WO2023048656A2 - Human monoclonal antibodies against the receptor-binding domain of sars-cov-2 spike protein - Google Patents

Human monoclonal antibodies against the receptor-binding domain of sars-cov-2 spike protein Download PDF

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Publication number
WO2023048656A2
WO2023048656A2 PCT/TH2022/000037 TH2022000037W WO2023048656A2 WO 2023048656 A2 WO2023048656 A2 WO 2023048656A2 TH 2022000037 W TH2022000037 W TH 2022000037W WO 2023048656 A2 WO2023048656 A2 WO 2023048656A2
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WIPO (PCT)
Prior art keywords
comprises seq
antibody
antigen
sequence
binding fragment
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PCT/TH2022/000037
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French (fr)
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WO2023048656A3 (en
Inventor
Trairak PISITKUN
Chatikorn BOONKRAI
Nattachai SRISAWAT
Tossapon WONGTANGPRASERT
Thittaya AUDOMSUN
Tanapati PHAKHAM
Chadaporn ATTAKITBANCHA
Watchadaporn CHAISURIYONG
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Chulalongkorn University
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Publication of WO2023048656A2 publication Critical patent/WO2023048656A2/en
Publication of WO2023048656A3 publication Critical patent/WO2023048656A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • 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
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to antibodies and antigen-binding fragments that bind specifically to the receptor-binding domain of SARS-CoV-2 and methods for treating or preventing SARS-CoV-2 infections with said antibodies and antigen-binding fragments.
  • Coronavirus disease 2019 is an infectious disease from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has rapidly spread globally, causing a pandemic.
  • SARS-CoV-2 is an RNA virus with the characteristic of multiple spike glycoproteins on its envelope.
  • the receptor-binding domain (RBD) on the spike proteins binds specifically with the cellular receptor angiotensin-converting enzyme 2 (ACE2) of its host cells, resulting in fusion cascade and virus entry.
  • ACE2 cellular receptor angiotensin-converting enzyme 2
  • a monoclonal antibody with neutralizing activity against SARS-CoV-2 could be used to block the virus entry and replication.
  • the disclosure provides an antibody or antigen-binding fragment that binds to a SARS-CoV-2 spike protein.
  • the SARS-CoV-2 spike protein comprises any one of SEQ ID NOs: 129-136, or a sequence that shares at least 90%, at least 95%, or least 98% identity thereto.
  • the antibody or antigen-binding fragment binds the SARS- CoV-2 spike protein with a KD value of about 10-12 M, about 10-11 M, about 10-10 M, about 10-9 M, or about 10-8 M.
  • the antibody or antigen-binding fragment inhibits the binding of the SARS-CoV-2 spike protein to an ACE2 receptor with an IC50 of about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL, about 65 ng/mL, about 70 ng/mL, about 75 ng/mL, or about 80 ng/mL.
  • the antibody or antigen-binding fragment described herein comprises a heavy-chain complementarity-determining region 1 (HCDR1), a heavy-chain complementarity-determining region 2 (HCDR2), a heavy-chain complementaritydetermining region 3 (HCDR3), a light-chain complementarity determining region 1 (LCDR1), a light-chain complementarity determining region 2 (LCDR2), and a light-chain complementarity determining region 3 (LCDR3).
  • HCDR1 heavy-chain complementarity-determining region 1
  • HCDR2 heavy-chain complementarity-determining region 2
  • HCDR3 heavy-chain complementarity determining region 3
  • LCDR1 light-chain complementarity determining region 1
  • LCDR2 light-chain complementarity determining region 2
  • LCDR3 light-chain complementarity determining region 3
  • the antibody or antigen-binding fragment described herein comprises: a) any one of the HCDR1 sequences set forth in Table 2; b) any one of the HCDR2 sequences set forth in Table 2; c) any one of the HCDR3 sequences set forth in Table 2; d) any one of the LCDR1 sequences set forth in Table 2; e) any one of the LCDR2 sequences set forth in Table 2; and f) any one of the LCDR3 sequences set forth in Table 2, wherein no more than 1, 2, or 3, amino acids in the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and/or LCDR3 sequences are substituted, deleted or inserted.
  • the HCDR1 comprises SEQ ID NO: 6; the HCDR2 comprises SEQ ID NO: 7; and the HCDR3 comprises SEQ ID NO: 8.
  • the HCDR1 comprises SEQ ID NO: 14; the HCDR2 comprises SEQ ID NO: 15; and the HCDR3 comprises SEQ ID NO: 16.
  • the HCDR1 comprises SEQ ID NO: 22; the HCDR2 comprises SEQ ID NO: 23; and the HCDR3 comprises SEQ ID NO: 24.
  • the HCDR1 comprises SEQ ID NO: 30; the HCDR2 comprises SEQ ID NO: 31; and the HCDR3 comprises SEQ ID NO: 32.
  • the HCDR1 comprises SEQ ID NO: 38; the HCDR2 comprises SEQ ID NO: 39; and the HCDR3 comprises SEQ ID NO: 40.
  • the HCDR1 comprises SEQ ID NO: 46; the HCDR2 comprises SEQ ID NO: 47; and the HCDR3 comprises SEQ ID NO: 48.
  • the HCDR1 comprises SEQ ID NO: 54; the HCDR2 comprises SEQ ID NO: 55; and the HCDR3 comprises SEQ ID NO: 56.
  • the HCDR1 comprises SEQ ID NO: 62; the HCDR2 comprises SEQ ID NO: 63; and the HCDR3 comprises SEQ ID NO: 64
  • the LCDR1 comprises SEQ ID NO: 2; the LCDR2 comprises SEQ ID NO: 3; and the LCDR3 comprises SEQ ID NO: 4.
  • the LCDR1 comprises SEQ ID NO: 10; the LCDR2 comprises SEQ ID NO: 11; and the LCDR3 comprises SEQ ID NO: 12.
  • the LCDR1 comprises SEQ ID NO: 18; the LCDR2 comprises SEQ ID NO: 19; and the LCDR3 comprises SEQ ID NO: 20.
  • the LCDR1 comprises SEQ ID NO: 26; the LCDR2 comprises SEQ ID NO: 27; and the LCDR3 comprises SEQ ID NO: 28.
  • the LCDR1 comprises SEQ ID NO: 34; the LCDR2 comprises SEQ ID NO: 35; and the LCDR3 comprises SEQ ID NO: 36.
  • the LCDR1 comprises SEQ ID NO: 42; the LCDR2 comprises SEQ ID NO: 43; and the LCDR3 comprises SEQ ID NO: 44.
  • the LCDR1 comprises SEQ ID NO: 50; the LCDR2 comprises SEQ ID NO: 51; and the LCDR3 comprises SEQ ID NO: 52.
  • the LCDR1 comprises SEQ ID NO: 58; the LCDR2 comprises SEQ ID NO: 59; and the LCDR3 comprises SEQ ID NO: 60.
  • the antibody or antigen-binding fragment described herein comprises: a) a heavy-chain variable region (VH) comprising a sequence set forth in Table 2, or a sequence at least that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto; and b) a light-chain variable region (VL) comprising a sequence set forth in Table 2, or a sequence at least that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto.
  • VH heavy-chain variable region
  • VL light-chain variable region
  • the antibody or antigen-binding fragment described herein comprises: a) the VH comprises SEQ ID NO: 5, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 1, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the antibody or antigen-binding fragment described herein comprises: a) the VH comprises SEQ ID NO: 13, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 9, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the antibody or antigen-binding fragment described herein comprises: a) the VH comprises SEQ ID NO: 21, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 17, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the antibody or antigen-binding fragment described herein comprises: a) the VH comprises SEQ ID NO: 29, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 25, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the antibody or antigen-binding fragment described herein comprises: a) the VH comprises SEQ ID NO: 37, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 33, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the antibody or antigen-binding fragment described herein comprises: a) the VH comprises SEQ ID NO: 45, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 41, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the antibody or antigen-binding fragment described herein comprises: a) the VH comprises SEQ ID NO: 53, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 49, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the antibody or antigen-binding fragment described herein comprises: a) the VH comprises SEQ ID NO: 61, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 57, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the antibody or antigen-binding fragment described herein further comprises a constant region.
  • the constant region is selected from an IgA, IgM, IgGl, IgG2, IgG3, or IgG4 constant region.
  • the antibody or antigen-binding fragment described herein is humanized.
  • the antibody or antigen-binding fragment described herein is recombinant.
  • the antibody or antigen-binding fragment described herein is multispecific.
  • the antibody or antigen-binding fragment described herein neutralizes a SARS-CoV-2 virus or a variant thereof.
  • the disclosure provides a pharmaceutical composition, comprising an antibody or antigen-binding fragment described herein and a pharmaceutically acceptable carrier, excipient, and/or diluent.
  • the disclosure further provides a pharmaceutical composition comprising the antibody or antigen-binding fragment described herein, for use in the treatment of COVID-19.
  • the pharmaceutical composition further comprises one or more additional therapeutic agents.
  • the one or more additional therapeutic agents is selected from an antibody, an antiviral, a steroid, an antiinflammatory, an immune booster, vitamin C, vitamin D, vitamin E, or any combination thereof.
  • the pharmaceutical composition is formulated for parenteral administration, enteral administration, mucosal administration, nasal administration, oral administration, or intra-articular administration.
  • the administration comprises inhalation, intravenous injection, infusion, subcutaneous injection, or intramuscular injection.
  • the disclosure provides a kit comprising an antibody or antigenbinding fragment described herein or a pharmaceutical composition described herein and instructions for use.
