇ g/kg to about 50 mg/kg; about 10 ⁇ g/kg to about 5 mg/kg; about 10 ⁇ g/kg to about 0.5 mg/kg; about 1 ⁇ g/kg to about 50 ⁇ g/kg; about 1 mg/kg to about 10 mg/kg; or about 1 mg/kg to about 5 mg/kg) .
  • weight e.g., about 1 ⁇ g/kg to about 500 mg/kg; about 100 ⁇ g/kg to about 500 mg/kg; about 100 ⁇ g/kg to about 50 mg/kg; about 10 ⁇ g/kg to about 5 mg/kg; about 10 ⁇ g/kg to about 0.5 mg/kg; about 1 ⁇ g/kg to about 50 ⁇ g/kg; about 1
  • therapeutic agents including antibodies and antigen-binding fragments thereof, vary in their potency, and effective amounts can be determined by methods known in the art.
  • relatively low doses are administered at first, and the attending health care professional or veterinary professional (in the case of therapeutic application) or a researcher (when still working at the development stage) can subsequently and gradually increase the dose until an appropriate response is obtained.
  • the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the half-life of the antibody or antibody fragment in vivo.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • disclosure also provides methods of manufacturing the antibodies or antigen binding fragments thereof for various uses as described herein.
  • RBD-mFc protein SARS-CoV-2 (2019-nCoV) Spike RBD-mFc Recombinant Protein (HPLC-verified)
  • Sino Biological Inc. Catalog number: 40592-V05H
  • Recombinant 2019-nCoV S protein RBD (C-6His) was purchased from Sino Biological Inc. (Catalog number: 40592-V08B) .
  • Plasmid expressing full-length SARS-CoV-2 spike protein was obtained from National Institutes for Food and Drug Control (China) .
  • ACE2-hFc (recombinant human ACE2 protein-hFc) was purchased from Kactus Biosystems Co. Ltd (Catalog number: ACE-HM501) .
  • ACE2-mFc (recombinant human ACE2 protein-mFc) was purchased from Sino Biological Inc. (Catalog number: 10108-H05H) .
  • S(S1+S2) -his protein SARS-CoV-2 (COVID-19) S protein (R683A, R685A) , His Tag) was purchased from ACROBiosystems (Catalog number: SPN-C52H4) .
  • Anti-hIgG PE R-Phycoerythrin AffiniPure F (ab') 2 Fragment Goat Anti-Human IgG, Fc ⁇ fragment specific
  • was purchased from Jackson ImmunoResearch Inc. Catalog number: 109-116-098) .
  • Anti-mIgG FITC Fluorescein (FITC) AffiniPure F (ab') 2 Fragment Goat Anti-Mouse IgG, Fc ⁇ fragment specific) was purchased from Jackson ImmunoResearch Inc. (Catalog number: 115-096-071) .
  • Anti-mouse IgG Fc-PE R-Phycoerythrin AffiniPure F (ab') 2 Fragment Goat Anti-Mouse IgG, Fc ⁇ fragment specific
  • was purchased from Jackson ImmunoResearch Inc. Catalog number: 115-116-071
  • ab' Royal ImmunoResearch Inc.
  • Anti-human IgG Fc-AF647 Alexa 647 AffiniPure F (ab') 2 Fragment Goat Anti-Human IgG, Fc ⁇ fragment specific
  • Jackson ImmunoResearch Inc. Catalog number: 109-606-170
  • HBS-EP+ buffer (10 ⁇ ) was purchased from GE Healthcare Life Sciences (Catalog number: BR-1006-69) .
  • Glycine-HCl, pH 1.5 buffer was purchased from GE Healthcare Life Sciences (Catalog number: BR-1003-54) .
  • TEE TM His Tag Antibody was purchased from GenScript Biotech (Catalog number: A00186) .
  • the anti-SARS-CoV-2 antibodies were collected by the methods as described below.
  • mice with homozygous humanized heavy chain immunoglobulin locus and homozygous humanized light chain immunoglobulin locus (hVH/hVL mice) (Biocytogen RenMab TM Mouse) were immunized with an RBD-mFc protein at least five times.
  • the RBD-mFc protein contains the SARS-CoV-2 Spike protein receptor-binding domain (amino acid 319-541 of NCBI Reference Sequence: YP_009724390.1 (SEQ ID NO: 141) ) and is fused to the Fc region of mouse IgG1 at C-terminus.
  • the RBD-mFc protein was emulsified with adjuvant and injected at several positions on the back of the mice. Procedures to enhance immunization were also performed after the fifth immunization.
  • CHO-S-SDNA (T) cells and the RBD-mFc protein were intravenously injected into the mice through tail veins. Spleen was then collected four days after the injection.
  • CHO-S-SDNA (T) were CHO-K cells transfected with a plasmid expressing full-length SARS-CoV-2 spike protein on the cell surface.
  • the expressed SARS-CoV-2 spike protein includes both S1 and S2 subunits of the spike protein (e.g., the extracellular domain of the S1 and S2 subunits) .
  • Spleen tissues were grinded. Spleen cells were first selected by CD3 ⁇ Microbeads and Anti-Mouse IgM Microbeads, and then fused with SP2/0 cells. The cells were then plated in 96-well plates with hypoxanthine-aminopterin-thymidine (HAT) medium.
  • HAT hypoxanthine-aminopterin-thymidine
  • FACS Fluorescence-Activated Cell Sorting
  • Sub-cloning was performed using ClonePix2. In short, the positive wells identified during the primary screening were transferred to semisolid medium, and IgG positive clones were identified and tested. FITC anti-mouse IgG Fc antibody was used.
  • Antibodies in ascites fluid were purified using GE AKTA protein chromatography (GE Healthcare, Chicago, Illinois, United States) . At least 17 chimeric antibodieswere produced. Specifically, each chimeric antibody heavy chain comprised a human variable region (VH) and a mouse constant region (CH) , and each chimeric antibody light chain comprised a human variable region (VL) and a mouse constant region (CL) . A few antibodies were selected because of the desired properties.
  • mouse antibodies produced by the methods described above include e.g., 03-9A8 ( “9A8” ) , 01-2H10 ( “2H10” ) , 03-1F9 ( “1F9” ) , 03-10D12 ( “10D12” ) , 03-10F9 ( “10F9” ) , 05-8G6 ( “8G6” ) , 05-9G11 ( “9G11” ) , 09-2F7 ( “2F7” ) , 09-4E5 ( “4E5” ) , and 09-7B8 ( “7B8” ) .
  • the amino acid sequences of the heavy chain variable region (VH) , light chain variable region (VL) , and complementarity determining regions (CDRs) of the antibodies were determined.
  • the heavy chain CDR1, CDR2, CDR3, and light chain CDR1, CDR2, and CDR3 amino acid sequences of these antibodies are shown in FIGS. 13-14.
  • the VH and VL amino acid sequences of these antibodies are shown in FIG. 15.
  • Consensus sequences in CDR1, CDR2, CDR3 in VH are shown in FIGS. 11A-11C.
  • Consensus sequences in CDR1, CDR2, CDR3 in VL are shown in FIGS. 12A-12C.
  • Example 2 In vitro testing of anti-SARS-CoV-2 antibodies in ascites fluid or hybridoma supernatant: blocking human ACE2 binding to SARS-CoV-2 S protein
  • Blocking effects of ascites fluid or hybridoma supernatant were detected by flow cytometry.
  • Detailed methods are as follows. First, the immunized mouse serum and unimmunized mouse serum were diluted using phosphate-buffered saline (PBS) at 1: 100 and 1: 500, respectively. The ACE2-hFc protein was diluted to 0.5 ⁇ g/ml by PBS. Next, CHO-S-SDNA (T) cells were washed to remove residual culture medium and resuspended in PBS to a cell density of 5 ⁇ 10 4 per 10 ⁇ l. The resuspended cells were added to a 96-well plate (10 ⁇ l/well) and 40 ⁇ l ascites fluid (or hybridoma supernatant) was added to the corresponding wells.
  • PBS phosphate-buffered saline
  • a row in the 96-well plate was reserved for control samples.
  • two wells were selected to add diluted immunized mouse serum (labelled as PC1) and diluted unimmunized mouse serum (labeled as NC1) to the CHO-S-SDNA (T) cells, respectively (40 ⁇ l/well) .
  • Another well was selected to add ascites fluid (or hybridoma supernatant) containing antibodies against other antigens (labelled as NC2) .
  • 50 ⁇ l diluted ACE2-hFc was added to each well, followed by an incubation at 4°C for 30 minutes.
  • 03-9A8, 01-2H10, 03-1F9, 03-10D12, 03-10F9, 05-8G6, 05-9G11, 09-2F7, 09-4E5, and 09-7B8 are chimeric anti-SARS-CoV-2 antibodies generated by the hVH/hVL mice.
  • the chimeric antibodies have the heavy chain variable domain (VH) and the light chain variable domain (VL) from human, and the constant domains from mouse IgG antibodies.
  • the human anti-SARS-CoV-2 antibodies including e.g., 03-9A8-IgG1-LALA, 01-2H10-IgG1-LALA, 03-1F9-IgG1-LALA, 03-10D12-IgG1-LALA, 03-10F9-IgG1-LALA, 05-8G6-IgG1-LALA, 05-9G11-IgG1-LALA, 09-2F7-IgG1-LALA, 09-4E5-IgG1-LALA, and 09-7B8-IgG1-LALA were generated.
  • 03-9A8-IgG1-LALA 01-2H10-IgG1-LALA, 03-1F9-IgG1-LALA, 03-10D12-IgG1-LALA, 03-10F9-IgG1-LALA, 05-8G6-IgG1-LALA, 05-9G11-IgG1-LALA, 09-2F7-Ig
  • the human antibodies have the same heavy chain variable domain and the light chain variable domain from the corresponding chimeric anti-SARS-CoV-2 antibodies, and the constant domains from human IgG1 antibodies (including, e.g., the CL, CH1, CH2, and CH3 domains of human IgG1) with LALA (Leu234Ala/Leu235Ala) mutations.
  • the binding affinity ofanti-SARS-CoV-2 antibodies against the SARS-CoV-2 spike protein RBD were measured by surface plasmon resonance (SPR) using Biacore 8K SPR System (GE) equipped with pre-immobilized Series S Sensor Chip Protein A at 25°C.
  • the human anti-SARS-CoV-2 antibodies exhibited high binding affinities against the SARS-CoV-2 spike protein RBD.
  • Example 4 IC 50 determination ofpurified human anti-SARS-CoV-2 antibodies: blocking human ACE2 binding to SARS-CoV-2 S protein
  • Blocking effects of purified human anti-SARS-CoV-2 antibodies were detected by fluorescence-activated cell sorting (FACS) using the CHO-S-SDNA (T) cells.
  • FACS fluorescence-activated cell sorting
  • the purified antibodies were diluted by serial dilution to 60 ⁇ g/ml, 20 ⁇ g/ml, 6.667 ⁇ g/ml, 2.222 ⁇ g/ml, 0.741 ⁇ g/ml, 0.247 ⁇ g/ml, 0.082 ⁇ g/ml, 0.027 ⁇ g/ml, 0.009 ⁇ g/ml, and 0.003 ⁇ g/ml.
  • the ACE2-mFc protein was diluted to 10 ⁇ g/ml.
  • CHO-S-SDNA (T) cells were washed to remove residual culture medium and resuspended in PBS to a cell density of 1 ⁇ 10 6 cells/ml.
  • the resuspended cells were added to a 96-well plate (50 ⁇ l/well) and the plate was centrifuged at 1600 rpm for 6 minutes. Supernatant in each well was discarded.
  • 40 ⁇ l diluted antibodies were added to the corresponding wells, followed by an incubation at 4°C for 30 minutes. Afterwards, 50 ⁇ l diluted ACE2-mFc were added to the corresponding wells, and the plate was further incubated at 4°C for 30 minutes.
  • the raw data were analyzed to generatethe mean fluorescence intensity (MFI) reflectingantibody-CHO-S-SDNA (T) cell binding at different antibody concentrations.
  • MFI values were used to generate a fitting curve with respect to the antibody concentrations. More specifically, logarithm of antibody concentrations was calculated and was used as the X-axis variable, while the corresponding MFI was used as the Y-axis variable.
  • An exemplary fitting curves of 03-9A8-IgG1-LALA and 09-7B8-IgG1-LALA are shown in FIG. 3.
  • the IC 50 value was determined. Two independent experiments were performed and the determined IC 50 is shown in the tables below.
  • Anti-SARS-CoV-2 Antibody IC 50 ( ⁇ g/mL) R 2 03-9A8-IgG1-LALA 1.363 0.8940 03-1F9-IgG1-LALA 1.975 0.8041 05-10B7-IgG1-LALA Not applicable No binding 09-4E5-IgG1-LALA 7.298 0.7436 09-7B8-IgG1-LALA 0.8011 0.8844
  • 09-7B8-IgG1-LALA had the best blocking effects.
  • the blocking effects of 03-9A8-IgG1-LALA and 03-1F9-IgG1-LALA are also very good.
  • Anti-SARS-CoV-2 Antibody IC 50 ( ⁇ g/mL) R 2 03-9A8-IgG1-LALA 3.148 0.8374 01-2H10-IgG1-LALA 2.711 0.8308 02-5A8-IgG1-LALA Not applicable No binding 02-6G4-IgG1-LALA Not applicable No binding 03-10D12-IgG1-LALA 10.39 0.9305 03-10F9-IgG1-LALA 19.37 0.8927 05-8G6-IgG1-LALA Not applicable No binding 05-9G11-IgG1-LALA 53.2 0.8348 09-2F7-IgG1-LALA 9.272 0.9192
  • 01-2H10-IgG1-LALA and 03-9A8-IgG1-LALA had the best blocking effects.
  • the blocking effects of 03-10D12-IgG1-LALA and 09-2F7-IgG1-LALA are also good.
  • Relative positions of target protein epitope between a pair of purified anti-SARS-CoV-2 monoclonal antibodies were analyzed through a surface plasmon resonance (SPR) competition experiment.
  • a total of 100 combination pairs ofthe 10 monoclonal antibodies (01-2H10-IgG1-LALA, 03-10D12-IgG1-LALA, 03-10F9-IgG1-LALA, 03-1F9-IgG1-LALA, 05-8G6-IgG1-LALA, 05-9G11-IgG1-LALA, 09-2F7-IgG1-LALA, 09-4E5-IgG1-LALA, 09-7B8-IgG1-LALA, and 03-9A8-IgG1-LALA) were used to study the binding inhibition (blocking) effect of each antibody on another antibody.
  • HBS-EP+ buffer (10mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) , 150mM NaCl, 3mM ethylenediaminetetraacetic acid (EDTA) and 0.05%P20, pH7.4) was diluted from HBS-EP+ buffer (10 ⁇ ) as the running buffer throughout the experiment.
  • Anti-His antibodies were fixed on the surface of a Series S sensor Chip CM5 by amino group coupling to generate an anti-His chip (i.e., CM5-Anti-His-Channel 1, 8-Chip) .
  • the chip was regenerated twice with a glycine buffer (pH 1.7; 30 ⁇ L/min for 30 seconds, followed by 30 ⁇ L/min for 20 seconds) .
  • a glycine buffer pH 1.7; 30 ⁇ L/min for 30 seconds, followed by 30 ⁇ L/min for 20 seconds.
  • Each pair of monoclonal antibodies was subjected to the same experimental steps to obtain the binding inhibition data when each monoclonal antibody was paired with another antibody.
  • the binding value of each antibody was obtained using Biacore 8K Evaluation Software. To quantify the interference of one antibody binding to another, a binding ratio was calculated to compare each pair of antibodies. The binding ratio is defined as the binding value of the second antibody (analyte 2) , divided by the binding value of the first antibody (analyte 1) . A statistical software wasalso used for cluster analysis. The binding ratio of each monoclonal antibody pair was summarized in a matrix table as shown in FIG. 4. More specifically, the binding ratio was between -0.1 to 0.5, if analyte 1 exhibited a blocking effect to analyte 2. The binding ratio was between 0.5-1.2, if analyte 1 did not exhibit a blocking effect to analyte 2.
  • epitope correlation was analyzed (FIG. 5) and the 10 human anti-SARS-CoV-2 antibodies were categorized into 5 epitope clusters (FIG. 6) .
  • 01-2H10-IgG1-LALA, 03-10F9-IgG1-LALA, and 03-10D12-IgG1-LALA shared the same or overlapping epitopes
  • 05-8G6-IgG1-LALA and 05-9G11-IgG1-LALA shared the same or overlapping epitopes
  • 09-2F7-IgG1-LALA and 09-4E5-IgG1-LALA shared the same or overlapping epitopes
  • 03-1F9-IgG1-LALA and 03-9A8-IgG1-LALA shared the same or overlapping epitopes
  • 09-7B8-IgG1-LALA did not exhibit epitope correlation with the other 9 antibodies.
  • the cluster including 09-2F7-IgG1-LALA and 09-4E5-IgG1-LALA exhibited a strong correlation with the cluster including 03-1F9-IgG1-LALA and 03-9A8-IgG1-LALA.
  • Example 6 In vitro testing of purified human anti-SARS-CoV-2 antibodies: blocking human ACE2 binding to SARS-CoV-2 S protein
  • Blocking effects of purified human anti-SARS-CoV-2 antibodies were detected by surface plasmon resonance (SPR) using a Biacore 8K SPR System equipped with CM5-Anti-His-Channel 1, 8-Chip.
  • SPR surface plasmon resonance
  • a purified human anti-SARS-CoV-2 antibody and recombinant ACE2 were sequentially injected to interact with captured 2019-nCoV S protein RBD, therefore to verify the ACE2 blocking effect of each antibody.
  • HBS-EP+ buffer 10 mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) , 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid (EDTA) and 0.05%P20, pH 7.4) was diluted from HBS-EP+ buffer (10 ⁇ ) as the running buffer throughout the experiment.
  • Anti-His antibodies were fixed on the surface of a Series S sensor Chip CM5 to generate an anti-His chip (i.e., CM5-Anti-His-Channel 1, 8-Chip) .
  • the Biacore system was equilibrated with the HBS-EP+ buffer.
  • Recombinant 2019-nCoV S protein RBD with His-tag (1 ⁇ g/ml) were injected into the Biacore 8K biosensor at 10 ⁇ L/min for 50 seconds and captured on the anti-His chip to achieve a desired protein density (about 50 RU) . Then, 1M ethanolamine, pH 8.5 was injected to block the remaining active carboxyl groups on the chip surface, followed by equilibration using the HBS-EP+ buffer for 2 hours.
  • HBS-EP+ buffer (10 mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) , 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid (EDTA) and 0.05%P20, pH 7.4) was diluted from HBS-EP+ buffer (10 ⁇ ) as the running buffer throughout the experiment.
  • Anti-His antibodies were fixed on the surface of a Series S sensor Chip CM5 by amino group coupling to generate an anti-His chip (i.e., CM5-Anti-His-Channel 1, 8-Chip) .
  • the Biacore system was equilibrated with the HBS-EP+ buffer.
  • Recombinant 2019-nCoV S protein RBD with His-tag (1 ⁇ g/ml) were injected into the Biacore 8K biosensor at 10 ⁇ L/min for 50 seconds and captured on the anti-His chip to achieve a desired protein density (about 50 RU) .
  • 1M ethanolamine, pH 8.5 was injected to block the remaining active carboxyl groups on the chip surface, followed by equilibration using the HBS-EP+ buffer for 2 hours.
  • a purified anti-SARS-CoV-2 antibody 100 nM was continuously injected at 30 ⁇ L/min into the chip.
  • the injected antibody (analyte 1) had a binding time of 300 seconds.
  • ACE2-hFc (200 nM; analyte 2) was injected with a binding time of 200 seconds. After injection of the antibodies in each analysis cycle, the chip was regenerated with a glycine buffer (pH 1.7; 30 ⁇ L/min for 30 seconds, followed by 30 ⁇ L/min for 20 seconds) .
  • COVID-19 pseudovirus (or pseudovirus particles) was used for testing neutralizing effects.
  • the pseudovirus preserves the virus infecting ability but cannot replicate.
  • the function and activities of pseudovirus have been verified at the cellular level and in humanized mice.