  • the disclosure provides a nucleic acid comprising a polynucleotide sequence encoding the antibody or antigen-binding fragment described herein.
  • the disclosure provides a nucleic acid comprising: a) any one of the sequences encoding a heavy-chain complementarity-determining region 1 (HCDR1) set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; b) any one of the sequences encoding a heavy-chain complementarity-determining region 2 (HCDR2) set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; c) any one of the sequences encoding a heavy-chain complementarity-determining region 3 (HCDR3) set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; d) any one of the sequences encoding a light-chain complementarity determining region 1 (LCDR1) set forth in Table 3, or
  • the nucleic acid described herein comprises: a) the sequence encoding the HCDR1 comprises SEQ ID NO: 70; b) the sequence encoding the HCDR2 comprises SEQ ID NO: 71; and c) the sequence encoding the HCDR3 comprises SEQ ID NO: 72.
  • the nucleic acid described herein comprises: a) the sequence encoding the HCDR1 comprises SEQ ID NO: 78; b) the sequence encoding the HCDR2 comprises SEQ ID NO: 79; and c) the sequence encoding the HCDR3 comprises SEQ ID NO: 80.
  • the nucleic acid described herein comprises: a) the sequence encoding the HCDR1 comprises SEQ ID NO: 86; b) the sequence encoding the HCDR2 comprises SEQ ID NO: 87; and c) the sequence encoding the HCDR3 comprises SEQ ID NO: 88.
  • the nucleic acid described herein comprises: a) the sequence encoding the HCDR1 comprises SEQ ID NO: 94; b) the sequence encoding the HCDR2 comprises SEQ ID NO: 95; and c) the sequence encoding the HCDR3 comprises SEQ ID NO: 96.
  • the nucleic acid described herein comprises: a) the sequence encoding the HCDR1 comprises SEQ ID NO: 102; b) the sequence encoding the HCDR2 comprises SEQ ID NO: 103; and c) the sequence encoding the HCDR3 comprises SEQ ID NO: 104.
  • the nucleic acid described herein comprises: a) the sequence encoding the HCDR1 comprises SEQ ID NO: 110; b) the sequence encoding the HCDR2 comprises SEQ ID NO: 111; and c) the sequence encoding the HCDR3 comprises SEQ ID NO: 112.
  • the nucleic acid described herein comprises: a) the sequence encoding the HCDR1 comprises SEQ ID NO: 118; b) the sequence encoding the HCDR2 comprises SEQ ID NO: 119; and c) the sequence encoding the HCDR3 comprises SEQ ID NO: 120.
  • the nucleic acid described herein comprises: a) the sequence encoding the HCDR1 comprises SEQ ID NO: 126; b) the sequence encoding the HCDR2 comprises SEQ ID NO: 127; and c) the sequence encoding the HCDR3 comprises SEQ ID NO: 128.
  • the nucleic acid described herein comprises: a) the sequence encoding the LCDR1 comprises SEQ ID NO: 66; b) the sequence encoding the LCDR2 comprises SEQ ID NO: 67; and c) the sequence encoding the LCDR3 comprises SEQ ID NO: 68.
  • the nucleic acid described herein comprises: a) the sequence encoding the LCDR1 comprises SEQ ID NO: 74; b) the sequence encoding the LCDR2 comprises SEQ ID NO: 75; and c) the sequence encoding the LCDR3 comprises SEQ ID NO: 76.
  • the nucleic acid described herein comprises: a) the sequence encoding the LCDR1 comprises SEQ ID NO: 82; b) the sequence encoding the LCDR2 comprises SEQ ID NO: 83; and c) the sequence encoding the LCDR3 comprises SEQ ID NO: 84.
  • the nucleic acid described herein comprises: a) the sequence encoding the LCDR1 comprises SEQ ID NO: 90; b) the sequence encoding the LCDR2 comprises SEQ ID NO: 91; and c) the sequence encoding the LCDR3 comprises SEQ ID NO: 92.
  • the nucleic acid described herein comprises: a) the sequence encoding the LCDR1 comprises SEQ ID NO: 98; b) the sequence encoding the LCDR2 comprises SEQ ID NO: 99; and c) the sequence encoding the LCDR3 comprises SEQ ID NO: 100.
  • the nucleic acid described herein comprises: a) the sequence encoding the LCDR1 comprises SEQ ID NO: 106; b) the sequence encoding the LCDR2 comprises SEQ ID NO: 107; and c) the sequence encoding the LCDR3 comprises SEQ ID NO: 108.
  • the nucleic acid described herein comprises: a) the sequence encoding the LCDR1 comprises SEQ ID NO: 114; b) the sequence encoding the LCDR2 comprises SEQ ID NO: 115; and c) the sequence encoding the LCDR3 comprises SEQ ID NO: 116.
  • the nucleic acid described herein comprises: a) the sequence encoding the LCDR1 comprises SEQ ID NO: 122; b) the sequence encoding the LCDR2 comprises SEQ ID NO: 123; and c) the sequence encoding the LCDR3 comprises SEQ ID NO: 124.
  • the nucleic acid described herein comprises: a) a sequence encoding a heavy-chain variable region (VH) comprising a sequence set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto; and b) a sequence encoding a light-chain variable region (VL) comprising a sequence set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto.
  • VH heavy-chain variable region
  • VL light-chain variable region
  • the nucleic acid described herein comprises: a) the VH comprises SEQ ID NO: 69, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 65, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the nucleic acid described herein comprises: a) the VH comprises SEQ ID NO: 77, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 73, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the nucleic acid described herein comprises: a) the VH comprises SEQ ID NO: 85, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 81, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the nucleic acid described herein comprises: a) the VH comprises SEQ ID NO: 93, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 89, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the nucleic acid described herein comprises: a) the VH comprises SEQ ID NO: 101, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 97, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the nucleic acid described herein comprises: a) the VH comprises SEQ ID NO: 109, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 105, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the nucleic acid described herein comprises: a) the VH comprises SEQ ID NO: 117, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 113, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the nucleic acid described herein comprises: a) the VH comprises SEQ ID NO: 125, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 121, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the nucleic acid provided herein is codon optimized.
  • the disclosure provides a vector comprising a nucleic acid described herein.
  • the nucleic acid is operably linked to a promoter.
  • the disclosure provides a cell line for producing an antibody or antigen-binding fragment described herein, a nucleic acid described herein, or a vector described herein.
  • the disclosure provides a method of producing an antibody or antigen-binding fragment described herein, comprising a) culturing a cell line described herein under conditions sufficient for expression of the antibody or antigen-binding fragment; and b) purifying the antibody or antigen-binding fragment.
  • the disclosure provides a method of producing an antibody or antigen-binding fragment that binds to a SARS-CoV-2 spike protein comprising: a) contacting a cell with a nucleic acid described herein or a vector described herein; b) culturing the cell under conditions sufficient to express the antibody or antigen-binding fragment; and c) purifying the antibody or antigen-binding fragment.
  • the disclosure provides a method of treating COVID-19 in a subject in need thereof, comprising administering to the subject an antibody or antigen-binding fragment described herein or a pharmaceutical composition described herein.
  • the disclosure provides a method of preventing COVID-19 in a subject in need thereof, comprising administering to the subject an antibody or antigenbinding fragment described herein or a pharmaceutical composition described herein.
  • the disclosure provides a method of reducing one or more symptoms associated with COVID-19 in a subject in need thereof, comprising administering to the subject an antibody or antigen-binding fragment described herein or a pharmaceutical composition described herein.
  • the severity of the one or more symptoms is reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or eliminated completely.
  • the duration of the one or more symptoms is reduced by at least one day, at least two days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days.
  • the disclosure provides a method of treating, preventing, or reducing symptoms associated with COVID-19 in a subject in need thereof, comprising: a) detecting a SARS-CoV-2 viral genome or a SARS-CoV-2 antigen in a sample collected from the subject; and b) administering a treatment to the subject, wherein the treatment i) prevents, reduces, or eliminates a SARS-CoV-2 infection or a variant thereof; and/or ii) prevents, reduces, or eliminates symptoms associated with COVID-19, wherein the treatment comprises an antibody or antigen-binding fragment described herein or a pharmaceutical composition described herein.
  • the subject has or is suspected of having a SARS-CoV-2 infection or a variant thereof. In some embodiments, the subject has at least one symptom associated with a SARS-CoV-2 infection or a variant thereof. In some embodiments, the subject is a human.
  • administration is enteral, parenteral, mucosal, nasal, oral, by inhalation, by intravenous injection, by infusion, by subcutaneous injection, by intramuscular injection, or intra-articular.
  • the disclosure provides a method for detecting a SARS-CoV-2 spike protein in a sample, comprising contacting a sample with the antibody or antigen-binding fragment described herein.
  • the sample is an environmental sample, sewage, wastewater, urine, feces, saliva, nasal swab, mucous, blood, plasma, bronchoalveolar lavage, or one or more cells.
  • the sample is collected from a subject.
  • the subject has or is suspected of having a SARS-CoV-2 infection or COVID-19.
  • the antibody or antigen-binding fragment is labeled with a tag, detection dye or a secondary antibody.
  • the method further comprises use of an electrochemical assay, biosensor, or immunoassay.
  • the disclosure provides a method of diagnosing an individual as having COVID-19, comprising detecting a SARS-CoV-2 spike protein in a sample, comprising contacting a sample with the antibody or antigen-binding fragment described herein.
  • the antibody or antigen-binding fragment is labeled with a detection dye or a secondary antibody.