  • the anti-SARS-CoV-2 antibodies were serial diluted in a 96-well plate. Then, the COVID-19 pseudovirus was diluted to 1-2 ⁇ 10 4 TCID 50 /ml and mixed with the antibodies, followed by an incubation at 37°C, 5%CO 2 for 1 hour. Next, Huh-7 cells (2 ⁇ 10 4 cells/well) were added to the corresponding wells, and the plate was incubated at 37°C, 5%CO 2 for 20-28 hours. After the incubation, 150 ⁇ l supernatant was discarded by pipetting, and 100 ⁇ l luciferase detection reagent was added to each well. The plate was then incubated at room temperature in dark for 2 minutes. Afterwards, 150 ⁇ l solution in each well was transferred to a new plate, which was placed in a plate reader to measure chemiluminescence signals. The neutralization inhibition ratio was calculated as follows:
  • Inhibition ratio [1- (Ab-CC) / (VC-CC) ] ⁇ 100%
  • IC 50 can be calculated by the Reed-Muench algorithm. Details of this method can be found, e.g., in Reed, Lowell Jacob, and Hugo Muench. "A simple method of estimating fifty per cent endpoints. " American Journal of Epidemiology 27.3 (1938) : 493-497; and Nie et al., "Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2.
  • Anti-SARS-CoV-2 Antibody IC 50 (ng/ml) 05-9G11-IgG1-LALA 79 03-10D12-IgG1-LALA 20 01-2H10-IgG1-LALA 26 03-10F9-IgG1-LALA 16 03-9A8-IgG1-LALA 38 03-1F9-IgG1-LALA 74 09-7B8-IgG1-LALA 37 09-4E5-IgG1-LALA 112 05-8G6-IgG1-LALA - 09-2F7-IgG1-LALA 593
  • Example 8 IC 50 determination of purified human anti-SARS-CoV-2 antibodies in combination: blocking human ACE2 binding to SARS-CoV-2 S protein
  • Blocking effects of purified human anti-SARS-CoV-2 antibodies were detected by fluorescence-activated cell sorting (FACS) using the CHO-S-ACE2 cells.
  • FACS fluorescence-activated cell sorting
  • the purified antibodies were diluted by serial dilution to 9000 ng/mL, 3000 ng/mL, 1000 ng/mL, 333.3 ng/mL, 111.1 ng/mL, 37.0 ng/mL, 12.3 ng/mL, 4.1ng/mL, 1.4ng/mL, and 0.5ng/mL.
  • the PE labeled Recombinant SARA-Cov-2 spike Protein (RBD, His Tag) protein was diluted to 200 ng/ml.
  • CHO-S-ACE2 cells were washed to remove residual culture medium and resuspended in PBS to a desired cell density (5 ⁇ 10 4 cells for 30 ul) .
  • the resuspended cells were added to a 96-well plate (30 ⁇ l/well) .
  • 30 ⁇ l diluted antibodies were added to the corresponding wells, followed by an incubation at 4°C for 30 minutes.
  • 30 ⁇ l diluted RBD were added to the corresponding wells, and the plate was further incubated at 4°C for 1 hour.
  • 200 ⁇ l PBS was added to each well, and then the plate was centrifuged at 1600 rpm for 6 minutes. Supernatant in each well was discarded.
  • cells in each well were washed as described above and then resuspended in 30 ⁇ l PBS for FACS analysis.
  • Raw data was analyzed to generate the mean fluorescence intensity (MFI) reflecting RBD-cell binding at different antibody concentrations.
  • MFI values were then used to generate a fitting curve with respect to the antibody concentrations. More specifically, logarithm of antibody concentrations were calculated and used as the X-axis variable, while the corresponding MFI was used as the Y-axis variable.
  • FIGS. 8A-8E The IC 50 value was determined and the determined IC 50 is shown in the tables below and FIG. 9.
  • Antibody Name IC 50 (ng/mL) R 2 30 ⁇ L Ab. 01 (9A8) 296.4 0.9809 30 ⁇ L Ab. 02 (03-10D12) 216.6 0.9771 30 ⁇ L Ab. 03 (05-9G11) 137.4 0.9809 30 ⁇ L Ab. 04 (09-4E5) 447.3 0.9897 30 ⁇ L Ab. 05 (09-7B8) 488.8 0.9892 15 ⁇ L Ab. 01 (9A8) +15 ⁇ L Ab. 02 (03-10D12) 269.6 0.9963 15 ⁇ L Ab. 01 (9A8) +15 ⁇ L Ab. 03 (05-9G11) 210.0 0.9870 15 ⁇ L Ab. 01 (9A8) +15 ⁇ L Ab. 04 (09-4E5) 251.3 0.9956 15 ⁇ L Ab.
  • anti-SARS-CoV-2 antibodies can be combined to have an improved blocking effect (or lower IC 50 ) .
  • the IC 50 of 03-9A8-IgG1-LALA and09-4E5-IgG1-LALA were determined as 296.4ng/mL and 488.8ng/mL, respectively.
  • the IC 50 was reduced to 245.5 ng/mL.
  • Example 9 IC 50 determination of purified human anti-SARS-CoV-2 antibodies in combination: neutralizing COVID-19 pseudovirus
  • COVID-19 pseudovirus was prepared and the neutralizing effects of the anti-SARS-CoV-2 antibodies were determined as described in Example 7.
  • the IC 50 of 03-10D12-IgG1-LALA (Ab1) , 03-9A8-IgG1-LALA (Ab2) , 09-4E5-IgG1-LALA (Ab3) , 09-7B8-IgG-LALA (Ab4) , 05-9G11-IgG1-LALA (Ab5) alone, or in combination were determined and the results are listed in the table below.
  • the results indicate that anti-SARS-CoV-2 antibodies can be combined to have an improved neutralizing activity (or lower IC 50 ) .
  • the IC 50 of 03-10D12-IgG1-LALA and 05-9G11-IgG1-LALA were determined as 0.016828 ⁇ g/mL and 0.1147 ⁇ g/mL, respectively.
  • the IC 50 was reduced to 0.010834 ⁇ g/mL.
  • Anti-SARS-CoV-2 Antibody IC 50 ( ⁇ g/ml) Ab1 0.016828 Ab2 0.213159 Ab3 0.137845 Ab4 0.032121 Ab5 0.1147 Ab1 + Ab2 0.031044 Ab1 + Ab3 0.020152 Ab1 + Ab4 0.021578 Ab1 + Ab5 0.010834 Ab2 + Ab3 0.161572 Ab2 + Ab4 0.07572 Ab2 + Ab5 0.125433 Ab3 + Ab4 0.092523 Ab3 + Ab5 0.051045 Ab4 + Ab5 0.02586
  • COVID-19 pseudovirus neutralizing activity titration was measured for the anti-SARS-CoV-2 antibodies alone, or in combination.
  • the results for 03-10D12-IgG1-LALA (Ab1) , 03-9A8-IgG1-LALA (Ab2) , 09-4E5-IgG1-LALA (Ab3) , 09-7B8-IgG-LALA (Ab4) , 05-9G11-IgG1-LALA (Ab5) , and Ab1 + Ab5 combination, are shown in FIG. 10.
  • the results are consistent with the IC 50 determination results as shown in Table 6.
  • the titration curve of combined 03-10D12-IgG1-LALA and 05-9G11-IgG1-LALA presents a shift towards left relative to the titration curve of each individual antibody.

Abstract

Relates to anti-coronavirus antibodies or antigen-binding fragments and uses thereof.

Description

ANTI-CORONAVIRUS ANTIBODIES AND USES THEREOF
CLAIM OF PRIORITY
This application claims the benefit of PCT Application No. PCT/CN2020/095959, filed on June 12, 2020. The entire contents of the foregoing are incorporated herein by reference.
TECHNICAL FIELD
This disclosure relates to anti-coronavirus antibodies or antigen-binding fragments and uses thereof.
BACKGROUND
Coronaviruses are a group of related RNA viruses that cause diseases in mammals and birds. In humans, these viruses cause respiratory tract infections that can range from mild to lethal. Mild illnesses include some cases of the common cold (which is also caused by other viruses, predominantly rhinoviruses) , while more lethal varieties can cause SARS, MERS, and COVID-19.
The high rate of infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) worldwide led the World Health Organization to declare COVID-19 a pandemic. As of June 2020, more than 7 million cases have been reported across 188 countries and territories, resulting in more than 400,000 deaths. At this moment, there are no vaccines nor effective antiviral treatments for COVID-19. Management of the disease is limited to the treatment of symptoms, supportive care, isolation, and experimental measures. There is an urgent need for diagnostic assays and therapeutics against SARS-CoV-2.
SUMMARY
This disclosure relates to anti-coronavirus S protein antibodies, antigen-binding fragment thereof, and the uses thereof.
In one aspect, provided herein is an antibody or antigen-binding fragment thereof that binds to a coronavirus spike protein, comprising: a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising  CDRs  1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR3 amino acid sequence,
In some embodiments, the selected  VH CDRs  1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are one of the following:
(1) the selected  VH CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 2, 3, respectively, and the selected  VL CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, 6, respectively;
(2) the selected  VH CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 7, 8, 9, respectively, and the selected  VL CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10, 11, 12, respectively;
(3) the selected  VH CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 13, 14, 15, respectively, and the selected  VL CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, 18, respectively;
(4) the selected  VH CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 19, 20, 21, respectively, and the selected  VL CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22, 23, 24, respectively;
(5) the selected  VH CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 25, 26, 27 respectively, and the selected  VL CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 28, 29, 30, respectively;
(6) the selected  VH CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 31, 32, 33, respectively, and the selected  VL CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 34, 35, 36, respectively;
(7) the selected  VH CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 37, 38, 39, respectively, and the selected  VL CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 40, 41, 42, respectively;
(8) the selected  VH CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 43, 44, 45, respectively, and the selected  VL CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47, 48, respectively;
(9) the selected  VH CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 49, 50, 51, respectively, and the selected  VL CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 52, 53, 54, respectively;
(10) the selected  VH CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 55, 56, 57, respectively, and the selected  VL CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 58, 59, 60, respectively;
(11) the selected  VH CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 61, 62, 63, respectively, and the selected  VL CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 64, 65, 66, respectively;
(12) the selected  VH CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 67, 68, 69, respectively, and the selected  VL CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 70, 71, 72, respectively;
(13) the selected  VH CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 73, 74, 75, respectively, and the selected  VL CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 76, 77, 78, respectively;
(14) the selected  VH CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 79, 80, 81, respectively, and the selected  VL CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 82, 83, 84, respectively;
(15) the selected  VH CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 85, 86, 87, respectively, and the selected  VL CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 88, 89, 90, respectively;
(16) the selected  VH CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 91, 92, 93, respectively, and the selected  VL CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 94, 95, 96, respectively;
(17) the selected  VH CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 97, 98, 99, respectively, and the selected  VL CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 100, 101, 102, respectively;
(18) the selected  VH CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 103, 104, 105, respectively, and the selected  VL CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 106, 107, 108, respectively;
(19) the selected  VH CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 109, 110, 111, respectively, and the selected  VL CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 112, 113, 114, respectively;
(20) the selected  VH CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 115, 116, 117, respectively, and the selected  VL CDRs  1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 118, 119, 120, respectively.
In some embodiments, the VH comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3 respectively, and the VL comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively according to Kabat numbering scheme.
In some embodiments, the VH comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, and the VL comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively according to Kabat numbering scheme.
In some embodiments, the VH comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively, and the VL comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively according to Kabat numbering scheme.
In some embodiments, the VH comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively, and the VL comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively according to Kabat numbering scheme.
In some embodiments, the VH comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 25, 26, and 27, respectively, and the VL comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively according to Kabat numbering scheme.
In some embodiments, the VH comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively, and the VL comprises  CDRs  1, 2, 3 with the  amino acid sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively according to Kabat numbering scheme.
In some embodiments, the VH comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 37, 38, and 39, respectively, and the VL comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 40, 41, and 42, respectively according to Kabat numbering scheme.
In some embodiments, the VH comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 43, 44, and 45, respectively, and the VL comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 46, 47, and 48, respectively according to Kabat numbering scheme.
In some embodiments, the VH comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively, and the VL comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 52, 53, and 54, respectively according to Kabat numbering scheme.
In some embodiments, the VH comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 55, 56, and 57, respectively, and the VL comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 58, 59, and 60, respectively according to Kabat numbering scheme.
In some embodiments, the VH comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 61, 62, and 63, respectively, and the VL comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 64, 65, and 66, respectively according to Chothia numbering scheme.
In some embodiments, the VH comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 67, 68, and 69, respectively, and the VL comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 70, 71, and 72, respectively according to Chothia numbering scheme.
In some embodiments, the VH comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 73, 74, and 75, respectively, and the VL comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 76, 77, and 78, respectively according to Chothia numbering scheme.
In some embodiments, the VH comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 79, 80, and 81, respectively, and the VL comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 82, 83, and 84, respectively according to Chothia numbering scheme.
In some embodiments, the VH comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 85, 86, and 87, respectively, and the VL comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 88, 89, and 90, respectively according to Chothia numbering scheme.
In some embodiments, the VH comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 91, 92, and 93, respectively, and the VL comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 94, 95, and 96, respectively according to Chothia numbering scheme.
In some embodiments, the VH comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 97, 98, and 99, respectively, and the VL comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 100, 101, and 102, respectively according to Chothia numbering scheme.
In some embodiments, the VH comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 103, 104, and 105, respectively, and the VL comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 106, 107, and 108, respectively according to Chothia numbering scheme.
In some embodiments, the VH comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 109, 110, and 111, respectively, and the VL comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 112, 113, and 114, respectively according to Chothia numbering scheme.
In some embodiments, the VH comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 115, 116, and 117, respectively, and the VL comprises  CDRs  1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 118, 119, and 120, respectively according to Chothia numbering scheme.
In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to a human coronavirus spike protein.
In some embodiments, the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof (e.g., a human IgG1 antibody) .
In some embodiments, the antibody or antigen-binding fragment thereof comprises a mouse constant domain. In some embodiments, the antibody or antigen-binding fragment thereof comprises a human constant domain. In some embodiments, the human constant domain comprises LALA mutations.
In one aspect, provided herein is a nucleic acid comprising a polynucleotide encoding a polypeptide comprising:
(1) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively; and in some embodiments, the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 122 binds to a coronavirus spike protein;
(2) an immunoglobulin light chain or a fragment thereof comprising a light chain variable region (VL) comprising  CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively; and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 121 binds to the coronavirus spike protein;
(3) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising  CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively; and in some embodiments, the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 124 binds to the coronavirus spike protein;
(4) an immunoglobulin light chain or a fragment thereof comprising a  VL comprising CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively; and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 123 binds to the coronavirus spike protein;
(5) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising  CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively; and in some embodiments, the VH, when  paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 126 binds to the coronavirus spike protein;
(6) an immunoglobulin light chain or a fragment thereof comprising a  VL comprising CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively, and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 125 binds to the coronavirus spike protein;
(7) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising  CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively, and in some embodiments, the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 128 binds to the coronavirus spike protein;
(8) an immunoglobulin light chain or a fragment thereof comprising a  VL comprising CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively, and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 127 binds to the coronavirus spike protein;
(9) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising  CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 25, 26, and 27, respectively, and in some embodiments, the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 130 binds to the coronavirus spike protein;
(10) an immunoglobulin light chain or a fragment thereof comprising a  VL comprising CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively, and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 129 binds to the coronavirus spike protein;
(11) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising  CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively, and in some embodiments, the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 132 binds to the coronavirus spike protein;
(12) an immunoglobulin light chain or a fragment thereof comprising a  VL comprising CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 34, 35, and 36,  respectively, and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 131 binds to the coronavirus spike protein;
(13) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising  CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 37, 38, and 39, respectively, and in some embodiments, the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 134 binds to the coronavirus spike protein;
(14) an immunoglobulin light chain or a fragment thereof comprising a  VL comprising CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 40, 41, and 42, respectively, and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 133 binds to the coronavirus spike protein;
(15) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising  CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 43, 44, and 45, respectively, and in some embodiments, the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 136 binds to the coronavirus spike protein;
(16) an immunoglobulin light chain or a fragment thereof comprising a  VL comprising CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 46, 47, and 48, respectively, and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 135 binds to the coronavirus spike protein;
(17) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising  CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively, and in some embodiments, the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 138 binds to the coronavirus spike protein;
(18) an immunoglobulin light chain or a fragment thereof comprising a  VL comprising CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 52, 53, and 54, respectively, and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 137 binds to the coronavirus spike protein;
(19) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising  CDRs  1, 2, and 3 comprising the amino acid sequences set  forth in SEQ ID NOs: 55, 56, and 57, respectively, and in some embodiments, the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 140 binds to the coronavirus spike protein; or
(20) an immunoglobulin light chain or a fragment thereof comprising a  VL comprising CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 58, 59, and 60, respectively, and in some embodiments, the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 139 binds to the coronavirus spike protein.
In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a  VH comprising CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively.
In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a  VL comprising CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively.
In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a  VH comprising CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively.
In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a  VL comprising CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively.
In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a  VH comprising CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively.
In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a  VL comprising CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively.
In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a  VH comprising CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively.
In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a  VL comprising CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively.
In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a  VH comprising CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 25, 26, and 27, respectively.
In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a  VL comprising CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively.
In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a  VH comprising CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively.
In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a  VL comprising CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively.
In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a  VH comprising CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 37, 38, and 39, respectively.
In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a  VL  comprising CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 40, 41, and 42, respectively.
In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a  VH comprising CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 43, 44, and 45, respectively.
In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a  VL comprising CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 46, 47, and 48, respectively.
In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a  VH comprising CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively.
In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a  VL comprising CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 52, 53, and 54, respectively.
In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a  VH comprising CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 55, 56, and 57, respectively.
In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a  VL comprising CDRs  1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 58, 59, and 60, respectively.
In some embodiments, the VH when paired with a VL specifically binds to the coronavirus spike protein, or the VL when paired with a VH specifically binds to the coronavirus spike protein.
In some embodiments, the immunoglobulin heavy chain or the fragment thereof is a human immunoglobulin heavy chain or a fragment thereof, and the immunoglobulin light chain or the fragment thereof is a human immunoglobulin light chain or a fragment thereof.
In some embodiments, the nucleic acid is cDNA.
In one aspect, provided herein is a vector comprising one or more of the nucleic acids as described herein.
In one aspect, provided herein is a vector comprising two of the nucleic acids as described herein. In some embodiments, the vector encodes the VH region and the VL region that together bind to the coronavirus spike protein.
In one aspect, provided herein is a pair of vectors. In some embodiments, each vector comprises one of the nucleic acids as described herein. In some embodiments, together the pair of vectors encodes the VH region and the VL region that together bind to the coronavirus spike protein.
In one aspect, provided herein is a cell comprising the vector or the pair of vectors as described herein.
In some embodiments, the cell is a CHO cell.
In one aspect, provided herein is a cell comprising one or more of the nucleic acids as described herein.
In one aspect, provided herein is a cell comprising two of the nucleic acids as described herein.
In some embodiments, the two nucleic acids together encode the VH region and the VL region that together bind to the coronavirus spike protein.
In one aspect, provided herein is an antibody or antigen-binding fragment thereof that binds to a coronavirus spike protein comprising a heavy chain variable region (VH) comprising an amino acid sequence that is at least 80%identical to a selected VH sequence, and a light chain variable region (VL) comprising an amino acid sequence that is at least 80%identical to a selected VL sequence.
In some embodiments, the selected VH sequence and the selected VL sequence are one of the following:
(1) the selected VH sequence is SEQ ID NO: 121, and the selected VL sequence is SEQ ID NO: 122;
(2) the selected VH sequence is SEQ ID NO: 123, and the selected VL sequence is SEQ ID NO: 124;
(3) the selected VH sequence is SEQ ID NO: 125, and the selected VL sequence is SEQ ID NO: 126;
(4) the selected VH sequence is SEQ ID NO: 127, and the selected VL sequence is SEQ ID NO: 128;
(5) the selected VH sequence is SEQ ID NO: 129, and the selected VL sequence is SEQ ID NO: 130;
(6) the selected VH sequence is SEQ ID NO: 131, and the selected VL sequence is SEQ ID NO: 132;
(7) the selected VH sequence is SEQ ID NO: 133, and the selected VL sequence is SEQ ID NO: 134;
(8) the selected VH sequence is SEQ ID NO: 135, and the selected VL sequence is SEQ ID NO: 136;
(9) the selected VH sequence is SEQ ID NO: 137, and the selected VL sequence is SEQ ID NO: 138;
(10) the selected VH sequence is SEQ ID NO: 139, and the selected VL sequence is SEQ ID NO: 140.
In some embodiments, the VH comprises the sequence of SEQ ID NO: 121 and the VL comprises the sequence of SEQ ID NO: 122.
In some embodiments, the VH comprises the sequence of SEQ ID NO: 123 and the VL comprises the sequence of SEQ ID NO: 124.
In some embodiments, the VH comprises the sequence of SEQ ID NO: 125 and the VL comprises the sequence of SEQ ID NO: 126.
In some embodiments, the VH comprises the sequence of SEQ ID NO: 127 and the VL comprises the sequence of SEQ ID NO: 128.
In some embodiments, the VH comprises the sequence of SEQ ID NO: 129 and the VL comprises the sequence of SEQ ID NO: 130.
In some embodiments, the VH comprises the sequence of SEQ ID NO: 131 and the VL comprises the sequence of SEQ ID NO: 132.
In some embodiments, the VH comprises the sequence of SEQ ID NO: 133 and the VL comprises the sequence of SEQ ID NO: 134.
In some embodiments, the VH comprises the sequence of SEQ ID NO: 135 and the VL comprises the sequence of SEQ ID NO: 136.
In some embodiments, the VH comprises the sequence of SEQ ID NO: 137 and the VL comprises the sequence of SEQ ID NO: 138.
In some embodiments, the VH comprises the sequence of SEQ ID NO: 139 and the VL comprises the sequence of SEQ ID NO: 140.
In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to a human coronavirus spike protein.
In some embodiments, the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof (e.g., a human IgG1 antibody) .
In some embodiments, the antibody or antigen-binding fragment thereof comprises a mouse constant domain. In some embodiments, the antibody or antigen-binding fragment thereof comprises a human constant domain. In some embodiments, the human constant domain comprises LALA mutations.
In one aspect, provided herein is an antibody or antigen-binding fragment thereof comprising the  VH CDRs  1, 2, 3, and the  VL CDRs  1, 2, 3 of the antibody or antigen-binding fragment thereof as described herein.
In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to an S1 subunit of the coronavirus spike protein.
In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to a receptor binding domain (RBD) of the S1 subunit of the coronavirus spike protein. In some embodiments, the amino acid sequence of the RBD is at least 80%identical to amino acids 319-541 of SEQ ID NO: 141.
In one aspect, provided herein is an antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof as described herein.
In one aspect, provided herein is a method of producing an antibody or an antigen-binding fragment thereof, the method comprising (a) culturing the cell as described herein under conditions sufficient for the cell to produce the antibody or the antigen-binding fragment thereof; and (b) collecting the antibody or the antigen-binding fragment thereof produced by the cell.
In one aspect, provided herein is a method of treating a subject having a coronavirus-related disease, the method comprising administering a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof as described herein to the subject.
In one aspect, provided herein is a method of neutralizing a coronavirus, the method comprising contacting the coronavirus with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof as described herein.
In one aspect, provided herein is a method of blocking internalization of a coronavirus by a cell, the method comprising contacting the coronavirus with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof as described herein.
In one aspect, provided herein is a method of identifying a subject as having a coronavirus disease, the method comprisingdetecting a sample collected from the subject as having the coronavirus by the antibody or antigen-binding fragment thereof as described herein, thereby identifying the subject as having a coronavirus infection. In some embodiments, the sample is a blood sample, a saliva sample, a stool sample, or a liquid sample from the respiratory tract of the subject.
In some embodiments, the coronavirus is SARS-CoV-2. In some embodiments, the coronavirus is MERS-CoV. In some embodiments, the coronavirus is SARS-CoV.
In one aspect, provided herein is a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described herein, and a pharmaceutically acceptable carrier.
In one aspect, provided herein is a method of treating a subject having a coronavirus-related disease or neutralizing coronavirus in the subject, the method comprising administering to the subject a therapeutically effective amount of a first antibody or antigen-binding fragment thereof, and a therapeutically effective amount of a second antibody or antigen-binding fragment thereof. In some embodiments, the first antibody or antigen-binding fragment thereof is the antibody or antigen-binding fragment thereof as described herein.
In some embodiments, the second antibody or antigen-binding fragment thereof is an anti-S protein antibody or antigen-binding fragment thereof.
In some embodiments, the method further comprises administering a third antibody or antigen-binding fragment thereof to the subject.
In some embodiments, the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof target different epitopes of an S protein.
In some embodiments, the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof are selected from FIG. 16.
In some embodiments, the first antibody or antigen-binding fragment thereof comprises  VH CDRs  1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 37, 38, 39, respectively, and  VL CDRs  1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 40, 41, 42, respectively; and the second antibody or antigen-binding fragment thereof comprises  VH CDRs  1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 31, 32, 33, respectively, and  VL CDRs  1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 34, 35, 36, respectively.
In some embodiments, the first antibody or antigen-binding fragment thereof comprises  VH CDRs  1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 13, 14, 15, respectively, and  VL CDRs  1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 16, 17, 18, respectively; and the second antibody or antigen-binding fragment thereof comprises  VH CDRs  1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 49, 50, 51, respectively, and  VL CDRs  1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 52, 53, 54, respectively.
In one aspect, provided herein is a pharmaceutical composition comprising two or more antibodies or antigen-binding fragment thereof. In some embodiments, one of the two or more antibodies or antigen-binding fragment thereof is the antibody or antigen-binding fragment thereof as described herein.
In some embodiments, the pharmaceutical composition further comprises a third antibody or antigen-binding fragment thereof.
In some embodiments, each antibody or antigen-binding fragment thereof targets different epitopes of an S protein.
In some embodiments, two antibodies or antigen-binding fragments thereof are selected from FIG. 16.
In some embodiments, a first antibody or antigen-binding fragment thereof comprises  VH CDRs  1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 37, 38, 39, respectively, and  VL CDRs  1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 40, 41, 42, respectively; and a second antibody or antigen-binding fragment thereof comprises  VH CDRs  1,  2, 3 amino acid sequences that are set forth in SEQ ID NOs: 31, 32, 33, respectively, and  VL CDRs  1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 34, 35, 36, respectively.
In some embodiments, a first antibody or antigen-binding fragment thereof comprises  VH CDRs  1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 13, 14, 15, respectively, and  VL CDRs  1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 16, 17, 18, respectively; and a second antibody or antigen-binding fragment thereof comprises  VH CDRs  1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 49, 50, 51, respectively, and  VL CDRs  1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 52, 53, 54, respectively.
In one aspect, provided herein is an anti-coronavirus spike protein antibody or antigen binding fragment thereof comprising: a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3; and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3.
In some embodiments, the VH CDR1 sequence is SX 1X 2X 3X 4X 5X 6 (SEQ ID NO: 142) . In some embodiments, X 1 is N, F, G, Y, or null; X 2 is G, or null; X 3 is Y, or null; X 4 is Y, A, or null; X 5 is M, L, or W; X 6 is S, N, T, A, or H. In some embodiments, the VH CDR2 sequence is X 7IX 8X 9X 10GX 11X 12X 13X 14YX 15X 16SX 17X 18X 19 (SEQ ID NO: 143) . In some embodiments, X 7 is V, S, or Y; X 8 is Y, S, W, or H; X 9 is Y, G, F, or null; X 10 is S, or D; X 11 is G, S, or null; X 12 is S, or N; X 13 is T, or K; X 14 is Y, F, or N; X 15 is A, or N; X 16 is D, or P; X 17 is V, or L; X 18 is K, E, or R; X 19 is G, A, S, or N. In some embodiments, the VH CDR3 sequence is X 20X 21X 22X 23X 24X 25X 26X 27X 28X 29X 30X 31DX 32 (SEQ ID NO: 144) . In some embodiments, X 20 is D, E, or Q; X 21 is R, L, V, T, or A; X 22 is G, or null; X 23 is Y, or null; X 24 is S, V, L, or null; X 25 is S, G, D, or null; X 26 is D, S, P, K, N, G, or null; X 27 is Y, N, V, L, W, or null; X 28 is N, S, L, T, or null; X 29 is Y, S, F, G, or D; X 30 is G, N, F, or S; X 31 is M, or F; X 32 is V, Y, or I.
In some embodiments, X 1 is N, X 2 is null, X 3 is null, X 4 is Y, X 5 is M, and X 6 is S. In some embodiments, X 7 is V, X 8 is Y, X 9 is Y, X 10 is S, X 11 is G, X 12 is S, X 13 is T, X 14 is Y, X 15 is A, X 16 is D, X 17 is V, X 18 is K, and X 19 is G. In some embodiments, X 20 is D, X 21 is R, X 22 is null, X 23 is null, X 24 is null, X 25 is null, X 27 is Y, X 28 is null, X 29 is Y, X 30 is G, X 31 is M, and X 32 is V.
In some embodiments, the VL CDR1 sequence is X 33AX 34QX 35IX 36X 37X 38LX 39 (SEQ ID NO: 145) . In some embodiments, X 33 is Q, or R; X 34 is S, or R; X 35 is D, or G; X 36 is S, N, or T; X 37 is N, I, S, or K; X 38 is Y, or F; X 39 is N, or A. In some embodiments, the VL CDR2 sequence is  X 40ASX 41LX 42X 43 (SEQ ID NO: 146) . In some embodiments, X 40 is D, or A; X 41 is N, T, or S; X 42 is E, Q, or L; X 43 is T, or S. In some embodiments, the VL CDR3 sequence is X 44X 45X 46X 47X 48X 49X 50X 51X 52T (SEQ ID NO: 147) . In some embodiments, X 44 is Q, or L; X 45 is Q, or H; X 46 is Y, L, or H; X 47 is D, H, or N; X 48 is N, H, or S; X 49 is L, I, or Y; X 50 is P, or L; X 51 is R, M, L, P, or null; X 52 is W, Y, F, L, or null.
In some embodiments, X 33 is Q, X 34 is S, X 35 is D, X 36 is S, X 37 is N, X 38 is Y, and X 39 is N. In some embodiments, X 40 is D, X 41 is N, X 42 is E, X 43 is T. In some embodiments, X 44 is Q, X 45 is Q, X 46 is Y, X 47 is D, X 48 is N, X 49 is L, X 50 is P, X 51 is null, and X 52 is null.
As used herein, the term “antibody” refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementary determining region (CDR) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope. Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bi-specific antibodies) , single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, an antibody can contain an Fc region of a human antibody. The term antibody also includes derivatives, e.g., bi-specific antibodies, single-chain antibodies, diabodies, linear antibodies, and multi-specific antibodies formed from antibody fragments.
As used herein, the term “antigen-binding fragment” refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of a heavy chain or a variable domain of light chain) . Non-limiting examples of antibody fragments include, e.g., Fab, Fab’, F (ab’)  2, and Fv fragments.
As used herein, the term “human antibody” refers to an antibody that is encoded by a nucleic acid (e.g., rearranged human immunoglobulin heavy or light chain locus sequence) derived from a human. In some embodiments, a human antibody is collected from a human or produced in a human cell culture (e.g., human hybridoma cells) . In some embodiments, a human antibody is produced in a non-human cell (e.g., a mouse or hamster cell line) . In some embodiments, a human antibody is produced in a bacterial or yeast cell. In some embodiments, a human antibody is derived from a transgenic non-human animal (e.g., a mouse) containing an  unrearranged or rearranged human immunoglobulin locus (e.g., heavy or light chain human immunoglobulin locus) .
As used herein, the term “chimeric antibody” refers to an antibody that contains a sequence present in at least two different species (e.g., antibodies from two different mammalian species such as a human and a mouse antibody) . A non-limiting example of a chimeric antibody is an antibody containing the variable domain sequences (e.g., all or part of a light chain and/or heavy chain variable domain sequence) of a human antibody and the constant domains of a non-human antibody. Additional examples of chimeric antibodies are described herein and are known in the art.
As used herein, the term “humanized antibody” refers to a non-human antibody which contains minimal sequence derived from a non-human (e.g., mouse) immunoglobulin and contains sequences derived from a human immunoglobulin. In non-limiting examples, humanized antibodies are human antibodies (recipient antibody) in which hypervariable (e.g., CDR) region residues of the recipient antibody are replaced by hypervariable (e.g., CDR) region residues from a non-human antibody (e.g., a donor antibody) , e.g., a mouse, rat, or rabbit antibody, having the desired specificity, affinity, and capacity. In some embodiments, the Fv framework residues of the human immunoglobulin are replaced by corresponding non-human (e.g., mouse) immunoglobulin residues. In some embodiments, humanized antibodies may contain residues which are not found in the recipient antibody or in the donor antibody. These modifications can be made to further refine antibody performance. In some embodiments, the humanized antibody contains substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops (CDRs) correspond to those of a non-human (e.g., mouse) immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin. The humanized antibody can also contain at least a portion of an immunoglobulin constant region (Fc) , typically, that of a human immunoglobulin. In some embodiments, the chimeric antibody has a human heavy chain variable domain and a human light chain variable domain, and mouse constant domains. The humanization can involve replace mouse constant domains with human constant domains, thereby making a full human antibody. Humanized antibodies can be produced using molecular biology methods known in the art. Non-limiting examples of methods for generating humanized antibodies are described herein.
As used herein, the term “single-chain antibody” refers to a single polypeptide that contains at least two immunoglobulin variable domains (e.g., a variable domain of a mammalian immunoglobulin heavy chain or light chain) that is capable of specifically binding to an antigen. Non-limiting examples of single-chain antibodies are described herein.
As used herein, the term “multimeric antibody” refers to an antibody that contains four or more (e.g., six, eight, or ten) immunoglobulin variable domains. In some embodiments, the multimeric antibody is able to crosslink one target molecule (e.g., S protein) to at least one second target molecule (e.g., CD3) on the surface of a mammalian cell (e.g., a human T-cell) .
As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present invention is provided. Veterinary and non-veterinary applications are contemplated in the present disclosure. Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old) . In addition to humans, patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, non-human primates (e.g., monkey, chimpanzee, gorilla, and the like) , rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits) , lagomorphs, swine (e.g., pig, miniature pig) , equine, canine, feline, bovine, and other domestic, farm, and zoo animals.
As used herein, when referring to an antibody, the phrases “specifically binding” and “specifically binds” mean that the antibody interacts with its target molecule preferably to other molecules, because the interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) on the target molecule; in other words, the reagent is recognizing and binding to molecules that include a specific structure rather than to all molecules in general. An antibody that specifically binds to the target molecule may be referred to as a target-specific antibody. For example, an antibody that specifically binds to a S protein molecule may be referred to as a S protein-specific antibody or an anti-S protein antibody.
As used herein, the terms “polypeptide, ” “peptide, ” and “protein” are used interchangeably to refer to polymers of amino acids of any length of at least two amino acids.
As used herein, the terms “polynucleotide, ” “nucleic acid molecule, ” and “nucleic acid sequence” are used interchangeably herein to refer to polymers of nucleotides of any length of at least two nucleotides, and include, without limitation, DNA, RNA, DNA/RNA hybrids, and modifications thereof.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
DESCRIPTION OF DRAWINGS
FIG. 1 is a set of flow cytometry results showing the blocking effects of anti-SARS-CoV-2 antibodies in hybridoma supernatant. PC1 is positive control. NC1 and NC2 are  negative control  1 and 2, respectively.
FIG. 2 shows antigen-binding curves of 09-4E5-IgG1-LALA at 0.78125nM, 1.5625nM, 3.125 nM, 6.25 nM, 12.5 nM, 25 nM and 50 nM, 100nM.
FIG. 3 shows fitting curves of 03-9A8-IgG1-LALA and 09-7B8-IgG1-LALA.
FIG. 4 is a matrix table showing binding ratios between analyte 1 and analyte 2. The antibody numbers 1-10 are purified human anti-SARS-CoV-2 antibodies (1) 01-2H10-IgG1-LALA, (2) 03-10D12-IgG1-LALA, (3) 03-10F9-IgG1-LALA, (4) 03-1F9-IgG1-LALA, (5) 05-8G6-IgG1-LALA, (6) 05-9G11-IgG1-LALA, (7) 09-2F7-IgG1-LALA, (8) 09-4E5-IgG1-LALA, (9) 09-7B8-IgG1-LALA, and (10) 03-9A8-IgG1-LALA, respectively.
FIG. 5 shows epitope correlation of the human anti-SARS-CoV-2 antibodies 01-2H10-IgG1-LALA (01-2H10) , 03-10F9-IgG1-LALA (03-10F9) , 03-10D12-IgG1-LALA (03-10D12) , 03-1F9-IgG1-LALA (03-1F9) , 03-9A8-IgG1-LALA (03-9A8) , 09-2F7-IgG1-LALA (09-2F7) , 09-4E5-IgG1-LALA (09-4E5) , 09-7B8-IgG1-LALA (09-7B8) , 05-8G6-IgG1-LALA (05-8G6) , and 05-9G11-IgG1-LALA (05-9G11) .
FIG. 6 shows epitope clusters of the human anti-SARS-CoV-2 antibodies 01-2H10-IgG1-LALA (2H10) , 03-10F9-IgG1-LALA (10F9) , 03-10D12-IgG1-LALA (10D12) , 03-1F9-IgG1-LALA (1F9) , 03-9A8-IgG1-LALA (9A8) , 09-2F7-IgG1-LALA (2F7) , 09-4E5-IgG1-LALA  (4E5) , 09-7B8-IgG1-LALA (7B8) , 05-8G6-IgG1-LALA (8G6) , and 05-9G11-IgG1-LALA (9G11) .
FIGS. 7A-7B show a set of binding curves of captured 2019-nCoV S protein RBD by ACE2 alone, or a human anti-SARS-CoV-2 antibody followed by ACE2.
FIGS. 8A-8E show a set of binding curves of 03-9A8-IgG1-LALA (Ab. 01) , 03-10D12-IgG1-LALA (Ab. 02) , 05-9G11-IgG1-LALA (Ab. 03) , 09-4E5-IgG1-LALA (Ab. 04) , and 09-7B8-IgG-LALA (Ab. 05) alone, or in combination.
FIG. 9 shows IC 50 of 03-9A8-IgG1-LALA (Ab. 01, or 9A8) , 03-10D12-IgG1-LALA (Ab. 02, or 03-10D12) , 05-9G11-IgG1-LALA (Ab. 03, or 05-9G11) , 09-4E5-IgG1-LALA (Ab. 04, or 09-4E5) , and 09-7B8-IgG-LALA (Ab. 05, or 09-7B8) alone, or in combination.
FIG. 10 shows COVID-10 pseudovirus neutralizing activity curves of 03-10D12-IgG1-LALA (Ab1) , 03-9A8-IgG1-LALA (Ab2) , 09-4E5-IgG1-LALA (Ab3) , 09-7B8-IgG-LALA (Ab4) , 05-9G11-IgG1-LALA (Ab5) , and Ab1 + Ab5 combination.
FIG. 11A shows CDR1 consensus sequence of the heavy chain variable region (VH) of anti-SARS-CoV-2 antibodies.
FIG. 11B shows CDR2 consensus sequence of the VH of anti-SARS-CoV-2 antibodies.
FIG. 11C shows CDR3 consensus sequence of the VH of anti-SARS-CoV-2 antibodies.
FIG. 12A shows CDR1 consensus sequence of the light chain variable region (VL) of anti-SARS-CoV-2 antibodies.
FIG. 12B shows CDR2 consensus sequence of the VL of anti-SARS-CoV-2 antibodies.
FIG. 12C shows CDR3 consensus sequence of the VL of anti-SARS-CoV-2 antibodies.
FIG. 13 lists CDR sequences of several anti-SARS-CoV-2 antibodies as defined by Kabat numbering.
FIG. 14 lists CDR sequences of several anti-SARS-CoV-2 antibodies as defined by Chothia numbering.
FIG. 15 lists sequences that are described in the present disclosure.
FIG. 16 lists different pairs of antibodies or antigen binding fragments thereof.
DETAILED DESCRIPTION
Coronavirus uses its spike glycoprotein (S) , a main target for neutralization antibody, to bind its receptor, and mediate membrane fusion and virus entry. Each monomer of trimeric S  protein is about 180 kDa, and contains two subunits, S1 and S2, mediating attachment and membrane fusion, respectively. The SARS-CoV-2 virion consists of a helical capsid formed by nucleocapsid (N) proteins bound to the RNA genome, which is enclosed by membrane (M) proteins, envelope (E) proteins and trimeric spike (S) proteins that render them their “corona-like” appearance (See Zhou P, Yang XL, Wang XG, et al., A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020 Mar; 579 (7798) : 270-273) . The S protein receptor binding domain (RBD) in the S1 subunit binds to the angiotensin converting enzyme (Angiotensin I Converting Enzyme 2; ACE2) on the cell membranes of type 2 pneumocytes and intestinal epithelial cells. Following binding, the S protein is cleaved by host cell transmembrane serine protease 2 (TMPRSS2) , which facilitates subsequent viral entry into host cell (See Hoffmann M, Kleine-Weber H, Schroeder S, et al., SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell. 2020 Mar 4) .