  • the method further comprises use of an ELISA technique or a lateral flow assay.
  • the sample is saliva, nasal swab, mucous, blood, plasma, bronchoalveolar lavage, or one or more cells.
  • the sample is collected from a subject.
  • the subject has or is suspected of having a SARS-CoV-2 infection or COVID-19.
  • FIG. 1 is a plot showing binding of human monoclonal antibodies disclosed herein against the receptor binding domain (RBD) of SARS-CoV-2 spike protein.
  • FIG. 2 is a plot showing neutralization of SARS-CoV-2 by the antibodies described herein using a surrogate virus neutralization test (sVNT) (GenScript ePassTM SARS-CoV- 2 Neutralization Antibody Detection Kit).
  • sVNT surrogate virus neutralization test
  • FIG. 3 is a plot showing neutralization of SARS-CoV-2 by the antibodies described herein using a pseudovirus neutralization assay.
  • FIG. 4 is a plot showing neutralization of SARS-CoV-2 (alpha (B. l.1.7) variant) by the antibodies described herein using a pseudovirus neutralization assay.
  • FIG. 5 is a plot showing neutralization of SARS-CoV-2 (beta ( B.1.351) variant) by the antibodies described herein using a pseudovirus neutralization assay.
  • FIG. 6 is a plot showing neutralization of SARS-CoV-2 (gamma (P. l) variant) by the antibodies described herein using a pseudovirus neutralization assay.
  • FIG. 7 is a plot showing neutralization of SARS-CoV-2 (delta (B.1.617.2) variant) by the antibodies described herein using a pseudovirus neutralization assay.
  • This disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to the SARS-CoV2 spike protein.
  • the antibodies and antigen-binding fragments may be isolated.
  • the antibodies and antigen-binding fragments can be used in pharmaceutical compositions and kits.
  • This disclosure provides nucleic acids and vectors encoding the antibodies and antigen-binding fragments described herein.
  • the disclosure provides cell lines used in the production of the antibodies and antigen-binding fragments described herein.
  • the disclosure provides methods for producing the antibodies and antigen-binding fragments described herein.
  • the disclosure provides methods of using the antibodies and binding fragments described herein.
  • the methods can be methods of treatment and prevention of a SARS- CoV-2 infection or COVID-19.
  • the antibodies and binding fragments thereof can be used in a method of preventing, reducing, or eliminating symptoms associated with a SARS- CoV-2 infection or COVID-19.
  • the antibodies and binding fragments thereof can be used in a method of detecting SARS-CoV-2 in a sample.
  • the coronavirus i.e. SARS-CoV-1, MERS-CoV, and SARS-CoV-2
  • viral genome encodes spike (S), envelope (E), membrane (M), and nucleocapsid (N) structural proteins, among which the surface glycoprotein i.e. spike protein) is responsible for binding the host receptor via the receptor-binding domain (RBD) in its SI subunit, as well as the subsequent membrane fusion and viral entry driven by its S2 subunit.
  • RBD contains a core subdomain and a receptor-binding motif (RBM).
  • SARS-CoV and SARS-CoV-2 recognize the angiotensin-converting enzyme 2 (ACE or ACE2), whereas MERS-CoV binds the dipeptidyl peptidase 4 (DPP4). Binding of the spike protein RBM to the ACE2 facilitates viral entry; a possible membrane fusion process has been proposed.
  • the spike protein, the RBD, and the RBM provide a set of unique protein structures to act as antigens with unique epitopes for antibody based targeting and binding of the coronavirus (e.g. SARS-CoV-2), for detection of the coronavirus, treatment of coronavirus infection, treatment disease phenotypes (e.g. COVID-19), and treatment of symptoms associated with disease phenotypes.
  • the SARS-CoV-2 spike protein has formed the basis for vaccines to immunize populations against SARS-CoV-2 infection and COVID-19 by acting as the antigen (i.e. immunogen) of the vaccine to which the host receiving the vaccine develops antibodies against.
  • the disclosure provides antibodies and binding fragments thereof that specifically bind a SARS-CoV-2 spike protein antigen.
  • Any region or epitope within a SARS-CoV-2 spike protein antigen is envisaged as a putative binding site for the antibodies and binding fragments thereof provided herein.
  • putative binding sites can be, without limitation, on the following SARS-CoV-2 spike protein regions: the SI subunit, S2 subunit, the SD-1 subdomain, the S1/S2 cleavage region, the S2 subunit, the receptor-binding domain, and the receptor binding motif.
  • the SARS-CoV-2 spike protein antigen comprises a sequence, or a portion thereof, set forth in Table 1.
  • the SARS-CoV-2 spike protein antigen comprises SEQ ID NO: 129, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto.
  • the SARS- CoV-2 spike protein antigen comprises SEQ ID NO: 130, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto.
  • the SARS-CoV-2 spike protein antigen comprises SEQ ID NO: 131, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto. In some embodiments, the SARS-CoV-2 spike protein antigen comprises SEQ ID NO: 132, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto. In some embodiments, the SARS-CoV-2 spike protein antigen comprises SEQ ID NO: 133, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto.
  • CoV-2 spike protein antigen comprises SEQ ID NO: 135, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto.
  • the antibody or antigen-binding fragment provided herein binds to a portion, or an epitope, of the SARS-CoV-2 spike protein. Put another way, the antibody or antigen-binding fragment provided herein can bind to any one or more amino acid subsequence(s) of the SARS-CoV-2 spike protein.
  • the disclosure provides antibodies and antigen-binding fragments that bind to a SARS-CoV-2 spike protein.
  • the antibody or antigenbinding fragment binds a sequence, or a portion thereof, set forth in Table 1.
  • the antibody or antigen-binding fragment binds a SARS-CoV-2 spike protein antigen comprises SEQ ID NO: 129, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto.
  • the antibody or antigen-binding fragment binds a SARS-CoV-2 spike protein antigen comprises SEQ ID NO: 130, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto. In some embodiments, the antibody or antigenbinding fragment binds a SARS-CoV-2 spike protein antigen comprises SEQ ID NO: 131, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto.
  • the antibody or antigen-binding fragment binds a SARS-CoV-2 spike protein antigen comprises SEQ ID NO: 132, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto. In some embodiments, the antibody or antigen-binding fragment binds a SARS-CoV-2 spike protein antigen comprises SEQ ID NO: 133, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto.
  • the antibody or antigen-binding fragment binds a SARS-CoV-2 spike protein antigen comprises SEQ ID NO: 134, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto. In some embodiments, the antibody or antigenbinding fragment binds a SARS-CoV-2 spike protein antigen comprises SEQ ID NO: 135, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto.
  • the antibody or antigen-binding fragment binds a SARS-CoV-2 spike protein antigen comprises SEQ ID NO: 136, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto.
  • the antibodies and binding fragments provided herein comprise a variable heavy chain (i.e. VH or HC) region and a variable light chain (i.e. VL or LC) region.
  • the antibody or binding fragment comprises two identical VH regions.
  • the antibody or binding fragment comprises two identical VL regions.
  • the antibody or fragment comprises two different VL regions.
  • the antibody or fragment comprises two different VH regions.
  • the two VH regions are covalently bound.
  • VH regions can be covalently be bound by, for example, disulfide bonds.
  • the VL regions are covalently bound to the VH regions.
  • VL and VH regions each have one variable region and three constant regions.
  • CDRs complementarity-determining regions
  • the binding specificity of an antibody is conferred by its combination of CDRs.
  • CDRs are hypervariable, a particular CDR on one antibody may also be found on antibodies with different specificities, as it is the total combination of CDRs regions that determine binding specificity.
  • Identification of the CDRs is useful for changing the “speciation” of an antibody, for example changing a mouse antibody to a humanized form suitable for human use, because one would want to preserve the CDRs so as not to eliminate the binding specificity.
  • the CDRs of the light chain are between amino acids 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (95 a-f, LCDR3).
  • the complementarity-determining regions of the heavy chain are between amino acids 31-35 (35 a-b, HCDR1), 50-65 (52 a-c, HCDR2), and 95-102 (100 a-k, HCDR3). Insertions of extra amino acids into the complementaritydetermining regions can be observed and their locations are represented above in parentheses, e.g. 95 a-f. Deletions are also observed, for example in CDR3 of some types of heavy chains. Illustrative CDRs of the disclosure are provided in Table 2.
  • the antibody is isolated. In some embodiments, the antibody is humanized. In some embodiments, the antibody is recombinant. In some embodiments, the antibody is multispecific
  • the antibodies and antigen-binding fragments provided herein comprises a heavychain complementarity-determining region 1 (HCDR1), a heavy-chain complementaritydetermining region 2 (HCDR2), a heavy-chain complementarity-determining region 3 (HCDR3), a light-chain complementarity determining region 1 (LCDR1), a light-chain complementarity determining region 2 (LCDR2), and a light-chain complementarity determining region 3 (LCDR3).
  • HCDR1 heavychain complementarity-determining region 1
  • HCDR2 heavy-chain complementarity determining region 2
  • HCDR3 heavy-chain complementarity determining region 3
  • LCDR1 light-chain complementarity determining region 1
  • LCDR2 light-chain complementarity determining region 2
  • LCDR3 light-chain complementarity determining region 3
  • the antibody or antigen-binding fragment comprises any one of the HCDR1 sequences set forth in Table 2; any one of the HCDR2 sequences set forth in Table 2; any one of the HCDR3 sequences set forth in Table 2; any one of the LCDR1 sequences set forth in Table 2; any one of the LCDR2 sequences set forth in Table 2; and any one of the LCDR3 sequences set forth in Table 2.