The present disclosure provides antibodies, antigen-binding fragments thereof that specifically bind to a coronavirus (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV) S protein. These antibodies or antigen-binding fragments thereof are high titer neutralizing antibodies or antigen-binding fragments thereof. Therapeutic antibodies can neutralize viral infections via two mechanisms of action, e.g., Fc-independent functions that block capsid/host receptor interaction, and induce virus aggregation, and/or Fc-dependent functions that cause Fc-FcR interaction to activate immune cells leading to killing of virus.
The disclosure also provides methods of treating COVID-19 using the antibodies or antigen-binding fragments thereof as described herein, and methods of diagnosing COVID-19 using the antibodies or antigen-binding fragments thereof as described herein.
Anti-Coronavirus Spike Protein Antibodies and Antigen-Binding Fragments
The disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to coronavirus spike proteins. The antibodies and antigen-binding fragments described herein are capable of binding to coronavirus spike proteins. In some embodiments, these antibodies can block human ACE2 binding to the coronavirus spike proteins. In some embodiments, these antibodies can neutralize the coronavirus.
The disclosure provides e.g., anti-coronavirus spike protein antibodies 01-2H10, 03-1F9, 03-9A8, 03-10F9, 05-8G6, 05-9G11, 03-10D12, 09-2F7, 09-7B8, and 09-4E5, and any  antibodies derived therefrom (e.g., the chimeric antibodies, the humanized antibodies, andthe full human antibodies) .
The CDR sequences for 01-2H10, and 01-2H10 derived antibodies (e.g., humanized antibodies and full human antibodies) include CDRs of the heavy chain variable domain, SEQ ID NOs: 1, 2, 3, and CDRs of the light chain variable domain, SEQ ID NOs: 4, 5, 6 as defined by Kabat numbering. The CDRs can also be defined by Chothia system. Under the Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 61, 62, 63, and CDR sequences of the light chain variable domain are set forth in SEQ ID NOs: 64, 65, 66.
The CDR sequences for 03-1F9, and 03-1F9 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 7, 8, 9, and CDRs of the light chain variable domain, SEQ ID NOs: 10, 11, 12, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 67, 68, 69, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 70, 71, 72.
The CDR sequences for 03-9A8, and 03-9A8 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 13, 14, 15, and CDRs of the light chain variable domain, SEQ ID NOs: 16, 17, 18, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 73, 74, 75, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 76, 77, 78.
The CDR sequences for 03-10F9, and 03-10F9 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 19, 20, 21, and CDRs of the light chain variable domain, SEQ ID NOs: 22, 23, 24, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 79, 80, 81, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 82, 83, 84.
The CDR sequences for 05-8G6, and 05-8G6 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 25, 26, 27, and CDRs of the light chain variable domain, SEQ ID NOs: 28, 29, 30, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 85, 86, 87, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 88, 89, 90.
The CDR sequences for 05-9G11, and 05-9G11 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 31, 32, 33, and CDRs of the light chain variable  domain, SEQ ID NOs: 34, 35, 36, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 91, 92, 93, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 94, 95, 96.
The CDR sequences for 03-10D12, and 03-10D12 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 37, 38, 39, and CDRs of the light chain variable domain, SEQ ID NOs: 40, 41, 42, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 97, 98, 99, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 100, 101, 102.
The CDR sequences for 09-2F7, and 09-2F7 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 43, 44, 45, and CDRs of the light chain variable domain, SEQ ID NOs: 46, 47, 48, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 103, 104, 105, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 106, 107, 108.
The CDR sequences for 09-7B8, and 09-7B8 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 49, 50, 51, and CDRs of the light chain variable domain, SEQ ID NOs: 52, 53, 54, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 109, 110, 111, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 112, 113, 114.
The CDR sequences for 09-4E5, and 09-4E5 derived antibodies include CDRs of the heavy chain variable domain, SEQ ID NOs: 55, 56, 57, and CDRs of the light chain variable domain, SEQ ID NOs: 58, 59, 60, as defined by Kabat numbering. Under Chothia numbering, the CDR sequences of the heavy chain variable domain are set forth in SEQ ID NOs: 115, 116, 117, and CDRs of the light chain variable domain are set forth in SEQ ID NOs: 118, 119, 120.
The amino acid sequence for the heavy chain variable region of 01-2H10 antibody is set forth in SEQ ID NO: 121. The amino acid sequence for the light chain variable region of 01-2H10 antibody is set forth in SEQ ID NO: 122. The amino acid sequence for the heavy chain variable region of 03-1F9 antibody is set forth in SEQ ID NO: 123. The amino acid sequence for the light chain variable region of 03-1F9 antibody is set forth in SEQ ID NO: 124. The amino acid sequence for the heavy chain variable region of 03-9A8 antibody is set forth in SEQ ID NO: 125. The amino acid sequence for the light chain variable region of 03-9A8 antibody is set forth in SEQ ID NO: 126. The amino acid sequence for the heavy chain variable region of 03-10F9  antibody is set forth in SEQ ID NO: 127. The amino acid sequence for the light chain variable region of 03-10F9 antibody is set forth in SEQ ID NO: 128. The amino acid sequence for the heavy chain variable region of 05-8G6 antibody is set forth in SEQ ID NO: 129. The amino acid sequence for the light chain variable region of 05-8G6 antibody is set forth in SEQ ID NO: 130. The amino acid sequence for the heavy chain variable region of 05-9G11 antibody is set forth in SEQ ID NO: 131. The amino acid sequence for the light chain variable region of 05-9G11 antibody is set forth in SEQ ID NO: 132. The amino acid sequence for the heavy chain variable region of 03-10D12 antibody is set forth in SEQ ID NO: 133. The amino acid sequence for the light chain variable region of 03-10D12 antibody is set forth in SEQ ID NO: 134. The amino acid sequence for the heavy chain variable region of 09-2F7 antibody is set forth in SEQ ID NO: 135. The amino acid sequence for the light chain variable region of 09-2F7 antibody is set forth in SEQ ID NO: 136. The amino acid sequence for the heavy chain variable region of 09-7B8 antibody is set forth in SEQ ID NO: 137. The amino acid sequence for the light chain variable region of 09-7B8 antibody is set forth in SEQ ID NO: 138. The amino acid sequence for the heavy chain variable region of 09-4E5 antibody is set forth in SEQ ID NO: 139. The amino acid sequence for the light chain variable region of 09-4E5 antibody is set forth in SEQ ID NO: 140.
As there are different ways to modify an antibody, the heavy chain and the light chain of an antibody can have one or more mutations. In some embodiments, the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 121, 123, 125, 127, 129, 131, 133, 135, 137, or 139. In some embodiments, the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 122, 124, 126, 128, 130, 132, 134, 136, 138, or 140. The heavy chain variable region sequence can be paired with the corresponding light chain variable region sequence, and together they bind to the coronavirus spike protein described herein. In some embodiments, the heavy chain variable region and the light chain variable region are completely obtained from a human sequence (e.g., human immunoglobulin heavy chain locus sequence and/or human immunoglobulin kappa chain locus sequence) .
Furthermore, in some embodiments, the antibodies or antigen-binding fragments thereof described herein can also contain one, two, or three heavy chain variable region CDRs selected from the group of SEQ ID NOs: 1-3, SEQ ID NOs: 7-9, SEQ ID NOs: 13-15, SEQ ID NOs: 19- 21, SEQ ID NOs: 25-27, SEQ ID NOs: 31-33, SEQ ID NOs: 37-39, SEQ ID NOs: 43-45, SEQ ID NOs: 49-51, and SEQ ID NOs: 55-57; and/or one, two, or three light chain variable region CDRs selected from the group of SEQ ID NOs: 4-6, SEQ ID NOs: 10-12, SEQ ID NOs: 16-18, SEQ ID NOs: 22-24, SEQ ID NOs: 28-30, SEQ ID NOs: 34-36, SEQ ID NOs: 40-42, SEQ ID NOs: 46-48, SEQ ID NOs: 52-54, and SEQ ID NOs: 58-60.
In some embodiments, the antibodies can have a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH CDR3 amino acid sequence. In some embodiments, the antibodies can have a light chain variable region (VL) comprising  CDRs  1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL CDR3 amino acid sequence. The selected  VH CDRs  1, 2, 3 amino acid sequences and the selected VL CDRs, 1, 2, 3 amino acid sequences are shown in FIG. 13 (Kabat CDR) and FIG. 14 (Chothia CDR) .
In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 1 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 2 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 3 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 7 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 8 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 9 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 13 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 14 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 15 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 19 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 20 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 21 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 25 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 26 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 27 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 31 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 32 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 33 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 37 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 38 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 39 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 43 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 44 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 45 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 49 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 50 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 51 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the antibody or an antigen-binding fragment described herein can contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 55 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 56 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 57 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 4 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 5 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 6 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 10 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 11 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 12 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 16 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 17 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 18 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 22 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 23 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 24 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 28 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 29 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 30 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 34 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 35 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 36 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 40 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 41 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 42 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 46 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 47 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 48 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 52 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 53 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 54 with zero, one or two amino acid insertions, deletions, or substitutions.
In some embodiments, the antibody or an antigen-binding fragment described herein can contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 58 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 59 with zero, one or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 60 with zero, one or two amino acid insertions, deletions, or substitutions.
The insertions, deletions, and substitutions can be within the CDR sequence, or at one or both terminal ends of the CDR sequence. In some embodiments, the CDR is determined based on Kabat numbering scheme. In some embodiments, the CDR is determined based on Chothia numbering scheme.
The disclosure also provides antibodies or antigen-binding fragments thereof that bind to coronavirus spike proteins. The antibodies or antigen-binding fragments thereof contain a heavy chain variable region (VH) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VH sequence, and a light chain variable region (VL) comprising or consisting of an amino acid sequence that is at least 80%, 85%, 90%, or 95%identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 121, and the selected VL sequence is SEQ ID NO: 122. In some embodiments, the selected VH sequence is SEQ ID NO: 123 and the selected VL sequence is SEQ ID NO: 124. In some embodiments, the selected VH sequence is SEQ ID NO: 125 and the selected VL sequence is SEQ ID NO: 126. In some embodiments, the selected VH sequence is SEQ ID NO: 127 and the selected VL sequence is SEQ ID NO: 128. In some embodiments, the selected VH sequence is SEQ ID NO: 129 and the selected VL sequence is SEQ ID NO: 130. In some embodiments, the selected VH sequence is SEQ ID NO: 131 and the selected VL sequence is SEQ ID NO: 132. In some embodiments, the selected VH sequence is SEQ ID NO: 133 and the selected VL sequence is SEQ ID NO: 134. In some embodiments, the selected VH sequence is SEQ ID NO: 135 and the selected VL sequence is SEQ ID NO: 136. In some embodiments, the selected VH sequence is SEQ ID NO: 137 and the selected VL sequence is SEQ ID NO: 138. In some embodiments, the selected VH sequence is SEQ ID NO: 139 and the selected VL sequence is SEQ ID NO: 140.
To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) . The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps,  and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For purposes of illustration, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
The disclosure also provides nucleic acid comprising a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin light chain. The immunoglobulin heavy chain or immunoglobulin light chain comprises CDRs as shown in FIG. 13 or FIG. 14, or have sequences as shown in FIG. 15. When the polypeptides are paired with corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region) , the paired polypeptides bind to the coronavirus spike protein described herein.
The anti-coronavirus spike protein antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments and multi-specific (e.g., bi-specific) antibodies or antibody fragments. Additional antibodies provided herein are polyclonal, monoclonal, multi-specific (multimeric, e.g., bi-specific) , human antibodies, chimeric antibodies (e.g., human-mouse chimera) , single-chain antibodies, intracellularly-made antibodies (i.e., intrabodies) , and antigen-binding fragments thereof. The antibodies or antigen-binding fragments thereof can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) , class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) , or subclass. In some embodiments, the antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof.
Fragments of antibodies are suitable for use in the methods provided so long as they retain the desired affinity and specificity of the full-length antibody. Thus, a fragment of an antibody that binds to the coronavirus spike protein described herein will retain an ability to bind to the coronavirus spike protein. An Fv fragment is an antibody fragment which contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight association, which can be covalent in nature, for example in scFv. It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs or a subset thereof confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) can have the  ability to recognize and bind antigen, although usually at a lower affinity than the entire binding site. Single-chain Fv or (scFv) antibody fragments comprise the VH and VL domains (or regions) of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.
The present disclosure also provides an antibody or antigen-binding fragment thereof that cross-competes with any antibody or antigen-binding fragment as described herein. The cross-competing assay is known in the art, and is described in the present disclosure (e.g., by testing the binding of two antibodies to Recombinant 2019-nCoV S protein) . In one aspect, the present disclosure also provides an antibody or antigen-binding fragment thereof that binds to the same epitope or region as any antibody or antigen-binding fragment as described herein. The epitope binning assay is known in the art, and is described e.g., in Estep et al., "High throughput solution-based measurement of antibody-antigen affinity and epitope binning. " MAbs. Vol. 5. No. 2. Taylor &Francis, 2013, which is incorporated herein reference in its entirety.
Antibodies and Antigen Binding Fragments
The present disclosure provides various antibodies and antigen-binding fragments thereof derived from anti-Sprotein antibodies described herein. In general, antibodies (also called immunoglobulins) are made up of two classes of polypeptide chains, light chains and heavy chains. A non-limiting examples of antibody of the present disclosure can be an intact, four immunoglobulin chain antibody comprising two heavy chains and two light chains. The heavy chain of the antibody can be of any isotype including IgM, IgG, IgE, IgA, or IgD or sub-isotype including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a kappa light chain or a lambda light chain. An antibody can comprise two identical copies of a light chain and two identical copies of a heavy chain. The heavy chains, which each contain one variable domain (or variable region, V H) and multiple constant domains (or constant regions) , bind to one another via disulfide bonding within their constant domains to form the “stem” of the antibody. The light chains, which each contain one variable domain (or variable region, V L) and one constant domain (or constant region) , each bind to one heavy chain via disulfide binding. The variable region of each light chain is aligned with the variable region of the heavy chain to  which it is bound. The variable regions of both the light chains and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FR) .
These hypervariable regions, known as the complementary determining regions (CDRs) , form loops that comprise the antigen binding surface of the antibody. The four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting the beta-sheet structure, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding region.
Methods for identifying the CDR regions of an antibody by analyzing the amino acid sequence of the antibody are well known, and a number of definitions of the CDRs are commonly used. The Kabat definition is based on sequence variability, and the Chothia definition is based on the location of the structural loop regions. These methods and definitions are described in, e.g., Martin, "Protein sequence and structure analysis of antibody variable domains, " Antibody engineering, Springer Berlin Heidelberg, 2001.422-439; Abhinandan, et al., "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains, " Molecular immunology 45.14 (2008) : 3832-3839; Wu, T.T. and Kabat, E.A. (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203: 121-53 (1991) ; Morea et al., Biophys Chem. 68 (1-3) : 9-16 (Oct. 1997) ; Morea et al., J Mol Biol. 275 (2) : 269-94 (Jan . 1998) ; Chothia et al., Nature 342 (6252) : 877-83 (Dec. 1989) ; Ponomarenko and Bourne, BMC Structural Biology 7: 64 (2007) ; each of which is incorporated herein by reference in its entirety.
The CDRs are important for recognizing an epitope of an antigen. As used herein, an “epitope” is the smallest portion of a target molecule capable of being specifically bound by the antigen binding domain of an antibody. The minimal size of an epitope may be about three, four, five, six, or seven amino acids, but these amino acids need not be in a consecutive linear sequence of the antigen’s primary structure, as the epitope may depend on an antigen’s three-dimensional configuration based on the antigen’s secondary and tertiary structure.
In some embodiments, the antibody is an intact immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA) . The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, differ in their constant region, particularly in their hinges and upper CH2 domains. The sequences and differences of the IgG subclasses are known in the art, and are described, e.g., in Vidarsson, et al, "IgG subclasses and allotypes: from structure to effector  functions. " Frontiers in immunology 5 (2014) ; Irani, et al., "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases. " Molecular immunology 67.2 (2015) : 171-182; Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016; each of which is incorporated herein by reference in its entirety.
The antibody can also be an immunoglobulin molecule that is derived from any species (e.g., human, rodent, mouse, camelid) . Antibodies disclosed herein also include, but are not limited to, polyclonal, monoclonal, monospecific, polyspecific antibodies, and chimeric antibodies that include an immunoglobulin binding domain fused to another polypeptide. The term “antigen binding domain” or “antigen binding fragment” is a portion of an antibody that retains specific binding activity of the intact antibody, i.e., any portion of an antibody that is capable of specific binding to an epitope on the intact antibody’s target molecule. It includes, e.g., Fab, Fab', F (ab') 2, and variants of these fragments. Thus, in some embodiments, an antibody or an antigen binding fragment thereof can be, e.g., a scFv, a Fv, a Fd, a dAb, a bispecific antibody, a bispecific scFv, a diabody, a linear antibody, a single-chain antibody molecule, a multi-specific antibody formed from antibody fragments, and any polypeptide that includes a binding domain which is, or is homologous to, an antibody binding domain. Non-limiting examples of antigen binding domains include, e.g., the heavy chain and/or light chain CDRs of an intact antibody, the heavy and/or light chain variable regions of an intact antibody, full length heavy or light chains of an intact antibody, or an individual CDR from either the heavy chain or the light chain of an intact antibody.
Fragments of antibodies are suitable for use in the methods described herein are also provided. The Fab fragment contains a variable and constant domain of the light chain and a variable domain and the first constant domain (CH1) of the heavy chain. F (ab') 2 antibody fragments comprise a pair of Fab fragments which are generally covalently linked near their carboxy termini by hinge cysteines between them. Other chemical couplings of antibody fragments are also known in the art.
Diabodies are small antibody fragments with two antigen-binding sites, which fragments comprise a VH connected to a VL in the same polypeptide chain (VH and VL) . By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are  forced to pair with the complementary domains of another chain and create two antigen-binding sites.
Linear antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.
Antibodies and antibody fragments of the present disclosure can be modified in the Fc region to provide desired effector functions or serum half-life.
Multimerization of antibodies may be accomplished through natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, some percentage of purified antibody preparations (e.g., purified IgG 1 molecules) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody multimers.
Alternatively, antibody homodimers may be formed through chemical linkage techniques known in the art. For example, heterobifunctional crosslinking agents including, but not limited to SMCC (succinimidyl 4- (maleimidomethyl) cyclohexane-1-carboxylate) and SATA (N-succinimidyl S-acethylthio-acetate) can be used to form antibody multimers. An exemplary protocol for the formation of antibody homodimers is described in Ghetie et al., (Proc. Natl. Acad. Sci. U.S.A. 94: 7509-7514, 1997) . Antibody homodimers can be converted to Fab’ 2 homodimers through digestion with pepsin. Another way to form antibody homodimers is through the use of the autophilic T15 peptide described in Zhao et al., (J. Immunol. 25: 396-404, 2002) .
In some embodiments, the multi-specific antibody is a bi-specific antibody. Bi-specific antibodies can be made by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers that are recovered from recombinant cell culture. For example, the interface can contain at least a part of the CH3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan) . Compensatory “cavities” of identical or similar size to the large side chain (s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine) . This provides a mechanism for increasing the yield of the  heterodimer over other unwanted end-products such as homodimers. This method is described, e.g., in WO 96/27011, which is incorporated by reference in its entirety.
Bi-specific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. Heteroconjugate antibodies can also be made using any convenient cross-linking methods. Suitable cross-linking agents and cross-linking techniques are well known in the art and are disclosed in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.
Methods for generating bi-specific antibodies from antibody fragments are also known in the art. For example, bi-specific antibodies can be prepared using chemical linkage. Brennan et al., (Science 229: 81, 1985) describes a procedure where intact antibodies are proteolytically cleaved to generate F (ab’)  2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab’ fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab’ TNB derivatives is then reconverted to the Fab’ thiol by reduction with mercaptoethylamine, and is mixed with an equimolar amount of another Fab’ TNB derivative to form the bi-specific antibody.