  • no more than 1, 2, or 3, amino acids in the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and/or LCDR3 sequences are substituted, deleted or inserted.
  • the antibody or antigen-binding fragment comprises a HCDR1 comprising SEQ ID NO: 6; a HCDR2 comprising SEQ ID NO: 7; and a HCDR3 comprising SEQ ID NO: 8.
  • the antibody or antigen-binding fragment comprises a HCDR1 comprising SEQ ID NO: 14; a HCDR2 comprising SEQ ID NO: 15; and a HCDR3 comprising SEQ ID NO: 16.
  • the antibody or antigen-binding fragment comprises a HCDR1 comprising SEQ ID NO: 22; a HCDR2 comprising SEQ ID NO: 23; and a HCDR3 comprising SEQ ID NO: 24.
  • the antibody or antigen-binding fragment comprises a HCDR1 comprising SEQ ID NO: 30; a HCDR2 comprising SEQ ID NO: 31; and a HCDR3 comprising SEQ ID NO: 32.
  • the antibody or antigen-binding fragment comprises a HCDR1 comprising SEQ ID NO: 38; a HCDR2 comprising SEQ ID NO: 39; and a HCDR3 comprising SEQ ID NO: 40.
  • the antibody or antigen-binding fragment comprises a HCDR1 comprising SEQ ID NO: 46; a HCDR2 comprising SEQ ID NO: 47; and a HCDR3 comprising SEQ ID NO: 48.
  • the antibody or antigen-binding fragment comprises a HCDR1 comprising SEQ ID NO: 54; a HCDR2 comprising SEQ ID NO: 55; and a HCDR3 comprising SEQ ID NO: 56.
  • the antibody or antigen-binding fragment comprises a HCDR1 comprising SEQ ID NO: 62; a HCDR2 comprising SEQ ID NO: 63; and a HCDR3 comprising SEQ ID NO: 64.
  • the antibody or antigen-binding fragment comprises a LCDR1 comprising SEQ ID NO: 2; a LCDR2 comprising SEQ ID NO: 3; and a LCDR3 comprising SEQ ID NO: 4.
  • the antibody or antigen-binding fragment comprises a LCDR1 comprising SEQ ID NO: 10; a LCDR2 comprising SEQ ID NO: 11; and aLCDR3 comprising SEQ ID NO: 12.
  • the antibody or antigen-binding fragment comprises a LCDR1 comprising SEQ ID NO: 18; a LCDR2 comprising SEQ ID NO: 19; and aLCDR3 comprising SEQ ID NO: 20.
  • the antibody or antigen-binding fragment comprises a LCDR1 comprising SEQ ID NO: 26; a LCDR2 comprising SEQ ID NO: 27; and a LCDR3 comprising SEQ ID NO: 28.
  • the antibody or antigen-binding fragment comprises a LCDR1 comprising SEQ ID NO: 34; a LCDR2 comprising SEQ ID NO: 35; and aLCDR3 comprising SEQ ID NO: 36.
  • the antibody or antigen-binding fragment comprises a LCDR1 comprising SEQ ID NO: 42; a LCDR2 comprising SEQ ID NO: 43; and a LCDR3 comprising SEQ ID NO: 44.
  • the antibody or antigen-binding fragment comprises a LCDR1 comprising SEQ ID NO: 50; a LCDR2 comprising SEQ ID NO: 51; and aLCDR3 comprising SEQ ID NO: 52.
  • the antibody or antigen-binding fragment comprises a LCDR1 comprising SEQ ID NO: 58; a LCDR2 comprises SEQ ID NO: 59; and a LCDR3 comprising SEQ ID NO: 60.
  • the antibody or antigen-binding fragment comprises a heavy-chain variable region (VH) comprising a sequence set forth in Table 2, or a sequence at least that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto; and a light-chain variable region (VL) comprising a sequence set forth in Table 2, or a sequence at least that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto.
  • VH heavy-chain variable region
  • VL light-chain variable region
  • the antibody or antigen-binding fragment comprises a VH comprising SEQ ID NO: 5, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and a VL comprising SEQ ID NO: 1, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the antibody or antigen-binding fragment comprises a VH comprising SEQ ID NO: 13, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and a VL comprising SEQ ID NO: 9, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the antibody or antigen-binding fragment comprise a VH comprising SEQ ID NO: 21, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and a VL comprising SEQ ID NO: 17, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the antibody or antigen-binding fragment comprises a VH comprising SEQ ID NO: 29, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and a VL comprising SEQ ID NO: 25, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the antibody or antigen-binding fragment comprises a VH comprising SEQ ID NO: 37, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and a VL comprising SEQ ID NO: 33, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the antibody or antigen-binding fragment comprises a VH comprising SEQ ID NO: 45, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and a VL comprising SEQ ID NO: 41, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the antibody or antigen-binding fragment comprises a VH comprising SEQ ID NO: 53, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and a VL comprising SEQ ID NO: 49, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the antibody or antigen-binding fragment comprises a VH comprising SEQ ID NO: 61, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and a VL comprising SEQ ID NO: 57, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto
  • the antibody or antigen-binding fragment provided herein may further comprise a constant region.
  • a constant region can be an Fc region.
  • An "Fc region” (fragment crystallizable region) or “Fc domain” or “Fc” refers to the C- terminal region of the heavy chain of an antibody that mediates the binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (Clq) of the classical complement system.
  • an Fc region of an antibody of isotype IgG comprises the heavy chain constant region of the antibody excluding the first constant region immunoglobulin domain (CHI).
  • the Fc region comprises CH2 and CH3 constant domains in each of the antibody’s two heavy chains; IgM and IgE Fc regions comprise three heavy chain constant domains (CH domains 2-4) in each polypeptide chain.
  • the Fc region comprises immunoglobulin domains consisting of the hinge, CH2 and CH3.
  • “Hinge”, “hinge domain”, or “hinge region” refers to the domain of a heavy chain constant region that joins the CHI domain to the CH2 domain and comprises upper, middle, and lower portions.
  • the hinge provides varying levels of flexibility between the antigenbinding domain and effector region of an antibody and also provides sites for intermolecular disulfide bonding between the two heavy chain constant regions.
  • the antibody or antigen-binding fragment comprises a hinge region.
  • the Fc region is defined as starting at amino acid 216 and ending at amino acid 447, wherein the numbering is according to the EU index as in Kabat. Kabat et al. (1991) Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD, and according to FIGs.3c-3f of U.S. Pat. App. Pub. No. 2008/0248028.
  • the Fc may be a native (or naturally-occurring or wildtype) Fc, including any allotypic variant, or a variant Fc (e.g., a non- naturally occurring Fc), comprising, e.g., 1, 2, 3, 4, 5, 1-5, 1-10 or 5-10 or more amino acid mutations, e.g., substitutions, additions or deletions.
  • a variant Fc may comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a wildtype Fc.
  • Modified or mutated Fes may have enhanced or reduced effector function and/or half-life.
  • the CH2 and CH3 regions are the primary site of effector functions and FcRn binding.
  • Fc may refer to this region in isolation or in the context of an Fc-comprising protein polypeptide such as a “binding protein comprising an Fc region,” also referred to as an “Fc fusion protein” (e.g., an antibody or immunoadhesin).
  • a binding protein comprising an Fc region also referred to as an “Fc fusion protein” (e.g., an antibody or immunoadhesin).
  • the constant regions of the antibodies provided herein may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • the constant region mediates an effector function, which refers to the interaction of an antibody Fc region with an Fc receptor or ligand, or a biochemical event that results therefrom.
  • effector functions include Clq binding, complement dependent cytotoxicity (CDC), Fc receptor binding, FcyR- mediated effector functions such as ADCC and antibody dependent cell-mediated phagocytosis (ADCP), and downregulation of a cell surface receptor (e.g., the B cell receptor; BCR).
  • Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain).
  • FcR Fc receptor
  • FcRs that bind to an IgG antibody comprise receptors of the FcyR family, including allelic variants and alternatively spliced forms of these receptors.
  • the FcyR family consists of three activating (FcyRI, FcyRIII, and FcyRIV in mice; FcyRIA, FcyRIIA, and FcyRIIIA in humans) and one inhibitory (FcyRIIB) receptor.
  • NK cells selectively express one activating Fc receptor (FcyRIII in mice and FcyRIIIA in humans) but not the inhibitory FcyRIIB in mice and humans.
  • Human IgGl binds to most human Fc receptors and is considered equivalent to murine IgG2a with respect to the types of activating Fc receptors that it binds to.
  • a constant region can be a constant region within the light chain.
  • the light chain constant region can be a lambda (t) light chain region or a kappa (K) light chain region.
  • the light chain constant region comprises a lambda light chain region.
  • the light chain constant region comprises a kappa light chain region.
  • the constant region may be from any of the commonly known immunoglobulin isotypes, including but not limited to IgA, secretory IgA, IgG and IgM.
  • the IgG isotype is divided in subclasses in certain species: IgGl, IgG2, IgG3 and IgG4 in humans, and IgGl, IgG2a, IgG2b and IgG3 in mice.
  • Immunoglobulins e.g., human IgGl, exist in several allotypes, which differ from each other in at most a few amino acids.
  • the constant region is selected from IgA, IgG, and IgM.
  • the constant region is selected from IgGl, IgG2, IgG3, and IgG4 constant regions.
  • the SARS-CoV-2 binding antibody or antigen-binding fragment comprises a constant region selected from a sequence in Table 2.1, or a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.
  • the antibody or antigen-binding fragment comprises a constant region comprises any one of SEQ ID NOs: 137-147, or a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.
  • Binding and binding affinities can be characterized using any method known in the art. Binding and binding affinities can be expressed as a dissociation constant (KD) value. Dissociation constant values can be determined using, for example and without limitation, surface plasmon resonance, surface plasmon resonance imaging, interferometry, ELISA, titration curves, radioligand binding assay, affinity chromatography, fluorescence energy resonance transfer, and/or isothermal titration calorimetry. It can be useful to determine the KD value using more than one method to validate the binding properties reported by a given characterization method.
  • the antibody or antigen-binding fragment binds the SARS- CoV-2 spike protein with a KD value of about 10' 12 M, about 1O' U M, about 10' 10 M, about 10' 9 M, or about 10' 8 M.
  • Biological activity refers to an effect the antibody has on a system of biological molecules.
  • a biological activity can be blocking a SARS-CoV-2 spike protein from binding a target receptor (e.g. an ACE2 receptor).
  • a target receptor e.g. an ACE2 receptor
  • an antibody or antigenbinding fragment in the presence of both a SARS-CoV-2 spike protein and an ACE2 receptor will bind the SARS-CoV-2 spike protein and form a complex such that the SARS- CoV-2 spike protein does not bind the ACE2 receptor, or has reduced or otherwise altered binding.
  • a biological activity is the prevention, reduction, or elimination of the ability of a SARS-CoV-2 virus to infect a cell.
  • An antibody or antigen-binding fragment of the disclosure is said to neutralize a SARS-CoV-2 virus if, when in the presence of a cell expressing an ACE2 receptor and a SARS-CoV-2 virus, the ability of the virus to infect the cell is prevented, reduced, or eliminated; said antibody or antigenbinding fragment can also be referred to as neutralizing, have neutralizing activity, or can neutralize SARS-CoV-2.
  • Neutralizing activity can arise from the antibody or antigenbinding fragment binding a SARS-CoV-2 spike protein on a SARS-CoV-2 virus and prevent, reduce or otherwise alter binding of the spike protein to an ACE2 receptor on a cell. Binding of the antibody or antigen-binding fragment to ACE2 receptor can thus be used as a surrogate measurement for classifying the antibody or antigen-binding fragment as having SARS-CoV-2 neutralizing activity, as described herein. In some embodiments, the antibody or antigen-binding fragment neutralizes a SARS-CoV-2 virus or a variant thereof.
  • Biological activity of the antibody or antigen-binding fragment can be determined using any method known in the art. Biological activity can be expressed in terms of IC50 or EC50 values. IC50 and EC50 values quantify the biological activity as the concentration of antibody or antigen-binding fragment required to achieve half of the maximum biological activity. For example, IC50 can be used to express the concentration of antibody or antigen-binding fragment required for reducing the binding of the spike protein to an ACE2 receptor by 50% of the binding observed in the absence of the antibody or antigenbinding fragment. In another example, EC50 can be used to express the concentration of antibody or antigen-binding fragment that is required to reduce infection of ACE2 expressing cells by SARS-CoV-2 by 50% of the infection observed in the absence of the antibody or antigen-binding fragment.
  • the antibody or antigen-binding fragment inhibits the binding of the SARS-CoV-2 spike protein to an ACE2 receptor with an IC50 of about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL, about 65 ng/mL, about 70 ng/mL, about 75 ng/mL, or about 80 ng/mL.
  • the disclosure provides a nucleic acid comprising a polynucleotide sequence encoding the antibody antigen-binding fragment described herein.
  • the sequence is codon optimized.
  • nucleic acids and polynucleotides described herein are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).
  • the disclosure provides a nucleic acid comprising: a.) any one of the sequences encoding a heavy-chain complementarity-determining region 1 (HCDR1) set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; b.) any one of the sequences encoding a heavy-chain complementarity-determining region 2 (HCDR2) set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; c.) any one of the sequences encoding a heavy-chain complementarity-determining region 3 (HCDR3) set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; d.) any one of the sequences encoding a light-chain complementarity determining region 1 (LCDR1) set forth in Table 3, or
  • the nucleic acid comprises a.) a sequence encoding the HCDR1 comprising SEQ ID NO: 70; b.) a sequence encoding the HCDR2 comprising SEQ ID NO: 71; and c.) a sequence encoding the HCDR3 comprising SEQ ID NO: 72.
  • the nucleic acid comprises a.) a sequence encoding the HCDR1 comprising SEQ ID NO: 78; b.) a sequence encoding the HCDR2 comprising SEQ ID NO: 79; and c.) a sequence encoding the HCDR3 comprising SEQ ID NO: 80.
  • the nucleic acid comprises a.) a sequence encoding the HCDR1 comprising SEQ ID NO: 86; b.) a sequence encoding the HCDR2 comprising SEQ ID NO: 87; and c.) a sequence encoding the HCDR3 comprising SEQ ID NO: 88.
  • the nucleic acid comprises a.) a sequence encoding the HCDR1 comprising SEQ ID NO: 94; b.) a sequence encoding the HCDR2 comprising SEQ ID NO: 95; and c.) a sequence encoding the HCDR3 comprising SEQ ID NO: 96.
  • the nucleic acid comprises a.) a sequence encoding the HCDR1 comprising SEQ ID NO: 102; b.) a sequence encoding the HCDR2 comprising SEQ ID NO: 103; and c.) a sequence encoding the HCDR3 comprising SEQ ID NO: 104.
  • the nucleic acid comprises a.) a sequence encoding the HCDR1 comprises SEQ ID NO: 110; b.) a sequence encoding the HCDR2 comprising SEQ ID NO: 111; and c.) a sequence encoding the HCDR3 comprising SEQ ID NO: 112.
  • the nucleic acid comprises a.) a sequence encoding the HCDR1 comprising SEQ ID NO: 118; b.) a sequence encoding the HCDR2 comprising SEQ ID NO: 119; and c.) a sequence encoding the HCDR3 comprising SEQ ID NO: 120.
  • the nucleic acid comprises a.) a sequence encoding the HCDR1 comprising SEQ ID NO: 126; a sequence encoding the HCDR2 comprising SEQ ID NO: 127; and c.) a sequence encoding the HCDR3 comprising SEQ ID NO: 128.
  • the nucleic acid comprises a.) a sequence encoding the LCDR1 comprising SEQ ID NO: 66; b.) a sequence encoding the LCDR2 comprising SEQ ID NO: 67; and c.) a sequence encoding the LCDR3 comprising SEQ ID NO: 68.
  • the nucleic acid comprises a.) a sequence encoding the LCDR1 comprising SEQ ID NO: 74; b.) a sequence encoding the LCDR2 comprising SEQ ID NO: 75; and c.) a sequence encoding the LCDR3 comprising SEQ ID NO: 76.
  • the nucleic acid comprises a.) a sequence encoding the LCDR1 comprising SEQ ID NO: 82; b.) a sequence encoding the LCDR2 comprising SEQ ID NO: 83; and c.) a sequence encoding the LCDR3 comprising SEQ ID NO: 84.
  • the nucleic acid comprises a.) a sequence encoding the LCDR1 comprising SEQ ID NO: 90; b.) a sequence encoding the LCDR2 comprising SEQ ID NO: 91; and c.) a sequence encoding the LCDR3 comprising SEQ ID NO: 92.
  • the nucleic acid comprises a.) a sequence encoding the LCDR1 comprising SEQ ID NO: 98; b.) a sequence encoding the LCDR2 comprising SEQ ID NO: 99; and c.) a sequence encoding the LCDR3 comprising SEQ ID NO: 100.
  • the nucleic acid comprises a.) a sequence encoding the LCDR1 comprising SEQ ID NO: 106; b.) a sequence encoding the LCDR2 comprising SEQ ID NO: 107; and c.) a sequence encoding the LCDR3 comprising SEQ ID NO: 108.
  • the nucleic acid comprises a.) a sequence encoding the LCDR1 comprises SEQ ID NO: 114; b.) a sequence encoding the LCDR2 comprising SEQ ID NO: 115; and c.) a sequence encoding the LCDR3 comprising SEQ ID NO: 116.
  • the nucleic acid comprises a.) a sequence encoding the LCDR1 comprising SEQ ID NO: 122; b.) a sequence encoding the LCDR2 comprising SEQ ID NO: 123; and c.) a sequence encoding the LCDR3 comprising SEQ ID NO: 124.
  • the disclosure provides a nucleic acid comprising a sequence encoding a heavychain variable region (VH) comprising a sequence set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto; and a sequence encoding a light-chain variable region (VL) comprising a sequence set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto.
  • VH heavychain variable region
  • VL light-chain variable region
  • the sequence encoding VH comprises SEQ ID NO: 69, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and the sequence encoding VL comprises SEQ ID NO: 65, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the sequence encoding VH comprises SEQ ID NO: 77, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and the sequence encoding VL comprises SEQ ID NO: 73, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the sequence encoding VH comprises SEQ ID NO: 85, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and the sequence encoding VL comprises SEQ ID NO: 81, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the sequence encoding VH comprises SEQ ID NO: 93, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and the sequence encoding VL comprises SEQ ID NO: 89, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the sequence encoding VH comprises SEQ ID NO: 101, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and the sequence encoding VL comprises SEQ ID NO: 97, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the sequence encoding VH comprises SEQ ID NO: 109, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and the sequence encoding VL comprises SEQ ID NO: 105, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the sequence encoding VH comprises SEQ ID NO: 117, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and the sequence encoding VL comprises SEQ ID NO: 113, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the sequence encoding VH comprises SEQ ID NO: 125, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and the sequence encoding VL comprises SEQ ID NO: 121, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
  • the term “vector” as used in the application refers to nucleic acid molecules, usually double-stranded DNA, which may have inserted into it another nucleic acid molecule (the insert nucleic acid molecule) such as, but not limited to, a polynucleotide encoding an antibody or antigen-binding fragment described herein.