Any of the antibodies or antigen-binding fragments described herein may be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or in solution) . Non-limiting examples of stabilizing molecules include: a polymer (e.g., a polyethylene glycol) or a protein (e.g., serum albumin, such as human serum albumin) . The conjugation of a stabilizing molecule can increase the half-life or extend the biological activity of an antibody or an antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in a human) .
In some embodiments, the antibodies or antigen-binding fragments described herein can be conjugated to a therapeutic agent. The antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof can covalently or non-covalently bind to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent.
In some embodiments, the scFV has one heavy chain variable domain, and one light chain variable domain. In some embodiments, the scFV has two heavy chain variable domains, and two light chain variable domains.
Antibody Characteristics
The antibodies or antigen-binding fragments thereof described herein can block the binding between the coronavirus (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV) S protein and ACE2. In some embodiments, by binding to coronavirus S protein, the antibody can neutralize coronavirus. In some embodiments, the antibody can promote virus aggregation. In some embodiments, the antibody can induce Fc-dependent antiviral functions. In some embodiments, the antibody can inhibit cleavage of the S protein by host cell TMPRSS2. In some embodiments, the antibody can block viral entry into host cell.
The disclosure provides antibodies or antigen-binding fragments thereof that neutralize the coronavirus (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV) such that the neutralized coronavirus is at least or about 5%, at least or about 10%, at least or about 15%, at least or about 20%, at least or about 25%, at least or about 30%, at least or about 35%, at least or about 40%, at least or about 45%, at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, or at least or about 95%of the total amount of the coronavirus.
The disclosure provides antibodies or antigen-binding fragments thereof that promote the coronavirus (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV) aggregation by at least or about 1 fold, at least or about 2 folds, at least or about 3 folds, at least or about 4 folds, at least or about 5 folds, at least or about 6 folds, at least or about 7 folds, at least or about 8 folds, at least or about 9 folds, at least or about 10 folds, at least or about 20 folds, at least or about 30 folds, at least or about 40 folds, at least or about 50 folds, or at least or about 100 folds as compared when no antibodies or antigen-binding fragments thereof as described herein are present.
The disclosure provides antibodies or antigen-binding fragments thereof comprising a human Fc domain, which induce Fc-dependent antiviral functions by at least or about at least or about 1 fold, at least or about 2 folds, at least or about 3 folds, at least or about 4 folds, at least or about 5 folds, at least or about 6 folds, at least or about 7 folds, at least or about 8 folds, at least or about 9 folds, at least or about 10 folds, at least or about 20 folds, at least or about 30 folds, at least or about 40 folds, at least or about 50 folds, or at least or about 100 folds as compared when no antibodies or antigen-binding fragments thereof as described herein are present.
The disclosure provides antibodies or antigen-binding fragments thereof comprising a human Fc domain, which induce host immune response by at least or about at least or about 1  fold, at least or about 2 folds, at least or about 3 folds, at least or about 4 folds, at least or about 5 folds, at least or about 6 folds, at least or about 7 folds, at least or about 8 folds, at least or about 9 folds, at least or about 10 folds, at least or about 20 folds, at least or about 30 folds, at least or about 40 folds, at least or about 50 folds, or at least or about 100 folds as compared when no antibodies or antigen-binding fragments thereof as described herein are present.
The disclosure provides antibodies or antigen-binding fragments thereof that block viral entry (or internalization) into host cell such that the internalization rate is less than or about 50%, less than or about 45%, less than or about 40%, less than or about 35%, less than or about 30%, less than or about 25%, less than or about 20%, less than or about 15%, less than or about 10%, less than or about 10%, or less than or about 5%of the internalization rate when no antibodies or antigen-binding fragments thereof as described herein are present.
In some embodiments, the antibodies or antigen-binding fragments thereof as described herein can increase immune response, activity or number of immune cells (e.g., T cells, CD8+ T cells, CD4+ T cells, macrophages, antigen presenting cells) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 folds, 3 folds, 5 folds, 10 folds, or 20 folds.
In some embodiments, the antibody (or antigen-binding fragment thereof) specifically binds to the S protein (e.g., SARS-CoV-2 S protein, SARS-CoV S protein, or MERS-CoV S protein) with a dissociation rate (koff) of less than 0.1 s -1, less than 0.01 s -1, less than 0.001 s -1, less than 0.0001 s -1, or less than 0.00001 s -1. In some embodiments, the dissociation rate (koff) is greater than 0.01 s -1, greater than 0.001 s -1, greater than 0.0001 s -1, greater than 0.00001 s -1, or greater than 0.000001 s -1.
In some embodiments, kinetic association rates (kon) is greater than 1 x 10 2/Ms, greater than 1 x 10 3/Ms, greater than 1 x 10 4/Ms, greater than 1 x 10 5/Ms, or greater than 1 x 10 6/Ms. In some embodiments, kinetic association rates (kon) is less than 1 x 10 5/Ms, less than 1 x 10 6/Ms, or less than 1 x 10 7/Ms.
Affinities can be deduced from the quotient of the kinetic rate constants (KD=koff/kon) . In some embodiments, KD is less than 1 x 10 -6 M, less than 1 x 10 -7 M, less than 1 x 10 -8 M, less than 1 x 10 -9 M, less than 1 x 10 -10 M, less than 1 x 10 -11 M, or less than 1 x 10 -12 M. In some embodiments, the KD is less than 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM or 0.05 nM. In some embodiments, KD is greater than 1 x 10 -7 M, greater  than 1 x 10 -8 M, greater than 1 x 10 -9 M, greater than 1 x 10 -10 M, greater than 1 x 10 -11 M, greater than 1 x 10 -12 M, or greater than 1 x 10 -13 M.
General techniques for measuring the affinity and/or avidity of an antibody for an antigen include, e.g., ELISA, RIA, and surface plasmon resonance (SPR) . In some embodiments, the antibody binds to SARS-CoV-2 S protein, SARS-CoV S protein, MERS-CoV S protein, or other coronavirus S proteins. In some embodiments, the antibody does not bind to other coronavirus S proteins.
In some embodiments, thermal stabilities are determined. The antibodies or antigen binding fragments as described herein can have a Tm greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 ℃. In some embodiments, Tm is less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 ℃.
In some embodiments, the antibodies or antigen binding fragments can enhance APC (e.g., DC cell) function, for example, inducing surface expression of costimulatory and MHC molecules, inducing production of proinflammatory cytokines, and/or enhancing T cell triggering function.
In some embodiments, the antibodies or antigen binding fragments can induce complement-dependent cytotoxicity (CMC) and/or antibody dependent cellular cytoxicity (ADCC) , and kill the infected cell.
In some embodiments, the antibodies or antigen binding fragments have a functional Fc region. In some embodiments, effector function of a functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC) . In some embodiments, effector function of a functional Fc region is phagocytosis. In some embodiments, effector function of a functional Fc region is ADCC and phagocytosis.
In some embodiments, the antibodies or antigen binding fragments can induce complement mediated cytotoxicity (CMC) .
In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the antibody is a human IgG1 antibody.
In some embodiments, the antibodies or antigen binding fragments do not have a functional Fc region. For example, the antibodies or antigen binding fragments are Fab, Fab’, F (ab’) 2, and Fv fragments. In some embodiments, the Fc region has LALA mutations (L234A  and L235A mutations in EU numbering) , or LALA-PG mutations (L234A, L235A, P329G mutations in EU numbering) .
Methods of Making Anti-S Protein Antibodies
An isolated fragment of S protein (e.g., RBD domain) can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be raised in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of an antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected with at least one adjuvant. In some embodiments, the antigenic peptide or protein can be conjugated to an agent that is immunogenic in the species to be immunized. Animals can be injected with the antigenic peptide or protein more than one time (e.g., twice, three times, or four times) .
The full-length polypeptide or protein can be used or, alternatively, antigenic peptide fragments thereof can be used as immunogens. The antigenic peptide of a protein comprises at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence of S protein and encompasses an epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with the protein. In some embodiments, the immunogen comprises or consists of RBD domain of the S protein (e.g., amino acid 319-541 of SEQ ID NO: 141) . In some embodiments, the immunogen polypeptide is linked to a Fc (e.g., a mouse Fc) .
An immunogen typically is used to prepare antibodies by immunizing a suitable subject (e.g., human or transgenic animal expressing at least one human immunoglobulin locus) . In some embodiments, the subject is a homozygous humanized heavy chain immunoglobulin locus and homozygous humanized light chain immunoglobulin locus (hVH/hVL mice) (Biocytogen RenMab TM Mouse) . The humanized mice are described e.g., in PCT/CN2020/075698, which is incorporated herein by reference in its entirety. An appropriate immunogenic preparation can contain, for example, a recombinantly-expressed or a chemically-synthesized polypeptide. The preparation can further include an adjuvant, such as Freund’s complete or incomplete adjuvant, or a similar immunostimulatory agent.
Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide, or an antigenic peptide thereof (e.g., part of S protein) as an  immunogen. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme-linked immunosorbent assay (ELISA) using the immobilized polypeptide or peptide. If desired, the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A of protein G chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the specific antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al., (Nature 256: 495-497, 1975) , the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4: 72, 1983) , the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985) , or trioma techniques. The technology for producing hybridomas is well known (see, generally, Current Protocols in Immunology, 1994, Coligan et al. (Eds. ) , John Wiley &Sons, Inc., New York, NY) . Hybridoma cells producing a monoclonal antibody are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide or epitope of interest, e.g., using a standard ELISA assay.
Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding a human, humanized, or chimeric antibody, or antigen-binding fragment thereof described herein, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acids sequences that make-up the antigen-binding site of the antibody or an antigen-binding domain. In a population of such variants, some antibodies or antigen-binding fragments will have increased affinity for the target protein. Any combination of deletions, insertions, and/or combinations can be made to arrive at an antibody or antigen-binding fragment thereof that has increased binding affinity for the target. The amino acid changes introduced into the antibody or antigen-binding fragment can also alter or introduce new post-translational modifications into the antibody or antigen-binding fragment, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence such that a different sugar is attached by enzymes present in a cell) , or introducing new glycosylation sites.
Antibodies disclosed herein can be derived from any species of animal, including mammals. Non-limiting examples of native antibodies include antibodies derived from humans,  primates, e.g., monkeys and apes, cows, pigs, horses, sheep, camelids (e.g., camels and llamas) , chicken, goats, and rodents (e.g., rats, mice, hamsters and rabbits) , including transgenic rodents genetically engineered to produce human antibodies.
Human and humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequence as those derived from) human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) , for example in the CDRs.
Identity or homology with respect to an original sequence is usually the percentage of amino acid residues present within the candidate sequence that are identical with a sequence present within the human, humanized, or chimeric antibody or fragment, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
Additional modifications to the antibodies or antigen-binding fragments can be made. For example, a cysteine residue (s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have any increased half-life in vitro and/or in vivo. Homodimeric antibodies with increased half-life in vitro and/or in vivo can also be prepared using heterobifunctional cross-linkers as described, for example, in Wolff et al., ( "Monoclonal antibody homodimers: enhanced antitumor activity in nude mice. " Cancer research 53.11 (1993) : 2560-2565) . Alternatively, an antibody can be engineered which has dual Fc regions.
In some embodiments, a covalent modification can be made to the antibody or antigen-binding fragment thereof. These covalent modifications can be made by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of the antibody or antibody fragment are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with an organic derivatization agent that is capable of reacting with selected side chains or the N-or C-terminal residues.
In some embodiments, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody composition may be from 1%to 80%, from 1%to 65%, from 5%to 65%or from 20%to 40%. The amount of fucose is determined by calculating the average amount of  fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues; or position 314 in Kabat numbering) ; however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody can be further engineered to replace the Asparagine at position 297 with Alanine (N297A) .
In some embodiments, to facilitate production efficiency by avoiding Fab-arm exchange, the Fc region of the antibodies was further engineered to replace the serine at position 228 (EU numbering) of IgG4 with proline (S228P) . A detailed description regarding S228 mutation is described, e.g., in Silva et al., "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation. " Journal of Biological Chemistry 290.9 (2015) : 5462-5469, which is incorporated by reference in its entirety.
Recombinant Vectors
The present disclosure also provides recombinant vectors (e.g., an expression vectors) that include an isolated polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein) , host cells into which are introduced the recombinant vectors (i.e., such that the host cells contain the polynucleotide and/or a vector comprising the polynucleotide) , and the production of recombinant antibody polypeptides or fragments thereof by recombinant techniques.
As used herein, a “vector” is any construct capable of delivering one or more polynucleotide (s) of interest to a host cell when the vector is introduced to the host cell. An “expression vector” is capable of delivering and expressing the one or more polynucleotide (s) of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by being operably linked with regulatory elements such as a promoter, enhancer, and/or a poly-A tail, either within the vector or in the genome of the host cell at or near  or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell introduced with the expression vector.
A vector can be introduced into the host cell by methods known in the art, e.g., electroporation, chemical transfection (e.g., DEAE-dextran) , transformation, transfection, and infection and/or transduction (e.g., with recombinant virus) . Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant virus) , naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.
In some implementations, a polynucleotide disclosed herein (e.g., a polynucleotide that encodes a polypeptide disclosed herein) is introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus) , which may involve the use of a non-pathogenic (defective) , replication competent virus, or may use a replication defective virus. In the latter case, viral propagation generally will occur only in complementing virus packaging cells. Suitable systems are disclosed, for example, in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86: 317-321; Flexner et al., 1989, Ann. N.Y. Acad Sci. 569: 86-103; Flexner et al., 1990, Vaccine, 8: 17-21; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner-Biotechniques, 6: 616-627, 1988; Rosenfeld et al., 1991, Science, 252: 431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91: 215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90: 11498-11502; Guzman et al., 1993, Circulation, 88: 2838-2848; and Guzman et al., 1993, Cir. Res., 73: 1202-1207. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be “naked, ” as described, for example, in Ulmer et al., 1993, Science, 259: 1745-1749, and Cohen, 1993, Science, 259: 1691-1692. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads that are efficiently transported into the cells.
For expression, the DNA insert comprising an antibody-encoding or polypeptide-encoding polynucleotide disclosed herein can be operatively linked to an appropriate promoter (e.g., a heterologous promoter) , such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters are known to the skilled artisan. In some embodiments, the promoter is a cytomegalovirus (CMV) promoter. The expression constructs can further contain  sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs may include a translation initiating at the beginning and a termination codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated.
As indicated, the expression vectors can include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces, and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells; and plant cells. Appropriate culture mediums and conditions for the host cells described herein are known in the art.
Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.
Non-limiting bacterial promoters suitable for use include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV) , and metallothionein promoters, such as the mouse metallothionein-I promoter.
In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used.
Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986) , which is incorporated herein by reference in its entirety.
Transcription of DNA encoding an antibody of the present disclosure by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. The signals may be endogenous to the polypeptide or they may be heterologous signals.
The polypeptide (e.g., antibody) can be expressed in a modified form, such as a fusion protein (e.g., a GST-fusion) or with a histidine-tag, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to the polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.
Methods of Treatment and Diagnosis
The antibodies or antigen-binding fragments thereof of the present disclosure can be used for various therapeutic purposes.
In one aspect, the disclosure provides methods for treating a coronavirus-related disease in a subject, methods of neutralizing a coronavirus, methods of blocking a coronavirus/ACE2 interaction, methods of promoting coronavirus aggregation, methods of inducing Fc-dependent antiviral functions, methods of blocking internalization of a coronavirus by a cell, methods of identifying a subject having a coronavirus-related disease. In some embodiments, the treatment can halt, slow, retard, or inhibit progression of a coronavirus-related disease. In some  embodiments, the treatment can result in the reduction of in the number, severity, and/or duration of one or more symptoms of the coronavirus-related disease in a subject.
In one aspect, the disclosure features methods that include administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein to a subject in need thereof (e.g., a subject having, or identified or diagnosed as having, a coronavirus-related disease) .
In some embodiments, the coronavirus-related disease is COVID-19 (Coronavirus disease 2019) , Severe acute respiratory syndrome (SARS) , or Middle East respiratory syndrome (MERS) .
In some embodiments, the coronavirus that causing the coronavirus-related disease is SARS-CoV, SARS-CoV-2, MERS-CoV, or other types of coronavirus having one or more S proteins. In some embodiments, the amino acid sequence of the S protein of the coronavirus described herein comprises a sequence that is at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, or at least or about 98%identical to the receptor-biding domain sequence of the SARS-CoV-2 S protein.
In some embodiments, the compositions and methods disclosed herein can be used for treatment of patients at risk for a coronavirus-related disease. Patients with coronavirus-related disease can be identified with various methods known in the art.
As used herein, by an “effective amount” is meant an amount or dosage sufficient to effect beneficial or desired results including halting, slowing, retarding, or inhibiting progression of a disease, e.g., a coronavirus-related disease. An effective amount will vary depending upon, e.g., an age and a body weight of a subject to which the antibody, antigen binding fragment, antibody-encoding polynucleotide, vector comprising the polynucleotide, and/or compositions thereof is to be administered, a severity of symptoms and a route of administration, and thus administration can be determined on an individual basis.
An effective amount can be administered in one or more administrations. By way of example, an effective amount of an antibody or an antigen binding fragment is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow and/or delay progression of a coronavirus-related disease in a patient. As is understood in the art, an effective amount of an  antibody or antigen binding fragment may vary, depending on, inter alia, patient history as well as other factors such as the type (and/or dosage) of antibody used.
Effective amounts and schedules for administering the antibodies, antibody-encoding polynucleotides, and/or compositions disclosed herein may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal that will receive the antibodies, antibody-encoding polynucleotides, and/or compositions disclosed herein, the route of administration, the particular type of antibodies, antibody-encoding polynucleotides, antigen binding fragments, and/or compositions disclosed herein used and other drugs being administered to the mammal.
A typical daily dosage of an effective amount of an antibody is 0.01 mg/kg to 100 mg/kg (mg per kg of patient weight) . In some embodiments, the dosage can be less than 100 mg/kg, 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, or 0.1 mg/kg. In some embodiments, the dosage can be greater than 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, 0.1 mg/kg, 0.05 mg/kg, or 0.01 mg/kg. In some embodiments, the dosage is about 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.9 mg/kg, 0.8 mg/kg, 0.7 mg/kg, 0.6 mg/kg, 0.5 mg/kg, 0.4 mg/kg, 0.3 mg/kg, 0.2 mg/kg, or 0.1 mg/kg.
In any of the methods described herein, the at least one antibody, antigen-binding fragment thereof, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding fragments, or pharmaceutical compositions described herein) and, optionally, at least one additional therapeutic agent can be administered to the subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day) . In some embodiments, at least two different antibodies and/or antigen-binding fragments are administered in the same composition (e.g., a liquid composition) . In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition) . In some embodiments, the at least one antibody or antigen-binding fragment and the at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition containing at least one antibody or antigen-binding fragment and a solid oral composition containing at least one additional therapeutic agent) . In some embodiments, the at  least one additional therapeutic agent is administered as a pill, tablet, or capsule. In some embodiments, the at least one additional therapeutic agent is administered in a sustained-release oral formulation.
In some embodiments, the one or more additional therapeutic agents can be administered to the subject prior to, or after administering the at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) . In some embodiments, the one or more additional therapeutic agents and the at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) are administered to the subject such that there is an overlap in the bioactive period of the one or more additional therapeutic agents and the at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein) in the subject.
In some embodiments, the subject can be administered the at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) over an extended period of time (e.g., over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years) . A skilled medical professional may determine the length of the treatment period using any of the methods described herein for diagnosing or following the effectiveness of treatment (e.g., the observation of at least one symptom of the coronavirus-related disease) . As described herein, a skilled medical professional can also change the identity and number (e.g., increase or decrease) of antibodies or antigen-binding antibody fragments (and/or one or more additional therapeutic agents) administered to the subject and can also adjust (e.g., increase or decrease) the dosage or frequency of administration of at least one antibody or antigen-binding antibody fragment (and/or one or more additional therapeutic agents) to the subject based on an assessment of the effectiveness of the treatment (e.g., using any of the methods described herein and known in the art) .
The antibodies, antigen-binding antibody fragments, or pharmaceutical compositions can be combined to have an improved therapeutic effect. In some embodiments, the subject can be administered the at least one, two, three, four, or five antibody, antigen-binding fragment thereof  selected from Table 1. In some embodiments, these antibodies or antigen-binding fragments thereof target different epitopes of an S protein. In some embodiments, these antibodies or antigen-binding fragments thereof do not cross-compete with each other. In some embodiments, at least two antibodies or antigen-binding fragments thereof are selected. Different pairs of antibodies or antigen-binding fragments thereof can be selected from FIG. 16. These pairs of antibodies or antigen-binding fragments include any antibodies or antigen-binding fragments derived from the antibodies as shown in FIG. 16, including human, humanized, and modified antibodies or antigen binding fragments thereof. In some embodiments, the subject is administered with 03-10D12-IgG1-LALA and 05-9G11-IgG1-LALA. In some embodiments, the subject is administered with 03-9A8-IgG1-LALA and 09-7B8-IgG-LALA..
In some embodiments, the antibodies or antigen-binding fragments thereof can be used for detecting coronavirus (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV) in a subject (e.g., a human) or diagnosing a coronavirus related disease. Methods known in the art can be designed, e.g., ELISA, to produce a diagnostic test kit. In some embodiments, one or more antibodies or antigen-binding fragments comprising any of the heavy chain single variable domains as described herein can be used.
Pharmaceutical Compositions and Routes of Administration
Also provided herein are pharmaceutical compositions that contain at least one (e.g., one, two, three, or four) of the antibodies or antigen-binding fragments described herein. Two or more (e.g., two, three, or four) of any of the antibodies or antigen-binding fragments described herein can be present in a pharmaceutical composition in any combination. The pharmaceutical compositions may be formulated in any manner known in the art.
In some embodiments, the pharmaceutical compositions can comprise one, two, three, four, five or more than five antibodies, antigen-binding fragments thereof as described herein (e.g., selected from Table 1) . In some embodiments, these antibodies or antigen-binding fragments thereof target different epitopes of an S protein. In some embodiments, these antibodies or antigen-binding fragments thereof do not cross-compete with each other. In some embodiments, at least two antibodies or antigen-binding fragments thereof are selected. Different pairs of antibodies or antigen-binding fragments thereof can be selected from FIG. 16. These pairs of antibodies or antigen-binding fragments include any antibodies or antigen-binding  fragments derived from the antibodies as shown in FIG. 16, including human (e.g., IgG1) , humanized, and modified antibodies (e.g., LALA mutations) or antigen binding fragments thereof. In some embodiments, the pharmaceutical composition comprises 03-10D12-IgG1-LALA and 05-9G11-IgG1-LALA. In some embodiments, the pharmaceutical composition comprises 03-9A8-IgG1-LALA and 09-7B8-IgG-LALA..
Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) . The compositions can include a sterile diluent (e.g., sterile water or saline) , a fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvents, antibacterial or antifungal agents, such as benzyl alcohol or methyl parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like, antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid, buffers, such as acetates, citrates, or phosphates, and isotonic agents, such as sugars (e.g., dextrose) , polyalcohols (e.g., mannitol or sorbitol) , or salts (e.g., sodium chloride) , or any combination thereof. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. Preparations of the compositions can be formulated and enclosed in ampules, disposable syringes, or multiple dose vials. Where required (as in, for example, injectable formulations) , proper fluidity can be maintained by, for example, the use of a coating, such as lecithin, or a surfactant. Absorption of the antibody or antigen-binding fragment thereof can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin) . Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid) .
Compositions containing one or more of any of the antibodies or antigen-binding fragments described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined quantity of active compound for ease of administration and uniformity of dosage) .
Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single  administration) . Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries. The formulation depends on the route of administration chosen. For injection, antibodies can be formulated in aqueous solutions, preferably in physiologically-compatible buffers to reduce discomfort at the site of injection. The solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively antibodies can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compositions as described herein can be administered through respiratory tract by various means, for example, nasal administration, nasal instillation, insufflation (e.g., nasal sprays) , inhalation (through nose or mouth) , intrapulmonary administration, intratracheal administration, or any combinations thereof. As used herein, the term “nasal instillation” refers to a procedure that delivers a therapeutic agent directly into the nose and onto the nasal membranes, wherein a portion of the therapeutic agent can pass through tracheas and is delivered into the lung.
Toxicity and therapeutic efficacy of compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys) . One can, for example, determine the LD50 (the dose lethal to 50%of the population) and the ED50 (the dose therapeutically effective in 50%of the population) : the therapeutic index being the ratio of LD50: ED50. Agents that exhibit high therapeutic indices are preferred. Where an agent exhibits an undesirable side effect, care should be taken to minimize potential damage (i.e., reduce unwanted side effects) . Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.
Data obtained from cell culture assays and animal studies can be used in formulating an appropriate dosage of any given agent for use in a subject (e.g., a human) . A therapeutically effective amount of the one or more (e.g., one, two, three, or four) antibodies or antigen-binding fragments thereof (e.g., any of the antibodies or antibody fragments described herein) will be an amount that treats the disease in a subject (e.g., inhibits coronavirus) in a subject (e.g., a human subject identified as having COVID-19) , or a subject identified as being at risk of developing the disease (e.g., a subject who has previously infected by coronavirus but now has been cured) , decreases the severity, frequency, and/or duration of one or more symptoms of a disease in a subject (e.g., a human) . The effectiveness and dosing of any of the antibodies or antigen-binding  fragments described herein can be determined by a health care professional or veterinary professional using methods known in the art, as well as by the observation of one or more symptoms of disease in a subject (e.g., a human) . Certain factors may influence the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and the presence of other diseases) .
Exemplary doses include milligram or microgram amounts of any of the antibodies or antigen-binding fragments described herein per kilogram of the subject’s weight (e.g., about 1 μg/kg to about 500 mg/kg; about 100 μg/kg to about 500 mg/kg; about 100 μg/kg to about 50 mg/kg; about 10 μg/kg to about 5 mg/kg; about 10 μg/kg to about 0.5 mg/kg; about 1 μg/kg to about 50 μg/kg; about 1 mg/kg to about 10 mg/kg; or about 1 mg/kg to about 5 mg/kg) . While these doses cover a broad range, one of ordinary skill in the art will understand that therapeutic agents, including antibodies and antigen-binding fragments thereof, vary in their potency, and effective amounts can be determined by methods known in the art. Typically, relatively low doses are administered at first, and the attending health care professional or veterinary professional (in the case of therapeutic application) or a researcher (when still working at the development stage) can subsequently and gradually increase the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the half-life of the antibody or antibody fragment in vivo.
The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. The disclosure also provides methods of manufacturing the antibodies or antigen binding fragments thereof for various uses as described herein.
EXAMPLES
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
Materials and Methods
The following materials were used in the following examples.
RBD-mFc protein (SARS-CoV-2 (2019-nCoV) Spike RBD-mFc Recombinant Protein (HPLC-verified) ) was purchased from Sino Biological Inc. (Catalog number: 40592-V05H) .
Recombinant 2019-nCoV S protein RBD (C-6His) was purchased from Sino Biological Inc. (Catalog number: 40592-V08B) .
Plasmid expressing full-length SARS-CoV-2 spike protein was obtained from National Institutes for Food and Drug Control (China) .
ACE2-hFc (recombinant human ACE2 protein-hFc) was purchased from Kactus Biosystems Co. Ltd (Catalog number: ACE-HM501) .
ACE2-mFc (recombinant human ACE2 protein-mFc) was purchased from Sino Biological Inc. (Catalog number: 10108-H05H) .
S(S1+S2) -his protein (SARS-CoV-2 (COVID-19) S protein (R683A, R685A) , His Tag) was purchased from ACROBiosystems (Catalog number: SPN-C52H4) .
Anti-hIgG PE (R-Phycoerythrin AffiniPure F (ab')  2 Fragment Goat Anti-Human IgG, Fcγ fragment specific) was purchased from Jackson ImmunoResearch Inc. (Catalog number: 109-116-098) .
Anti-mIgG FITC (Fluorescein (FITC) AffiniPure F (ab')  2 Fragment Goat Anti-Mouse IgG, Fcγ fragment specific) was purchased from Jackson ImmunoResearch Inc. (Catalog number: 115-096-071) .
Anti-mouse IgG Fc-PE (R-Phycoerythrin AffiniPure F (ab')  2 Fragment Goat Anti-Mouse IgG, Fcγ fragment specific) was purchased from Jackson ImmunoResearch Inc. (Catalog number: 115-116-071) .
Anti-human IgG Fc-AF647 (Alexa 
Figure PCTCN2021099779-appb-000001
647 AffiniPure F (ab')  2 Fragment Goat Anti-Human IgG, Fcγ fragment specific) was purchased from Jackson ImmunoResearch Inc. (Catalog number: 109-606-170) .
Series S Sensor Chip Protein A was purchased from GE Healthcare Life Sciences (Catalog number: 29127556) .
Series S sensor Chip CM5 was purchased from GE Healthcare Life Sciences (Catalog number: 29149603) .
HBS-EP+ buffer (10×) was purchased from GE Healthcare Life Sciences (Catalog number: BR-1006-69) .
Glycine-HCl, pH 1.5 buffer was purchased from GE Healthcare Life Sciences (Catalog number: BR-1003-54) .
Anti-His antibody (THE TM His Tag Antibody, mAb, mouse) was purchased from GenScript Biotech (Catalog number: A00186) .
Example 1. Generating anti-SARS-CoV-2antibodies
The anti-SARS-CoV-2 antibodies were collected by the methods as described below.
Immunization of mice
To generate human antibodies against SARS-CoV-2 spike protein receptor-binding domain (RBD) , ten mice with homozygous humanized heavy chain immunoglobulin locus and homozygous humanized light chain immunoglobulin locus (hVH/hVL mice) (Biocytogen RenMab TM Mouse) were immunized with an RBD-mFc protein at least five times. The RBD-mFc protein contains the SARS-CoV-2 Spike protein receptor-binding domain (amino acid 319-541 of NCBI Reference Sequence: YP_009724390.1 (SEQ ID NO: 141) ) and is fused to the Fc region of mouse IgG1 at C-terminus.
The RBD-mFc protein was emulsified with adjuvant and injected at several positions on the back of the mice. Procedures to enhance immunization were also performed after the fifth immunization. CHO-S-SDNA (T) cells and the RBD-mFc protein were intravenously injected into the mice through tail veins. Spleen was then collected four days after the injection. Specifically, CHO-S-SDNA (T) were CHO-K cells transfected with a plasmid expressing full-length SARS-CoV-2 spike protein on the cell surface. The expressed SARS-CoV-2 spike protein includes both S1 and S2 subunits of the spike protein (e.g., the extracellular domain of the S1 and S2 subunits) .
Fusion of SP2/0 cells and spleen cells
Spleen tissues were grinded. Spleen cells were first selected by CD3ε Microbeads and Anti-Mouse IgM Microbeads, and then fused with SP2/0 cells. The cells were then plated in 96-well plates with hypoxanthine-aminopterin-thymidine (HAT) medium.
Primary screening of hybridoma
Primary screening of the hybridoma supernatant in the 96-well plates was performed using Fluorescence-Activated Cell Sorting (FACS) pursuant to standard procedures. Chinese hamster ovary (CHO) cells were added to 96-well plates (2 × 10 4 cells per well) before the screening. 50 ul of supernatant was used.
Sub-cloning
Sub-cloning was performed using ClonePix2. In short, the positive wells identified during the primary screening were transferred to semisolid medium, and IgG positive clones were identified and tested. FITC anti-mouse IgG Fc antibody was used.
Ascites fluid antibodies
1 × 10 6 positive hybridoma cells were injected intraperitoneally to 
Figure PCTCN2021099779-appb-000002
mice (Beijing Biocytogen, Beijing, China) . Monoclonal antibodies were produced by growing hybridoma cells within the peritoneal cavity of the mouse. The hybridoma cells multiplied and produced ascites fluid in the abdomens of the mice. The fluid contained a high concentration of antibody which can be harvested for later use.
Purification of antibodies
Antibodies in ascites fluid were purified using GE AKTA protein chromatography (GE Healthcare, Chicago, Illinois, United States) . At least 17 chimeric antibodieswere produced. Specifically, each chimeric antibody heavy chain comprised a human variable region (VH) and a mouse constant region (CH) , and each chimeric antibody light chain comprised a human variable region (VL) and a mouse constant region (CL) . A few antibodies were selected because of the desired properties. These selected mouse antibodies produced by the methods described above include e.g., 03-9A8 ( “9A8” ) , 01-2H10 ( “2H10” ) , 03-1F9 ( “1F9” ) , 03-10D12 ( “10D12” ) , 03-10F9 ( “10F9” ) , 05-8G6 ( “8G6” ) , 05-9G11 ( “9G11” ) , 09-2F7 ( “2F7” ) , 09-4E5 ( “4E5” ) , and 09-7B8 ( “7B8” ) .
The amino acid sequences of the heavy chain variable region (VH) , light chain variable region (VL) , and complementarity determining regions (CDRs) of the antibodies were determined. The heavy chain CDR1, CDR2, CDR3, and light chain CDR1, CDR2, and CDR3 amino acid sequences of these antibodies are shown in FIGS. 13-14. The VH and VL amino acid  sequences of these antibodies are shown in FIG. 15. Consensus sequences in CDR1, CDR2, CDR3 in VH are shown in FIGS. 11A-11C. Consensus sequences in CDR1, CDR2, CDR3 in VL are shown in FIGS. 12A-12C.
Example 2. In vitro testing of anti-SARS-CoV-2 antibodies in ascites fluid or hybridoma supernatant: blocking human ACE2 binding to SARS-CoV-2 S protein
Blocking effects of ascites fluid or hybridoma supernatant were detected by flow cytometry. Detailed methods are as follows. First, the immunized mouse serum and unimmunized mouse serum were diluted using phosphate-buffered saline (PBS) at 1: 100 and 1: 500, respectively. The ACE2-hFc protein was diluted to 0.5 μg/ml by PBS. Next, CHO-S-SDNA (T) cells were washed to remove residual culture medium and resuspended in PBS to a cell density of 5 × 10 4 per 10 μl. The resuspended cells were added to a 96-well plate (10 μl/well) and 40 μl ascites fluid (or hybridoma supernatant) was added to the corresponding wells.
A row in the 96-well plate was reserved for control samples. For example, two wells were selected to add diluted immunized mouse serum (labelled as PC1) and diluted unimmunized mouse serum (labeled as NC1) to the CHO-S-SDNA (T) cells, respectively (40 μl/well) . Another well was selected to add ascites fluid (or hybridoma supernatant) containing antibodies against other antigens (labelled as NC2) . 50 μl diluted ACE2-hFc was added to each well, followed by an incubation at 4℃ for 30 minutes.
Next, 200 μl PBS was added to each well, and then the plate was centrifuged at 1500 rpm for 3 minutes. Supernatant in each well was discarded and the plate was gently tapped to release the cells from cell pellets. The above procedures were repeated twice to wash the cells in the plate. Next, 50 μl of pre-mixed secondary antibodies (1: 100 diluted anti-hIgG PE and 1: 500 diluted anti-mIgG FITC) were added to each well and the plate was incubated at 4℃ for 15 minutes. Afterwards, cells in each well were washed by 200 μl PBS as described above and then resuspended in 30 μl PBS for flow cytometry analysis.
As shown in FIG. 1, the binding between human ACE2 and SARS-CoV-2 spike protein was blocked by the 03-1F9, 09-4E5, 09-7B8, 03-9A8, 03-10D12, 05-8G6, 05-9G11, 03-10F9, and 01-2H10 antibodies. Immunized mouse serum (PC1) exhibited a similar blocking effect. However, unimmunized mouse serum (NC1) did not exhibit any blocking effect.
Example 3. Determination of binding affinity of purified human anti-SARS-CoV-2 antibodies to SARS-CoV-2 spike protein RBD
As described in Example 1, 03-9A8, 01-2H10, 03-1F9, 03-10D12, 03-10F9, 05-8G6, 05-9G11, 09-2F7, 09-4E5, and 09-7B8 are chimeric anti-SARS-CoV-2 antibodies generated by the hVH/hVL mice. The chimeric antibodies have the heavy chain variable domain (VH) and the light chain variable domain (VL) from human, and the constant domains from mouse IgG antibodies. Based on these chimeric anti-SARS-CoV-2 antibodies, the human anti-SARS-CoV-2 antibodies including e.g., 03-9A8-IgG1-LALA, 01-2H10-IgG1-LALA, 03-1F9-IgG1-LALA, 03-10D12-IgG1-LALA, 03-10F9-IgG1-LALA, 05-8G6-IgG1-LALA, 05-9G11-IgG1-LALA, 09-2F7-IgG1-LALA, 09-4E5-IgG1-LALA, and 09-7B8-IgG1-LALA were generated. The human antibodies have the same heavy chain variable domain and the light chain variable domain from the corresponding chimeric anti-SARS-CoV-2 antibodies, and the constant domains from human IgG1 antibodies (including, e.g., the CL, CH1, CH2, and CH3 domains of human IgG1) with LALA (Leu234Ala/Leu235Ala) mutations.
The binding affinity ofanti-SARS-CoV-2 antibodies against the SARS-CoV-2 spike protein RBD were measured by surface plasmon resonance (SPR) using Biacore 8K SPR System (GE) equipped with pre-immobilized Series S Sensor Chip Protein A at 25℃.
Purified anti-SARS-CoV-2 antibodies (1 μg/mL) were injected into Biacore 8K biosensor at 10 μL/min for 37 seconds to achieve to a desired protein density (about 52 response units (RU) ) . Histidine-tagged recombinant 2019-nCoV S protein RBD (C-6His) at the concentration of 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, or 0.78125 nM were then injected at 30 μL/min for 180 seconds. Dissociation was monitored for 600 seconds. The chip was regenerated after the last injection of each titration with a glycine buffer (pH 2.0, 30μL/min for 20 seconds) . For example, the results of 09-4E5-IgG1-LALA are shown in FIG. 2.
Kinetic association rates (kon) and dissociation rates (koff) were obtained simultaneously by fitting the data globally to a 1: 1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6.99-110) using Biacore 8K Evaluation Software. Affinities were calculated from the quotient of the kinetic rate constants (KD=koff/kon) .
As a person of ordinary skill in the art would understand, the same method with appropriate adjustments for parameters (e.g., antibody concentration) was performed for each tested antibody. The results for the tested antibodies are summarized in the table below.
According to the KD values, the human anti-SARS-CoV-2 antibodies exhibited high binding affinities against the SARS-CoV-2 spike protein RBD.
Table 1
Figure PCTCN2021099779-appb-000003
Example 4. IC 50 determination ofpurified human anti-SARS-CoV-2 antibodies: blocking human ACE2 binding to SARS-CoV-2 S protein
Blocking effects of purified human anti-SARS-CoV-2 antibodies were detected by fluorescence-activated cell sorting (FACS) using the CHO-S-SDNA (T) cells. Detailed methods are as follows. First, the purified antibodies were diluted by serial dilution to 60 μg/ml, 20 μg/ml, 6.667 μg/ml, 2.222 μg/ml, 0.741 μg/ml, 0.247 μg/ml, 0.082 μg/ml, 0.027 μg/ml, 0.009 μg/ml, and 0.003 μg/ml. The ACE2-mFc protein was diluted to 10 μg/ml. Next, CHO-S-SDNA (T) cells were washed to remove residual culture medium and resuspended in PBS to a cell density of 1 ×10 6 cells/ml. The resuspended cells were added to a 96-well plate (50 μl/well) and the plate was centrifuged at 1600 rpm for 6 minutes. Supernatant in each well was discarded. Next, 40 μl diluted antibodies were added to the corresponding wells, followed by an incubation at 4℃ for 30 minutes. Afterwards, 50 μl diluted ACE2-mFc were added to the corresponding wells, and the plate was further incubated at 4℃ for 30 minutes. Next, 200 μl PBS was added to each well, and then the plate was centrifuged at 1600 rpm for 6 minutes. Supernatant in each well was discarded. Next, 50 μl of pre-mixed secondary antibodies (anti-mouse IgG Fc-PE and anti-human IgG Fc-AF647) were added to each well and the plate was incubated at 4℃ for 30 minutes. Afterwards,  cells in each well were washed as described above and then resuspended in 30 μl PBS for FACS analysis.