  • the vector is used to transport the insert nucleic acid molecule into a suitable host cell.
  • a vector may contain the necessary elements that permit transcribing the insert nucleic acid molecule, and, optionally, translating the transcript into a polypeptide.
  • the insert nucleic acid molecule may be derived from the host cell, or may be derived from a different cell or organism. Once in the host cell, the vector can replicate independently of, or coincidental with, the host chromosomal DNA, and several copies of the vector and its inserted nucleic acid molecule may be generated.
  • vector may thus also be defined as a gene delivery vehicle that facilitates gene transfer into a target cell.
  • This definition includes both non-viral and viral vectors.
  • Non-viral vectors include but are not limited to cationic lipids, liposomes, nanoparticles, polyethylene glycol (PEG), polyethylenimine (PEI), etc.
  • Viral vectors are derived from viruses and include but are not limited to retroviral, lentiviral, adeno- associated viral, adenoviral, herpesviral, hepatitis viral vectors or the like.
  • viral vectors are replication-deficient as they have lost the ability to propagate in a given cell since viral genes essential for replication have been eliminated from the viral vector.
  • some viral vectors can also be adapted to replicate specifically in a given cell, such as, e.g., a cancer cell, and are typically used to trigger the (cancer) cell-specific (onco)lysis
  • the disclosure provides vectors comprising the polynucleotides described herein.
  • the vector comprises a polynucleotide described herein.
  • the vector comprises a promoter operably linked to the polynucleotide described herein.
  • a promoter is said to be operably linked to a sequence (e.g. a polynucleotide described herein) if the promoter can initiate transcription of the sequence.
  • a promoter operably linked to a polynucleotide encoding an antibody or antigen-binding fragment described herein will recruit transcriptional initiating and promoting proteins (e.g.
  • RNA polymerase and transcription factors initiate transcription of the polynucleotide to form an mRNA transcript encoding the antibody or antigen-binding fragment.
  • the mRNA transcript may then be translated to produce a polypeptide comprising the antibody or antigen-binding fragment.
  • the vector can be any vector known in the art suitable for expressing the antibody or antigen-binding fragments described herein.
  • the vector is an expression vector.
  • the expression vector comprises a promoter. Any suitable promoter known in the art can be used. The promoter will be selected based on the target host cell. For example, a vector will promote expression of the polynucleotides encoding the antibody or antigen-binding fragments described herein in a mammalian cell if the promoter is mammalian cell specific. A suitable promoter can promote strong expression of the polynucleotide encoding an antibody or antigen-binding fragment. In some embodiments, the promoter is inducible.
  • the promoter is constitutive.
  • suitable promoters for expression in a cell include, without limitation, a cytomegalovirus (CMV) promoter or a elongation factor alpha (EFla) promoter.
  • CMV cytomegalovirus
  • EFla elongation factor alpha
  • the vector comprises an enhancer sequence.
  • An enhancer sequence is a nucleic acid sequences that regulates, either directly or indirectly, the transcription of corresponding nucleic acid coding sequences to which they are operably linked (e.g., a sequence encoding an antibody or antigen-binding fragment described herein).
  • An enhancer may function alone to regulate transcription or may act in concert with one or more other regulatory sequences (e.g., promoters). Any suitable enhancer sequence known in the art may be used.
  • the vector comprises a sequence encoding a selectable marker.
  • a selectable marker can be a polypeptide that serves as a visualization marker of positive transduction or transfection of the vector described herein.
  • Illustrative examples of a polypeptide that serves as a visualization marker include, without limitation, a fluorescent protein (e.g. green fluorescent protein or red fluorescent protein) or a protein that catalyzes a visualization reagent (e.g. beta-galactosidase or luciferase).
  • a cell that has been transfected or transduced with a vector encoding a selectable marker comprising GFP will show a GFP -mediated fluorescent signal when visualized in a fluorescence microscope
  • a cell that is not transfected or transduced with a vector encoding a selectable marker comprising GFP will not show a GFP-mediated fluorescence signal when visualized in a fluorescence microscope.
  • a selectable marker can also be a polypeptide that allows selective killing of cells using that do not contain the vector.
  • Illustrative examples of a polypeptide that serves as a polypeptide that allows selective killing of cells that do not contain the vector include, without limitation, puromycin acetyltransferase, hygromycin phosphotransferase, and glutamine synthase.
  • puromycin acetyltransferase hygromycin phosphotransferase
  • glutamine synthase glutamine synthase.
  • a cell that has been transfected or transduced with a vector encoding a selectable marker comprising puromycin acetyltransferase will not be killed when contacted with puromycin
  • a cell that is not transfected or transduced with a vector encoding a selectable marker comprising puromycin acetyltransferase will be killed when contacted with puromycin.
  • Any suitable selective markers known in the art may be used in the vectors described herein.
  • the vector comprises a sequence encoding an intron sequence.
  • An intron sequence can be included in a 5' untranslated region of the polynucleotide encoding an antibody or antigen-binding fragment following the promoter/enhancer to increase export of transcribed mRNA to the cytoplasm from the nucleus. Any suitable intron sequence known in the art may be used in the vectors described herein.
  • the vector comprises a sequence encoding a polyadenylation (poly(A)) signal.
  • the poly(A) signal is a region is a polynucleotide sequence on the 3’ end of the polynucleotide sequence encoding a polypeptide (e.g. an antibody or antigen-binding fragment).
  • the poly(A) signal can regulate mRNA transport, stability, translation, and can promote expression of the polypeptide. Any suitable poly(A) signal sequence known in the art may be used in the vectors described herein.
  • the vector comprises a sequence encoding a Kozak sequence. In some embodiments, the vector comprises a sequence encoding a ribosomal binding site. In some embodiments, the vector comprises a sequence encoding an internal ribosome entry site.
  • the Kozak sequence, ribosomal binding site, and internal ribosome entry sites are nucleic acids that function as a translation initiation and/or ribosomal recruitment sites to ensure that a polypeptide is correctly translated from an mRNA transcript. Any suitable Kozak sequence known in the art may be used in the vectors described herein.
  • the vector comprises a sequence encoding a signal peptide.
  • the sequence encoding a signal peptide can be a polynucleotide sequence located on the 5’ end of the polynucleotide sequence encoding a polypeptide (e.g. an antibody or antigenbinding fragment).
  • the signal peptide facilitates export of the polypeptide from the cell in which they are expressed, and is cleaved from the polypeptide prior to export from the cell. Any suitable sequence encoding a signal peptide may be used in the vectors described herein.
  • the disclosure methods of producing the antibody or antigen-binding fragment described herein.
  • the disclosure provides a method of producing the antibody of antigenbinding fragment that binds to a SARS-CoV-2 spike protein comprising: a.) contacting a cell with the nucleic acid described herein or a vector described herein; b.) culturing the cell under conditions sufficient to express the antibody or antigen-binding fragment; and purifying the antigen-binding fragment.
  • the disclosure provides a cell line for producing the antibody or antigen-binding fragment.
  • the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art.
  • the expression vector can be transferred into a host cell by physical, chemical, or biological means.
  • Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). One method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.
  • Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.
  • Viral vectors, and especially retroviral vectors have become the most widely used method for inserting genes into mammalian, e.g., human cells.
  • Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.
  • colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).
  • assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the disclosure.
  • molecular biological assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR
  • biochemical assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the disclosure.
  • Host cells for use in cell line generation can be any cell known in the art for expressing an antibody or antigen-binding fragment.
  • the host cell is a mammalian cell.
  • the host cell is a Chinese hamster ovary (CHO) cell.
  • the host cell is a human embryonic kidney cell 293 (HEK293) cell.
  • the host cell is a baby hamster kidney (BKH) cell.
  • the host cell is a mouse myeloma cell.
  • the host cell is a human embryonic retinal cell.
  • the host cell is a bacterial cell.
  • the host cell is a yeast cell.
  • Host cells may be allowed to grow for 1-2 days in an enriched media following transduction or transfection with the nucleic acids or vectors described herein, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to generate cell lines which express the antibody molecule. Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al.
  • Established cell lines for producing the antibody or antigen-binding fragments described herein can be expanded and banked indefinitely under cryogenic conditions. Banked cells can be thawed and further expanded under suitable conditions for production of the antibody or antigen-binding fragments at a desired scale.
  • the disclosure provides a method of producing the antibody or antigen-binding fragment described herein, comprising culturing a cell line comprising a nucleic acid described herein or a vector described herein under conditions sufficient for expression of the antibody or antigen-binding fragment, and purifying the antibody or antigen-binding fragment.
  • a cell comprising the nucleic acids and/or vectors described herein can be cultured under suitable conditions to express the antibody or antigen-binding fragment.
  • Expression of an antibody or antigen-binding fragment described herein in the cell is referred to as “recombinant” expression, and the produced antibody or antigen-binding fragment can be referred to as “recombinant.”
  • the antibody or antigen-binding fragment is recombinant.
  • For long-term, high-yield production of recombinant polypeptide e.g. an antibody or antigen-binding fragment described herein
  • Stable expression of the recombinant polypeptide can be achieved with use of a constitutive promoter.