The raw data were analyzed to generatethe mean fluorescence intensity (MFI) reflectingantibody-CHO-S-SDNA (T) cell binding at different antibody concentrations. The MFI values were used to generate a fitting curve with respect to the antibody concentrations. More specifically, logarithm of antibody concentrations was calculated and was used as the X-axis variable, while the corresponding MFI was used as the Y-axis variable. An exemplary fitting curves of 03-9A8-IgG1-LALA and 09-7B8-IgG1-LALA are shown in FIG. 3. The IC 50 value was determined. Two independent experiments were performed and the determined IC 50 is shown in the tables below.
Table 2
Anti-SARS-CoV-2 Antibody IC 50 (μg/mL) R 2
03-9A8-IgG1-LALA 1.363 0.8940
03-1F9-IgG1-LALA 1.975 0.8041
05-10B7-IgG1-LALA Not applicable No binding
09-4E5-IgG1-LALA 7.298 0.7436
09-7B8-IgG1-LALA 0.8011 0.8844
As shown in Table 2, 09-7B8-IgG1-LALA had the best blocking effects. The blocking effects of 03-9A8-IgG1-LALA and 03-1F9-IgG1-LALA are also very good.
Table 3
Anti-SARS-CoV-2 Antibody IC 50 (μg/mL) R 2
03-9A8-IgG1-LALA 3.148 0.8374
01-2H10-IgG1-LALA 2.711 0.8308
02-5A8-IgG1-LALA Not applicable No binding
02-6G4-IgG1-LALA Not applicable No binding
03-10D12-IgG1-LALA 10.39 0.9305
03-10F9-IgG1-LALA 19.37 0.8927
05-8G6-IgG1-LALA Not applicable No binding
05-9G11-IgG1-LALA 53.2 0.8348
09-2F7-IgG1-LALA 9.272 0.9192
As shown in Table 3, 01-2H10-IgG1-LALA and 03-9A8-IgG1-LALA had the best blocking effects. The blocking effects of 03-10D12-IgG1-LALA and 09-2F7-IgG1-LALA are also good.
Example 5. Epitope correlation analysis of purified human anti-SARS-CoV-2 antibodies
Relative positions of target protein epitope between a pair of purified anti-SARS-CoV-2 monoclonal antibodies were analyzed through a surface plasmon resonance (SPR) competition experiment. A total of 100 combination pairs ofthe 10 monoclonal antibodies (01-2H10-IgG1-LALA, 03-10D12-IgG1-LALA, 03-10F9-IgG1-LALA, 03-1F9-IgG1-LALA, 05-8G6-IgG1-LALA, 05-9G11-IgG1-LALA, 09-2F7-IgG1-LALA, 09-4E5-IgG1-LALA, 09-7B8-IgG1-LALA, and 03-9A8-IgG1-LALA) were used to study the binding inhibition (blocking) effect of each antibody on another antibody.
HBS-EP+ buffer (10mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) , 150mM NaCl, 3mM ethylenediaminetetraacetic acid (EDTA) and 0.05%P20, pH7.4) was diluted from HBS-EP+ buffer (10×) as the running buffer throughout the experiment. Anti-His antibodies were fixed on the surface of a Series S sensor Chip CM5 by amino group coupling to generate an anti-His chip (i.e., CM5-Anti-His-Channel 1, 8-Chip) . Then, 1M ethanolamine, pH 8.5 was injected to block the remaining active carboxyl groups on the chip surface, followed by equilibration using the HBS-EP+ buffer for 2 hours. Recombinant 2019-nCoV S protein RBD with His-tag (0.2 μg/ml) were injected into the Biacore 8K biosensor at 10 μL/min for 50 seconds and captured on the anti-His chip to achieve a desired protein density (about 50 RU) . A pair of antibodies (100 nM each) was continuously injected at 30 μL/min into the chip. The first injected antibody (analyte 1) had a binding time of 300 seconds, and then the second antibody (analyte 2) was injected with a binding time of 300 seconds. After injection of the antibodies in each analysis cycle, the chip was regenerated twice with a glycine buffer (pH 1.7; 30 μL/min for 30 seconds, followed by 30 μL/min for 20 seconds) . Each pair of monoclonal antibodies was subjected to the same experimental steps to obtain the binding inhibition data when each monoclonal antibody was paired with another antibody.
The binding value of each antibody was obtained using Biacore 8K Evaluation Software. To quantify the interference of one antibody binding to another, a binding ratio was calculated to compare each pair of antibodies. The binding ratio is defined as the binding value of the second antibody (analyte 2) , divided by the binding value of the first antibody (analyte 1) . A statistical software wasalso used for cluster analysis. The binding ratio of each monoclonal antibody pair was summarized in a matrix table as shown in FIG. 4. More specifically, the binding ratio was  between -0.1 to 0.5, if analyte 1 exhibited a blocking effect to analyte 2. The binding ratio was between 0.5-1.2, if analyte 1 did not exhibit a blocking effect to analyte 2. When analyte 1 and analyte 2were the same monoclonal antibody, the second injection was regarded as a self-blocking and used as a control. In general, antibody pairs that interfere with each other have the same or overlapping epitopes.
Accordingly, epitope correlation was analyzed (FIG. 5) and the 10 human anti-SARS-CoV-2 antibodies were categorized into 5 epitope clusters (FIG. 6) . In summary, 01-2H10-IgG1-LALA, 03-10F9-IgG1-LALA, and 03-10D12-IgG1-LALA shared the same or overlapping epitopes; 05-8G6-IgG1-LALA and 05-9G11-IgG1-LALA shared the same or overlapping epitopes; 09-2F7-IgG1-LALA and 09-4E5-IgG1-LALA shared the same or overlapping epitopes; 03-1F9-IgG1-LALA and 03-9A8-IgG1-LALA shared the same or overlapping epitopes; 09-7B8-IgG1-LALA did not exhibit epitope correlation with the other 9 antibodies. In addition, the cluster including 09-2F7-IgG1-LALA and 09-4E5-IgG1-LALA exhibited a strong correlation with the cluster including 03-1F9-IgG1-LALA and 03-9A8-IgG1-LALA.
Example 6. In vitro testing of purified human anti-SARS-CoV-2 antibodies: blocking human ACE2 binding to SARS-CoV-2 S protein
Blocking effects of purified human anti-SARS-CoV-2 antibodies were detected by surface plasmon resonance (SPR) using a Biacore 8K SPR System equipped with CM5-Anti-His-Channel 1, 8-Chip. A purified human anti-SARS-CoV-2 antibody and recombinant ACE2 were sequentially injected to interact with captured 2019-nCoV S protein RBD, therefore to verify the ACE2 blocking effect of each antibody.
1× HBS-EP+ buffer (10 mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) , 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid (EDTA) and 0.05%P20, pH 7.4) was diluted from HBS-EP+ buffer (10×) as the running buffer throughout the experiment. Anti-His antibodies were fixed on the surface of a Series S sensor Chip CM5 to generate an anti-His chip (i.e., CM5-Anti-His-Channel 1, 8-Chip) . Next, the Biacore system was equilibrated with the HBS-EP+ buffer. Recombinant 2019-nCoV S protein RBD with His-tag (1 μg/ml) were injected into the Biacore 8K biosensor at 10 μL/min for 50 seconds and captured on the anti-His chip to achieve a desired protein density (about 50 RU) . Then, 1M ethanolamine, pH 8.5 was  injected to block the remaining active carboxyl groups on the chip surface, followed by equilibration using the HBS-EP+ buffer for 2 hours.
Detailed methods are as follows. 1× HBS-EP+ buffer (10 mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) , 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid (EDTA) and 0.05%P20, pH 7.4) was diluted from HBS-EP+ buffer (10×) as the running buffer throughout the experiment. Anti-His antibodies were fixed on the surface of a Series S sensor Chip CM5 by amino group coupling to generate an anti-His chip (i.e., CM5-Anti-His-Channel 1, 8-Chip) . Next, the Biacore system was equilibrated with the HBS-EP+ buffer. Recombinant 2019-nCoV S protein RBD with His-tag (1 μg/ml) were injected into the Biacore 8K biosensor at 10 μL/min for 50 seconds and captured on the anti-His chip to achieve a desired protein density (about 50 RU) . Then, 1M ethanolamine, pH 8.5 was injected to block the remaining active carboxyl groups on the chip surface, followed by equilibration using the HBS-EP+ buffer for 2 hours. A purified anti-SARS-CoV-2 antibody (100 nM) was continuously injected at 30 μL/min into the chip. The injected antibody (analyte 1) had a binding time of 300 seconds. ACE2-hFc (200 nM; analyte 2) was injected with a binding time of 200 seconds. After injection of the antibodies in each analysis cycle, the chip was regenerated with a glycine buffer (pH 1.7; 30 μL/min for 30 seconds, followed by 30 μL/min for 20 seconds) .
As shown in FIGS. 7A-7B, all 10 purified human anti-SARS-CoV-2 antibodies except 05-8G6-IgG1-LALA exhibited a strong AEC2 blocking effect.
Example 7. IC 50 determination for neutralizing COVID-19 pseudovirus
COVID-19 pseudovirus (or pseudovirus particles) was used for testing neutralizing effects. The pseudovirus preserves the virus infecting ability but cannot replicate. The function and activities of pseudovirus have been verified at the cellular level and in humanized mice.
Briefly, the anti-SARS-CoV-2 antibodieswere serial diluted in a 96-well plate. Then, the COVID-19 pseudovirus was diluted to 1-2 × 10 4 TCID 50/ml and mixed with the antibodies, followed by an incubation at 37℃, 5%CO 2 for 1 hour. Next, Huh-7 cells (2 × 10 4 cells/well) were added to the corresponding wells, and the plate was incubated at 37℃, 5%CO 2 for 20-28 hours. After the incubation, 150 μl supernatant was discarded by pipetting, and 100 μl luciferase detection reagent was added to each well. The plate was then incubated at room temperature in dark for 2 minutes. Afterwards, 150 μl solution in each well was transferred to a new plate,  which was placed in a plate reader to measure chemiluminescence signals. The neutralization inhibition ratio was calculated as follows:
Inhibition ratio = [1- (Ab-CC) / (VC-CC) ] × 100%
wherein Ab is the average signal of the antibody wells; CC is the average signal of the cell control wells (culture medium only) ; VC is the average signal of the virus control wells (culture medium with the pseudovirus added) . According to the inhibition ratio, IC 50 can be calculated by the Reed-Muench algorithm. Details of this method can be found, e.g., in Reed, Lowell Jacob, and Hugo Muench. "A simple method of estimating fifty per cent endpoints. " American Journal of Epidemiology 27.3 (1938) : 493-497; and Nie et al., "Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2. " Emerging microbes &infections 9.1 (2020) : 680-686; each of which is incorporated herein by reference in the entirety. The IC 50 of all the anti-SARS-CoV-2 antibodies except 05-8G6-IgG1-LALA were determined and the results are listed in the table below.
Table 4
Anti-SARS-CoV-2 Antibody IC 50 (ng/ml)
05-9G11-IgG1-LALA 79
03-10D12-IgG1-LALA 20
01-2H10-IgG1-LALA 26
03-10F9-IgG1-LALA 16
03-9A8-IgG1-LALA 38
03-1F9-IgG1-LALA 74
09-7B8-IgG1-LALA 37
09-4E5-IgG1-LALA 112
05-8G6-IgG1-LALA -
09-2F7-IgG1-LALA 593
Example 8. IC 50 determination of purified human anti-SARS-CoV-2 antibodies in combination: blocking human ACE2 binding to SARS-CoV-2 S protein
Blocking effects of purified human anti-SARS-CoV-2 antibodies (alone or in combination) were detected by fluorescence-activated cell sorting (FACS) using the CHO-S-ACE2 cells. First, the purified antibodies were diluted by serial dilution to 9000 ng/mL, 3000 ng/mL, 1000 ng/mL, 333.3 ng/mL, 111.1 ng/mL, 37.0 ng/mL, 12.3 ng/mL, 4.1ng/mL, 1.4ng/mL, and 0.5ng/mL. The PE labeled Recombinant SARA-Cov-2 spike Protein (RBD, His Tag) protein  was diluted to 200 ng/ml. Next, CHO-S-ACE2 cells were washed to remove residual culture medium and resuspended in PBS to a desired cell density (5 × 10 4cells for 30 ul) . The resuspended cells were added to a 96-well plate (30 μl/well) . Next, 30 μl diluted antibodies were added to the corresponding wells, followed by an incubation at 4℃ for 30 minutes. Afterwards, 30 μl diluted RBD were added to the corresponding wells, and the plate was further incubated at 4℃ for 1 hour. Next, 200 μl PBS was added to each well, and then the plate was centrifuged at 1600 rpm for 6 minutes. Supernatant in each well was discarded. Afterwards, cells in each well were washed as described above and then resuspended in 30 μl PBS for FACS analysis.
Raw data was analyzed to generate the mean fluorescence intensity (MFI) reflecting RBD-cell binding at different antibody concentrations. The MFI values were then used to generate a fitting curve with respect to the antibody concentrations. More specifically, logarithm of antibody concentrations were calculated and used as the X-axis variable, while the corresponding MFI was used as the Y-axis variable. The fitting curves of 03-9A8-IgG1-LALA (Ab. 01, or 9A8) , 03-10D12-IgG1-LALA (Ab. 02, or 03-10D12) , 05-9G11-IgG1-LALA (Ab. 03, or 05-9G11) , 09-4E5-IgG1-LALA (Ab. 04, or 09-4E5) , and 09-7B8-IgG-LALA (Ab. 05, or 09-7B8) alone, or in combination are shown in FIGS. 8A-8E. The IC 50 value was determined and the determined IC 50 is shown in the tables below and FIG. 9.
Table 5
Antibody Name IC 50 (ng/mL) R 2
30μL Ab. 01 (9A8) 296.4 0.9809
30μL Ab. 02 (03-10D12) 216.6 0.9771
30μL Ab. 03 (05-9G11) 137.4 0.9809
30μL Ab. 04 (09-4E5) 447.3 0.9897
30μL Ab. 05 (09-7B8) 488.8 0.9892
15μL Ab. 01 (9A8) +15μL Ab. 02 (03-10D12) 269.6 0.9963
15μL Ab. 01 (9A8) +15μL Ab. 03 (05-9G11) 210.0 0.9870
15μL Ab. 01 (9A8) +15μL Ab. 04 (09-4E5) 251.3 0.9956
15μL Ab. 01 (9A8) +15μL Ab. 05 (09-7B8) 245.5 0.9918
15μL Ab. 02 (03-10D12) +15μL Ab. 03 (05-9G11) 149.8 0.9900
15μL Ab. 02 (03-10D12) +15μL Ab. 04 (09-4E5) 286.4 0.9961
15μL Ab. 02 (03-10D12) +15μL Ab. 05 (09-7B8) 338.0 0.9984
15μL Ab. 03 (05-9G11) +15μL Ab. 04 (09-4E5) 228.6 0.9957
15μL Ab. 03 (05-9G11) +15μL Ab. 05 (09-7B8) 184.5 0.9912
15μL Ab. 04 (09-4E5) +15μL Ab. 05 (09-7B8) 472.3 0.9971
The results indicate that anti-SARS-CoV-2 antibodies can be combined to have an improved blocking effect (or lower IC 50) . For example, the IC 50 of 03-9A8-IgG1-LALA and09-4E5-IgG1-LALA were determined as 296.4ng/mL and 488.8ng/mL, respectively. When the two antibodies were used in combination, the IC 50 was reduced to 245.5 ng/mL.
Example 9. IC 50 determination of purified human anti-SARS-CoV-2 antibodies in combination: neutralizing COVID-19 pseudovirus
COVID-19 pseudovirus was prepared and the neutralizing effects of the anti-SARS-CoV-2 antibodies were determined as described in Example 7. The IC 50 of 03-10D12-IgG1-LALA (Ab1) , 03-9A8-IgG1-LALA (Ab2) , 09-4E5-IgG1-LALA (Ab3) , 09-7B8-IgG-LALA (Ab4) , 05-9G11-IgG1-LALA (Ab5) alone, or in combination were determined and the results are listed in the table below. The results indicate that anti-SARS-CoV-2 antibodies can be combined to have an improved neutralizing activity (or lower IC 50) . For example, the IC 50 of 03-10D12-IgG1-LALA and 05-9G11-IgG1-LALA were determined as 0.016828 μg/mL and 0.1147 μg/mL, respectively. When the two antibodies were used in combination, the IC 50 was reduced to 0.010834 μg/mL.
Table 6
Anti-SARS-CoV-2 Antibody IC 50 (μg/ml)
Ab1 0.016828
Ab2 0.213159
Ab3 0.137845
Ab4 0.032121
Ab5 0.1147
Ab1 + Ab2 0.031044
Ab1 + Ab3 0.020152
Ab1 + Ab4 0.021578
Ab1 + Ab5 0.010834
Ab2 + Ab3 0.161572
Ab2 + Ab4 0.07572
Ab2 + Ab5 0.125433
Ab3 + Ab4 0.092523
Ab3 + Ab5 0.051045
Ab4 + Ab5 0.02586
Furthermore, COVID-19 pseudovirus neutralizing activity titration was measured for the anti-SARS-CoV-2 antibodies alone, or in combination. The results for 03-10D12-IgG1-LALA  (Ab1) , 03-9A8-IgG1-LALA (Ab2) , 09-4E5-IgG1-LALA (Ab3) , 09-7B8-IgG-LALA (Ab4) , 05-9G11-IgG1-LALA (Ab5) , and Ab1 + Ab5 combination, are shown in FIG. 10. The results are consistent with the IC 50 determination results as shown in Table 6. For example, the titration curve of combined 03-10D12-IgG1-LALA and 05-9G11-IgG1-LALA presents a shift towards left relative to the titration curve of each individual antibody.
OTHER EMBODIMENTS
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims (109)

  1. An antibody or antigen-binding fragment thereof that binds toa coronavirusspike protein, comprising:
    a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VH CDR3 amino acid sequence; and
    a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80%identical to a selected VL CDR3 amino acid sequence,
    wherein the selected VH CDRs 1, 2, and 3 amino acid sequences and the selected VL CDRs, 1, 2, and 3 amino acid sequences are one of the following:
    (1) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 1, 2, 3, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 4, 5, 6, respectively;
    (2) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 7, 8, 9, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 10, 11, 12, respectively;
    (3) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 13, 14, 15, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 16, 17, 18, respectively;
    (4) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 19, 20, 21, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 22, 23, 24, respectively;
    (5) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 25, 26, 27 respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 28, 29, 30, respectively;
    (6) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 31, 32, 33, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 34, 35, 36, respectively;
    (7) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 37, 38, 39, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 40, 41, 42, respectively;
    (8) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 43, 44, 45, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 46, 47, 48, respectively;
    (9) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 49, 50, 51, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 52, 53, 54, respectively;
    (10) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 55, 56, 57, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 58, 59, 60, respectively;
    (11) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 61, 62, 63, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 64, 65, 66, respectively;
    (12) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 67, 68, 69, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 70, 71, 72, respectively;
    (13) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 73, 74, 75, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 76, 77, 78, respectively;
    (14) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 79, 80, 81, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 82, 83, 84, respectively;
    (15) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 85, 86, 87, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 88, 89, 90, respectively;
    (16) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 91, 92, 93, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 94, 95, 96, respectively;
    (17) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 97, 98, 99, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 100, 101, 102, respectively;
    (18) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 103, 104, 105, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 106, 107, 108, respectively;
    (19) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 109, 110, 111, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 112, 113, 114, respectively;
    (20) the selected VH CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 115, 116, 117, respectively, and the selected VL CDRs 1, 2, 3 amino acid sequences are set forth in SEQ ID NOs: 118, 119, 120, respectively.
  2. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3 respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively according to Kabat numbering scheme.
  3. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively according to Kabat numbering scheme.
  4. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 13, 14,  and 15, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively according to Kabat numbering scheme.
  5. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively according to Kabat numbering scheme.
  6. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 25, 26, and 27, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively according to Kabat numbering scheme.
  7. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively according to Kabat numbering scheme.
  8. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 37, 38, and 39, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 40, 41, and 42, respectively according to Kabat numbering scheme.
  9. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 43, 44, and 45, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences  set forth in SEQ ID NOs: 46, 47, and 48, respectively according to Kabat numbering scheme.
  10. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 52, 53, and 54, respectively according to Kabat numbering scheme.
  11. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 55, 56, and 57, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 58, 59, and 60, respectively according to Kabat numbering scheme.
  12. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 61, 62, and 63, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 64, 65, and 66, respectively according to Chothia numbering scheme.
  13. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 67, 68, and 69, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 70, 71, and 72, respectively according to Chothia numbering scheme.
  14. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 73, 74, and 75, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences  set forth in SEQ ID NOs: 76, 77, and 78, respectively according to Chothia numbering scheme.
  15. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 79, 80, and 81, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 82, 83, and 84, respectively according to Chothia numbering scheme.
  16. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 85, 86, and 87, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 88, 89, and 90, respectively according to Chothia numbering scheme.
  17. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 91, 92, and 93, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 94, 95, and 96, respectively according to Chothia numbering scheme.
  18. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 97, 98, and 99, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 100, 101, and 102, respectively according to Chothia numbering scheme.
  19. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 103, 104, and 105, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid  sequences set forth in SEQ ID NOs: 106, 107, and 108, respectively according to Chothia numbering scheme.
  20. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 109, 110, and 111, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 112, 113, and 114, respectively according to Chothia numbering scheme.
  21. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 115, 116, and 117, respectively, and the VL comprises CDRs 1, 2, 3 with the amino acid sequences set forth in SEQ ID NOs: 118, 119, and 120, respectively according to Chothia numbering scheme.
  22. The antibody or antigen-binding fragment thereof of any one of claims 1-21, wherein the antibody or antigen-binding fragment thereof specifically binds toa human coronavirus spike protein.
  23. The antibody or antigen-binding fragment thereof of any one of claims 1-22, wherein the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof (e.g., a human IgG1 antibody) .
  24. The antibody or antigen-binding fragment thereof of any one of claims 1-23, wherein the antibody or antigen-binding fragment thereof comprises a mouse constant domain.
  25. The antibody or antigen-binding fragment thereof of any one of claims 1-23, wherein the antibody or antigen-binding fragment thereof comprises a human constant domain.
  26. The antibody or antigen-binding fragment thereof of claim 25, wherein the human constant domain comprises LALA mutations.
  27. A nucleic acid comprising a polynucleotide encoding a polypeptide comprising:
    (1) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 122 binds to a coronavirus spike protein;
    (2) an immunoglobulin light chain or a fragment thereof comprising a light chain variable region (VL) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 121 binds to thecoronavirus spike protein;
    (3) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 124 binds to the coronavirus spike protein;
    (4) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 123 binds to the coronavirus spike protein;
    (5) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 126 binds to the coronavirus spike protein;
    (6) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively, and wherein the VL, when paired with a VH  comprising the amino acid sequence set forth in SEQ ID NO: 125 binds to the coronavirus spike protein;
    (7) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 128 binds to the coronavirus spike protein;
    (8) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 127 binds to the coronavirus spike protein;
    (9) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 25, 26, and 27, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 130 binds to the coronavirus spike protein;
    (10) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 129 binds to the coronavirus spike protein;
    (11) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 132 binds to the coronavirus spike protein;
    (12) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively, and wherein the VL, when paired with  a VH comprising the amino acid sequence set forth in SEQ ID NO: 131 binds to the coronavirus spike protein;
    (13) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 37, 38, and 39, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 134 binds to the coronavirus spike protein;
    (14) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 40, 41, and 42, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 133 binds to the coronavirus spike protein;
    (15) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 43, 44, and 45, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 136 binds to the coronavirus spike protein;
    (16) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 46, 47, and 48, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 135 binds to the coronavirus spike protein;
    (17) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 138 binds to the coronavirus spike protein;
    (18) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 52, 53, and 54, respectively, and wherein the VL, when paired with  a VH comprising the amino acid sequence set forth in SEQ ID NO: 137 binds to the coronavirus spike protein;
    (19) an immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 55, 56, and 57, respectively, and wherein the VH, when paired with a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 140 binds to the coronavirus spike protein; or
    (20) an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 58, 59, and 60, respectively, and wherein the VL, when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 139 binds to the coronavirus spike protein.
  28. The nucleic acid of claim 27, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively.
  29. The nucleic acid of claim 27, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively.
  30. The nucleic acid of claim 27, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively.
  31. The nucleic acid of claim 27, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof  comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively.
  32. The nucleic acid of claim 27, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively.
  33. The nucleic acid of claim 27, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively.
  34. The nucleic acid of claim 27, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively.
  35. The nucleic acid of claim 27, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively.
  36. The nucleic acid of claim 27, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 25, 26, and 27, respectively.
  37. The nucleic acid of claim 27, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof  comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively.
  38. The nucleic acid of claim 27, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively.
  39. The nucleic acid of claim 27, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively.
  40. The nucleic acid of claim 27, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 37, 38, and 39, respectively.
  41. The nucleic acid of claim 27, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 40, 41, and 42, respectively.
  42. The nucleic acid of claim 27, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 43, 44, and 45, respectively.
  43. The nucleic acid of claim 27, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof  comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 46, 47, and 48, respectively.
  44. The nucleic acid of claim 27, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively.
  45. The nucleic acid of claim 27, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 52, 53, and 54, respectively.
  46. The nucleic acid of claim 27, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof comprising a VH comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 55, 56, and 57, respectively.
  47. The nucleic acid of claim 27, wherein the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof comprising a VL comprising CDRs 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 58, 59, and 60, respectively.
  48. The nucleic acid of any one of claims 27-47, wherein the VH when paired with a VL specifically binds to the coronavirus spike protein, or the VL when paired with a VH specifically binds to the coronavirus spike protein.
  49. The nucleic acid of any one of claims 27-48, wherein the immunoglobulin heavy chain or the fragment thereof is a human immunoglobulin heavy chain or a fragment thereof, and the immunoglobulin light chain or the fragment thereof is a human  immunoglobulin light chain or a fragment thereof.
  50. The nucleic acid of any one of claims 27-49, wherein the nucleic acid is cDNA.
  51. A vector comprising one or more of the nucleic acids of any one of claims 27-50.
  52. A vector comprising two of the nucleic acids of any one of claims 27-50, wherein the vector encodes the VH region and the VL region that together bind to the coronavirus spike protein.
  53. A pair of vectors, wherein each vector comprises one of the nucleic acids of any one of claims 27-50, wherein together the pair of vectors encodes the VH region and the VL region that together bind to the coronavirus spike protein.
  54. A cell comprising the vector of claim 51 or 52, or the pair of vectors of claim 53.
  55. The cell of claim 54, wherein the cell is a CHO cell.
  56. A cell comprising one or more of the nucleic acids of any one of claims 27-50.
  57. A cell comprising two of the nucleic acids of any one of claims 27-50.
  58. The cell of claim 57, wherein the two nucleic acids together encode the VH region and the VL region that together bind to the coronavirus spike protein.
  59. An antibody or antigen-binding fragment thereof that binds to a coronavirus spike proteincomprising
    a heavy chain variable region (VH) comprising an amino acid sequence that is at least 80%identical to a selected VH sequence, and a light chain variable region (VL) comprising an amino acid sequence that is at least 80%identical to a selected VL  sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:
    (1) the selected VH sequence is SEQ ID NO: 121, and the selected VL sequence is SEQ ID NO: 122;
    (2) the selected VH sequence is SEQ ID NO: 123, and the selected VL sequence is SEQ ID NO: 124;
    (3) the selected VH sequence is SEQ ID NO: 125, and the selected VL sequence is SEQ ID NO: 126;
    (4) the selected VH sequence is SEQ ID NO: 127, and the selected VL sequence is SEQ ID NO: 128;
    (5) the selected VH sequence is SEQ ID NO: 129, and the selected VL sequence is SEQ ID NO: 130;
    (6) the selected VH sequence is SEQ ID NO: 131, and the selected VL sequence is SEQ ID NO: 132;
    (7) the selected VH sequence is SEQ ID NO: 133, and the selected VL sequence is SEQ ID NO: 134;
    (8) the selected VH sequence is SEQ ID NO: 135, and the selected VL sequence is SEQ ID NO: 136;
    (9) the selected VH sequence is SEQ ID NO: 137, and the selected VL sequence is SEQ ID NO: 138;
    (10) the selected VH sequence is SEQ ID NO: 139, and the selected VL sequence is SEQ ID NO: 140.
  60. The antibody or antigen-binding fragment thereof of claim 59, wherein the VH comprises the sequence of SEQ ID NO: 121 and the VL comprises the sequence of SEQ ID NO: 122.
  61. The antibody or antigen-binding fragment thereof of claim 59, wherein the VH comprises the sequence of SEQ ID NO: 123 and the VL comprises the sequence of SEQ ID NO: 124.
  62. The antibody or antigen-binding fragment thereof of claim 59, wherein the VH comprises the sequence of SEQ ID NO: 125 and the VL comprises the sequence of SEQ ID NO: 126.
  63. The antibody or antigen-binding fragment thereof of claim 59, wherein the VH comprises the sequence of SEQ ID NO: 127 and the VL comprises the sequence of SEQ ID NO: 128.
  64. The antibody or antigen-binding fragment thereof of claim 59, wherein the VH comprises the sequence of SEQ ID NO: 129 and the VL comprises the sequence of SEQ ID NO: 130.
  65. The antibody or antigen-binding fragment thereof of claim 59, wherein the VH comprises the sequence of SEQ ID NO: 131 and the VL comprises the sequence of SEQ ID NO: 132.
  66. The antibody or antigen-binding fragment thereof of claim 59, wherein the VH comprises the sequence of SEQ ID NO: 133 and the VL comprises the sequence of SEQ ID NO: 134.
  67. The antibody or antigen-binding fragment thereof of claim 59, wherein the VH comprises the sequence of SEQ ID NO: 135 and the VL comprises the sequence of SEQ ID NO: 136.
  68. The antibody or antigen-binding fragment thereof of claim 59, wherein the VH comprises the sequence of SEQ ID NO: 137 and the VL comprises the sequence of SEQ ID NO: 138.
  69. The antibody or antigen-binding fragment thereof of claim 59, wherein the VH comprises the sequence of SEQ ID NO: 139 and the VL comprises the sequence of SEQ ID NO: 140.
  70. The antibody or antigen-binding fragment thereof of any one of claims 59-69, wherein the antibody or antigen-binding fragment thereof specifically binds to a human coronavirus spike protein.
  71. The antibody or antigen-binding fragment thereof of any one of claims 59-70, wherein the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof (e.g., a human IgG1 antibody) .
  72. The antibody or antigen-binding fragment thereof of any one of claims 59-71, wherein the antibody or antigen-binding fragment thereof comprises a mouse constant domain.
  73. The antibody or antigen-binding fragment thereof of any one of claims 59-71, wherein the antibody or antigen-binding fragment thereof comprises a human constant domain.
  74. The antibody or antigen-binding fragment thereof of claim 73, wherein the human constant domain comprises LALA mutations.
  75. An antibody or antigen-binding fragment thereof comprising the VH CDRs 1, 2, 3, and the VL CDRs 1, 2, 3 of the antibody or antigen-binding fragment thereof of any one of claims 1-26 and 59-74.
  76. The antibody or antigen-binding fragment thereof of any one of claims 1-26 and 59-75, wherein the antibody or antigen-binding fragment thereof specifically binds to an S1 subunit of the coronavirus spike protein.
  77. The antibody or antigen-binding fragment thereof of claim 76, wherein the antibody or antigen-binding fragment thereof specifically binds to a receptor binding domain  (RBD) of the S1 subunit of the coronavirus spike protein, wherein the amino acid sequence of the RBD isat least 80%identical to amino acids 319-541of SEQ ID NO: 141.
  78. An antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof of any one of claims 1-26 and 59-77.
  79. A method of producing an antibody or an antigen-binding fragment thereof, the method comprising
    (a) culturing the cell of any one of claims 54-58 under conditions sufficient for the cell to produce the antibody or the antigen-binding fragment thereof; and
    (b) collecting the antibody or the antigen-binding fragment thereof produced by the cell.
  80. A method of treating a subject having a coronavirus-related disease, the method comprising administering a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-26 and 59-78 to the subject.
  81. A method of neutralizing a coronavirus, the method comprising contacting the coronavirus with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof of any one of claims 1-26 and 59-78.
  82. A method of blocking internalization of a coronavirus by a cell, the method comprising
    contacting the coronavirus with an effective amount of a composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-26 and 59-78.
  83. A method of identifying a subject as having a coronavirus disease, the method comprising
    detecting a sample collected from the subject as having the coronavirus by the antibody or antigen-binding fragment thereof of any one of claims 1-26 and 59-78,  thereby identifying the subject as having a coronavirus infection.
  84. The method of claim 83, wherein the sample is a blood sample, a saliva sample, a stool sample, or a liquid sample from the respiratory tract of the subject.
  85. The method of any one of claims 1-84, wherein the coronavirus is SARS-CoV-2.
  86. The method of any one of claims 1-84, wherein the coronavirus is MERS-CoV.
  87. The method of any one of claims 1-84, wherein the coronavirus is SARS-CoV.
  88. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-26 and 59-78, and a pharmaceutically acceptable carrier.
  89. A method of treating a subject having a coronavirus-related disease or neutralizing coronavirus in the subject, the method comprising administering to the subject a therapeutically effective amount of a first antibody or antigen-binding fragment thereof, and a therapeutically effective amount of a second antibody or antigen-binding fragment thereof, wherein the first antibody or antigen-binding fragment thereof is the antibody or antigen-binding fragment thereof of any one of claims 1-26 and 59-78.
  90. The method of claim 89, wherein thesecond antibody or antigen-binding fragment thereof is an anti-Sprotein antibody or antigen-binding fragment thereof.
  91. The method of claim 89 or 90, wherein the method further comprises administering a third antibody or antigen-binding fragment thereof to the subject.
  92. The method of any one of claims 89-91, wherein the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof target different epitopes of an S protein.
  93. The method of any one of claims 89-92, wherein the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof are selected from FIG. 16.
  94. The method of any one of claims 89-92, wherein the first antibody or antigen-binding fragment thereof comprises VH CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 37, 38, 39, respectively, and VL CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 40, 41, 42, respectively; and
    the second antibody or antigen-binding fragment thereof comprises VH CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 31, 32, 33, respectively, and VL CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 34, 35, 36, respectively.
  95. The method of any one of claims 89-92, wherein the first antibody or antigen-binding fragment thereof comprises VH CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 13, 14, 15, respectively, and VL CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 16, 17, 18, respectively; and
    the second antibody or antigen-binding fragment thereof comprises VH CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 49, 50, 51, respectively, and VL CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 52, 53, 54, respectively.
  96. A pharmaceutical composition comprisingtwo or more antibodies or antigen-binding fragment thereof, wherein one of the two or more antibodies or antigen-binding fragment thereof is the antibody or antigen-binding fragment thereof of any one of claims 1-26 and 59-78.
  97. The pharmaceutical composition of claim 96, wherein the pharmaceutical composition further comprises a third antibody or antigen-binding fragment thereof.
  98. The pharmaceutical composition of any one of claims 96-97, wherein each antibody or antigen-binding fragment thereof targets different epitopes of an S protein.
  99. The pharmaceutical composition of any one of claims 96-98, wherein two antibodies or antigen-binding fragments thereof are selected from FIG. 16.
  100. The pharmaceutical composition of any one of claims 96-99, wherein a first antibody or antigen-binding fragment thereof comprises VH CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 37, 38, 39, respectively, and VL CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 40, 41, 42, respectively; and
    a second antibody or antigen-binding fragment thereof comprises VH CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 31, 32, 33, respectively, and VL CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 34, 35, 36, respectively.
  101. The pharmaceutical composition of any one of claims 96-99, wherein a first antibody or antigen-binding fragment thereof comprises VH CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 13, 14, 15, respectively, and VL CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 16, 17, 18, respectively; and
    a second antibody or antigen-binding fragment thereof comprises VH CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 49, 50, 51, respectively, and VL CDRs 1, 2, 3 amino acid sequences that are set forth in SEQ ID NOs: 52, 53, 54, respectively.
  102. An anti-coronavirus spike protein antibody or antigen binding fragment thereof comprising: a heavy chain variable region (VH) comprising VH CDR1, VH CDR2,  and VH CDR3; and a light chain variable region (VL) comprising VL CDR1, VL CDR2, and VL CDR3, wherein:
    (a) the VH CDR1 sequence is SX 1X 2X 3X 4X 5X 6 (SEQ ID NO: 142) ; wherein:
    X 1 is N, F, G, Y, or null;
    X 2 is G, or null;
    X 3 is Y, or null;
    X 4 is Y, A, or null;
    X 5 is M, L, or W;
    X 6 is S, N, T, A, or H;
    (b) the VH CDR2 sequence is X 7IX 8X 9X 10GX 11X 12X 13X 14YX 15X 16SX 17X 18X 19 (SEQ ID NO: 143) , wherein
    X 7 is V, S, or Y;
    X 8 is Y, S, W, or H;
    X 9 is Y, G, F, or null;
    X 10 is S, or D;
    X 11 is G, S, or null;
    X 12 is S, or N;
    X 13 is T, or K;
    X 14 is Y, F, or N;
    X 15 is A, or N;
    X 16 is D, or P;
    X 17 is V, or L;
    X 18 is K, E, or R;
    X 19 is G, A, S, or N;
    (c) the VH CDR3 sequence is X 20X 21X 22X 23X 24X 25X 26X 27X 28X 29X 30X 31DX 32 (SEQ ID NO: 144) , wherein
    X 20 is D, E, or Q;
    X 21 is R, L, V, T, or A;
    X 22 is G, or null;
    X 23 is Y, or null;
    X 24 is S, V, L, or null;
    X 25 is S, G, D, or null;
    X 26 is D, S, P, K, N, G, or null;
    X 27 is Y, N, V, L, W, or null;
    X 28 is N, S, L, T, or null;
    X 29 is Y, S, F, G, or D;
    X 30 is G, N, F, or S;
    X 31 is M, or F;
    X 32 is V, Y, or I.
  103. The anti-coronavirus spike protein antibody or antigen binding fragment thereof of claim 102, wherein X 1 is N, X 2 is null, X 3 is null, X 4 is Y, X 5 is M, and X 6 is S.
  104. The anti-coronavirus spike protein antibody or antigen binding fragment thereof of claim 102 or 103, wherein X 7 is V, X 8 is Y, X 9 is Y, X 10 is S, X 11 is G, X 12 is S, X 13 is T, X 14 is Y, X 15 is A, X 16 is D, X 17 is V, X 18 is K, and X 19 is G.
  105. The anti-coronavirus spike protein antibody or antigen binding fragment thereof of any one of claims 102-104, wherein X 20 is D, X 21 is R, X 22 is null, X 23 is null, X 24 is null, X 25 is null, X 27 is Y, X 28 is null, X 29 is Y, X 30 is G, X 31 is M, and X 32 is V.
  106. The anti-coronavirus spike protein antibody or antigen binding fragment thereof in any one of claims 102-105, wherein:
    (a) the VL CDR1 sequence is X 33AX 34QX 35IX 36X 37X 38LX 39 (SEQ ID NO: 145) ; wherein:
    X 33 is Q, or R;
    X 34 is S, or R;
    X 35 is D, or G;
    X 36 is S, N, or T;
    X 37 is N, I, S, or K;
    X 38 is Y, or F;
    X 39 is N, or A;
    (b) the VL CDR2 sequence is X 40ASX 41LX 42X 43 (SEQ ID NO: 146) ; wherein:
    X 40 is D, or A;
    X 41 is N, T, or S;
    X 42 is E, Q, or L;
    X 43 is T, or S;
    (c) the VL CDR3 sequence is X 44X 45X 46X 47X 48X 49X 50X 51X 52T (SEQ ID NO: 147) ; wherein:
    X 44 is Q, or L;
    X 45 is Q, or H;
    X 46 is Y, L, or H;
    X 47 is D, H, or N;
    X 48 is N, H, or S;
    X 49 is L, I, or Y;
    X 50 is P, or L;
    X 51 is R, M, L, P, or null;
    X 52 is W, Y, F, L, or null.
  107. The anti-coronavirus spike protein antibody or antigen binding fragment thereof of claim 106, wherein X 33 is Q, X 34 is S, X 35 is D, X 36 is S, X 37 is N, X 38 is Y, and X 39 is N.
  108. The anti-coronavirus spike protein antibody or antigen binding fragment thereof of claim 106 or 107, wherein X 40 is D, X 41 is N, X 42 is E, X 43 is T.
  109. The anti-coronavirus spike protein antibody or antigen binding fragment thereof of any one of claims 106-108, wherein X 44 is Q, X 45 is Q, X 46 is Y, X 47 is D, X 48 is N, X 49 is L, X 50 is P, X 51 is null, and X 52 is null.
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