  • any suitable cell culture medium for the culture of cell lines described herein such as mammalian cells expressing an antibody or antigen-binding fragment described herein, may be used.
  • the culture can be an in vitro cell culture.
  • Cell culture media formulations are well known in the art.
  • cell culture media are comprised of buffers, salts, carbohydrates, amino acids, vitamins and trace essential elements.
  • the cell culture medium may or may not contain serum, peptone, and/or proteins.
  • tissue culture media including serum-free and defined culture media, are commercially available.
  • Illustrative examples include, without limitation, one or a combination of the following cell culture medias: RPMI-1640 Medium, RPMI-1641 Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium Eagle, F-12K Medium, Ham's F12 Medium, Iscove's Modified Dulbecco's Medium, McCoy's 5 A Medium, Leibovitz's L- 15 Medium, and serum-free media such as EX-CELLTM 300 Series (JRH Biosciences, Lenexa, Kansas), among others.
  • DMEM Dulbecco's Modified Eagle's Medium
  • F-12K Minimum Essential Medium Eagle
  • Ham's F12 Medium Ham's F12 Medium
  • Iscove's Modified Dulbecco's Medium McCoy's 5 A Medium
  • Leibovitz's L- 15 Medium and serum-free media
  • serum-free media such as EX-CELLTM 300 Series (JRH Biosciences, Lenex
  • Cell culture media may be supplemented with additional or increased concentrations of components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements and the like, depending on the requirements of the cells to be cultured and/or the desired cell culture parameters.
  • cell culture media may be supplemented with polyamines such as putrescine, spermidine and spermine, to improve cell growth, cell viability, and/or recombinant protein production in association with a particular host cell.
  • Cell culture media may be serum-free, protein-free, and/or peptone-free media. "Serum- free" applies to a cell culture medium that does not contain animal sera, such as fetal bovine serum.
  • Protein-free applies to cell culture media free from exogenously added protein, such as transferring, protein growth factors IGF-I, or insulin. Protein-free media may or may not contain peptones.
  • Protein-free media may or may not contain peptones.
  • Peptone-free applies to cell culture media which contains no exogenous protein hydrolysates such as animal and/or plant protein hydrolysates. Eliminating serum and/or hydrolysates from cell culture media has the advantage of reducing lot to lot variability and enhancing processing steps, such as filtration. However, when serum and/or peptone are removed from the cell culture media, cell growth, viability and/or protein expression may be diminished or less than optimal. As such, serum-free and/or peptone-free cell culture medium may be highly enriched for amino acids, trace elements and the like. See, for example, US Patent Nos. 5,122,469 and 5,633,162. Although there are many media formulations, there is a need to develop defined media formulations that perform as well or preferably better than those containing
  • cell culture or “culture” is meant the growth and propagation of cells outside of a multicellular organism or tissue. Suitable culture conditions for mammalian cells are known in the art. See e.g. Animal cell culture: A Practical Approach, D. Rickwood, ed., Oxford University Press, New York (1992). Mammalian cells may be cultured in suspension or while attached to a solid substrate. Fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, shake flasks, or stirred tank bioreactors, with or without microcarriers, and operated in a batch, fed batch, continuous, semi-continuous, or perfusion mode are available for mammalian cell culture.
  • Cell culture media and/or concentrated feed media may be added to the culture continuously or at intervals during the culture.
  • a culture may be fed once per day, every other day, every three days, or may be fed when the concentration of a specific medium component, which is being monitored, falls outside a desired range.
  • Cells described herein may be cultured in small scale cultures, such as, for example, in 100 ml containers having about 30 ml of media, 250 ml containers having about 80 to about 90 ml of media, 250 ml containers having about 150 to about 200 ml of media.
  • the cultures can be large scale such as for example 1000 ml containers having about 300 to about 1000 ml of media, 3000 ml containers having about 500 ml to about 3000 ml of media, 8000 ml containers having about 2000 ml to about 8000 ml of media, and 15000 ml containers having about 4000 ml to about 15000 ml of media.
  • Concentrated feed medium may be based on just about any cell culture media formulation.
  • Such a concentrated feed medium can contain most of the components of the cell culture medium at, for example, about 5x, 6x, 7x, 8x, 9x, lOx, 12x, 14x, 16x, 20x, 3 Ox, 50x, lOOx, 200x, 400x, 600x, 800x, or about lOOOx of their normal amount.
  • Concentrated feed media are often used in fed batch culture processes.
  • methods according to the present invention may be used to improve the production of recombinant proteins in both single phase and multiple phase culture processes.
  • a single phase process cells are inoculated into a culture environment and the disclosed methods are employed during the single production phase.
  • a multiple stage process cells are cultured in two or more distinct phases. For example cells may be cultured first in one or more growth phases, under environmental conditions that maximize cell proliferation and viability, then transferred to a production phase, under conditions that maximize protein production.
  • a commercial process for production of a protein by mammalian cells there are commonly multiple, for example, at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 growth phases that occur in different culture vessels preceding a final production phase.
  • the growth and production phases may be preceded by, or separated by, one or more transition phases.
  • the methods according to the present invention can be employed at least during the production phase, although they may also be employed in a preceding growth phase.
  • a production phase can be conducted at large scale.
  • a large scale process can be conducted in a volume of at least about 100, about 500, about 1000, about 2000, about 3000, about 5,000, about 7,000, about 8,000, about 10,000, about 15,000, or about 20,000 liters.
  • a growth phase may occur at a higher temperature than a production phase.
  • a growth phase may occur at a first temperature from about 35°C to about 38°C
  • a production phase may occur at a second temperature from about 29°C to about 37°C, optionally from about 30°C to about 36°C or from about 30°C to about 34°C.
  • chemical inducers of protein production such as, for example, caffeine, butyrate, and hexamethylene bisacetamide (HMBA) may be added at the same time as, before, and/or after a temperature shift. If inducers are added after a temperature shift, they can be added from one hour to five days after the temperature shift, optionally from one to two days after the temperature shift.
  • the antibody and antigen-binding fragments expressed by cells described herein can be purified or isolated after expression.
  • Antibodies may be isolated or purified in a variety of ways known to those skilled in the art. Standard purification methods include chromatographic techniques, including ion exchange, hydrophobic interaction, affinity, sizing or gel filtration, and reversed-phase, carried out at atmospheric pressure or at high pressure using systems such as FPLC and HPLC. Purification methods also include electrophoretic, immunological, precipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful, variety of natural proteins bind antibodies, and these proteins can be used for purification of antibody and antigen-binding fragments described herein.
  • the bacterial proteins A and G bind to the Fc region
  • the bacterial protein L binds to the Fab region.
  • Purification can often be enabled by fusions or tags.
  • antibody and antigen-binding fragments may be purified using glutathione resin if a GST fusion is employed, Ni+2 affinity chromatography if a His tag is employed, or immobilized antiflag antibody if a flag tag is used.
  • suitable purification techniques see Protein Purification: Principles and Practice, 3rd Ed., Scopes, Springer- Verlag, NY, 1994, hereby expressly incorporated by reference.
  • the degree of purification necessary will vary depending on the use of the antibody or antigen-binding fragment. In some instances no purification is necessary. For example characterization of an antibody or antigen-binding fragment may take place directly from the media. In some instances, a high degree of purification is necessary. For example, high purity preparations of the antibody or antigen-binding fragment are required for use in a medicament or pharmaceutical compositions described herein, methods of detection described herein, and methods of treatment described herein.
  • compositions comprising the antibody or antigen-binding fragment described herein.
  • the pharmaceutical composition comprises a pharmaceutically acceptable carrier, excipient, and/or diluent.
  • kits comprising the antibody or antigen-binding fragment described herein.
  • the kit comprises a pharmaceutical composition described herein.
  • the kit comprises instructions for use.
  • the disclosure provides a pharmaceutical composition comprising the antibody or antigen-binding fragment described herein, for use in the treatment of COVID-19.
  • the pharmaceutical composition can be formulated for various types of administration or storage conditions.
  • the formulation will be optimized, often in a non- obvious way, according to the desired administration or storage conditions using a subset of carriers, excipients, and/or diluents, each having at specific concentration or range of acceptable concentrations.
  • the formulation can include, for example, one or more salts (a buffering salt), one or more polyols and one or more excipients.
  • the formulations of the present disclosure may also contain buffering agents, or preservatives.
  • the pH range of the formulation can be buffered in a solution at a pH in the range of about 4 to 8; in the range of about 4 to 7; in the range of about 4 to 6; in the range of about 5 to 6; or in the range of about 5.5 to 6.5.
  • salts include those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, boric, formic, malonic, succinic, and the like. Such salts can also be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.
  • buffering agents include phosphate, citrate, acetate, and 2-(N-morpholino)ethanesulfonic acid (MES).
  • the formulations of the present disclosure may include a buffer system.
  • buffer or “buffer system” is meant a compound that, usually in combination with at least one other compound, provides a buffering system in solution that exhibits buffering capacity, that is, the capacity to neutralize, within limits, either acids or bases (alkali) with relatively little or no change in the original pH.
  • Buffers include borate buffers, phosphate buffers, calcium buffers, and combinations and mixtures thereof.
  • Borate buffers include, for example, boric acid and its salts, for example, sodium borate or potassium borate. Borate buffers also include compounds such as potassium tetraborate or potassium metaborate that produce borate acid or its salt in solutions.
  • a phosphate buffer system includes one or more monobasic phosphates, dibasic phosphates and the like.
  • Particularly useful phosphate buffers are those selected from phosphate salts of alkali and/or alkaline earth metals.
  • suitable phosphate buffers include one or more of sodium dibasic phosphate (ISfel PCU), sodium monobasic phosphate (Na ⁇ PCE) and potassium monobasic phosphate (KH2PO4).
  • the phosphate buffer components frequently are used in amounts from 0.01% or to 0.5% (w/v), calculated as phosphate ion.
  • buffer compounds can optionally be added to the formulations, for example, citrates, sodium bicarbonate, TRIS, and the like.
  • Other ingredients in the solution while having other functions, may also affect the buffer capacity.
  • EDTA often used as a complexing agent, can have a noticeable effect on the buffer capacity of a solution.
  • Illustrative salts for use in the formulation of the disclosure include sodium chloride, sodium acetate, sodium acetate trihydrate and sodium citrate.
  • the concentration of salt in the formulations according to the disclosure is between about 10 mM and 500mM, between about 25m and 250 mM, between about 25nM and 150mM.
  • the sodium acetate trihydrate is at a concentration in the range of about 10 mM to 100 mM.
  • the sodium acetate trihydrate is at about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mM.
  • the sodium acetate trihydrate is at 25mM.
  • the sodium chloride at a concentration in the range of about 50 mM to 500 mM.
  • the sodium chloride is at about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 or 500 mM.
  • the sodium citrate is at a concentration in the range of about 10 mM to 100 mM
  • the sodium citrate is at about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mM.
  • the salt is sodium acetate trihydrate at a concentration in the range of about 25 mm to 100 mm and sodium chloride at a concentration in the range of about 150 mm to 500 mm.
  • the formulation can include one or more polyols as a bulking agent and/or stabilizing excipients.
  • Polyols include for example, trehalose, mannitol, maltose, lactose, sucrose, sorbitol, or glycerol.
  • the polyols is at a concentration in the range of about 0.1% to 50% or 5% to 25%.
  • the polyol is at about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50%.
  • the polyol is trehalose at a concentration in the range of about 1% to 50% or 5% to 25%.
  • the trehalose is at about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50%.
  • the trehalose can be at a concentration of about 10% or about 20%.
  • the trehalose can be at a concentration of about 20%.
  • the polyol is sorbitol at a concentration in the range of about 1% to about 10%. In some embodiments, the polyol is glycerol at a concentration in the range of about 1% to about 10%.
  • the polyol is mannitol at a concentration in the range of about 0.1% to about 10%. In some embodiments, the polyol is maltose at a concentration in the range of about 1% to about 10%.
  • the formulation includes one or more excipients and/ or surfactants to suppress or otherwise reduce antibody aggregation. Suitable excipients to reduce antibody aggregation include, by way of non-limiting example, a surfactant such as, by way of non-limiting example, Polysorbate 20 or Polysorbate 80. In some embodiments, the Polysorbate 20 or Polysorbate 80 is present at a concentration in the range of about 0.01 to 1 % or about 0.0 Ito 0.05%.
  • Polysorbate 20 or Polysorbate 80 is at a concentration of about 0.01. 0.02, 0.03, 0.04, 0.05, 0.06, 0.07. 0.08, 0.09, 0.1, 0.2, 0.3. 0.4, 0.5, 0.6, 0.7, 0.8. 0.9, or 1.0%.
  • the surfactant can be Polysorbate 80 at a concentration in the range of about 0.01 to 0.05%.
  • the Polysorbate 80 concentration can be 0.02%.
  • the formulation includes one or more excipients to reduce antibody oxidation.
  • Suitable excipients to reduce antibody oxidation include, by way of non-limiting example, antioxidants.
  • Antioxidants include for example, methionine, D-arginine, BHT or ascorbic acid.
  • the antioxidant is present at a concentration in the range of about 0.01 % to 1% ; 0.1% to 1%; or 0.1% to 0.5%.
  • the antioxidant is methionine.
  • the methionine is present at a concentration in the range of about 0.01 % to 1% ; 0.1% to 1%; or 0.1% to 0.5%.
  • the methionine is present at a concentration of about 0.01.
  • the methionine concentration can be about 0.1%.
  • the formulation includes one or more chelating agents, such as, for example, ethylenediaminetetraacetic acid (EDTA).
  • EDTA ethylenediaminetetraacetic acid
  • the chelating agent is at a concentration in the range of 0.01% to 1% ; 0.1% to 1%; or 0.1% to 0.5%.
  • the chelating agent is present at a concentration of about 0.01%. 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%.
  • the chelating agent can be EDTA at a concentration of about 0.1%.
  • the formulation includes one or more excipients to increase stability.
  • the excipient to increase stability is human serum albumin.
  • the human serum albumin is present in the range of about 1 mg to about 5 mg.
  • the formulation includes magnesium stearate (Mg stearate), an amino acid, or both mg-stearate and an amino acid. Suitable amino acids include for example, leucine, arginine, histidine, or combinations thereof.
  • the formulation includes low moisture microcrystalline cellulose, such as Avicel, polyethylene glycols (PEG), or a starch.
  • compositions of the present disclosure include, but are not limited to binders, fillers, disintegrants, lubricants, anti-microbial agents, antioxidant, and coating agents.
  • Binders can be corn starch, potato starch, other starches, gelatin, natural and synthetic gums such as acacia, xanthan, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone (e.g., povidone, crospovidone, copovidone, etc), methyl cellulose, Methocel, pre-gelatinized starch (e.g., STARCH 1500® and STARCH 1500 LM®, sold by Colorcon, Ltd.), hydroxypropyl methyl cellulose, microcrystalline cellulose (FMC Corporation, Marcus Hook, PA, USA), Emdex, Plasdone, or mixtures thereof.
  • natural and synthetic gums such as acacia, xanthan, sodium alginate, alginic acid
  • Fillers can be talc, calcium carbonate (e.g., granules or powder), dibasic calcium phosphate, tribasic calcium phosphate, calcium sulfate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, dextrose, fructose, honey, lactose anhydrate, lactose monohydrate, lactose and aspartame, lactose and cellulose, lactose and microcrystalline cellulose, maltodextrin, maltose, mannitol, microcrystalline cellulose & guar gum, molasses, sucrose, or mixtures thereof.
  • calcium carbonate e.g., granules or powder
  • dibasic calcium phosphate tribasic calcium phosphate
  • calcium sulfate e.g.
  • Disintegrants can be agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, (such as Explotab), potato or tapioca starch, other starches, pre-gelatinized starch, clays, other algins, other celluloses, gums (like gellan), low-substituted hydroxypropyl cellulose, ployplasdone, or mixtures thereof.
  • Lubricants can be calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, compritol, stearic acid, sodium lauryl sulfate, sodium stearyl fumarate, (such as Pruv), vegetable based fatty acids lubricant, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar, syloid silica gel (AEROSIL 200, W.R.
  • Anti-caking agents can be calcium silicate, magnesium silicate, silicon dioxide, colloidal silicon dioxide, talc, or mixtures thereof.
  • Antimicrobial agents can be benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, butyl paraben, cetylpyridinium chloride, cresol, chlorobutanol, dehydroacetic acid, ethylparaben, methylparaben, phenol, phenylethyl alcohol, phenoxyethanol, phenylmercuric acetate, phenylmercuric nitrate, potassium sorbate, propylparaben, sodium benzoate, sodium dehydroacetate, sodium propionate, sorbic acid, thimersol, thymo, or mixtures thereof.
  • Antioxidants can be ascorbic acid, BHA, BHT, EDTA, or mixture thereof, and COATING AGENTS: sodium carboxymethyl cellulose, cellulose acetate phthalate, ethylcellulose, gelatin, pharmaceutical glaze, hydroxypropyl cellulose, hydroxypropyl methylcellulose (hypromellose), hydroxypropyl methyl cellulose phthalate, methylcellulose, polyethylene glycol, polyvinyl acetate phthalate, shellac, sucrose, titanium dioxide, carnauba wax, microcrystalline wax, gellan gum, maltodextrin, methacrylates, microcrystalline cellulose and carrageenan or mixtures thereof.
  • the formulation can also include other excipients and categories thereof including but not limited to Pluronic®, Pol oxamers (such as Lutrol® and Pol oxamer 188), ascorbic acid, glutathione, protease inhibitors (e.g.
  • soybean trypsin inhibitor, organic acids), pH lowering agents, creams and lotions like maltodextrin and carrageenans
  • materials for chewable tablets like dextrose, fructose, lactose monohydrate, lactose and aspartame, lactose and cellulose, maltodextrin, maltose, mannitol, microcrystalline cellulose and guar gum, sorbitol crystalline); parenterals (like mannitol and povidone); plasticizers (like dibutyl sebacate, plasticizers for coatings, polyvinylacetate phthalate); powder lubricants (like glyceryl behenate); soft gelatin capsules (like sorbitol special solution); spheres for coating (like sugar spheres); spheronization agents (like glyceryl behenate and microcrystalline cellulose); suspending/gelling agents (like carrageenan, gellan gum, mannitol, microcrystalline cellulose, povidone, sodium
  • FD&C Yellow No.10 glycerol palmitostearate, glyceryl monostearate, indigo carmine, lecithin, manitol, methyl and propyl parabens, mono ammonium glycyrrhizinate, natural and artificial orange flavor, pharmaceutical glaze, pol oxamer 188, Polydextrose, polysorbate 20, polysorbate 80, polyvidone, pregelatinized com starch, pregelatinized starch, red iron oxide, saccharin sodium, sodium carboxymethyl ether, sodium chloride, sodium citrate, sodium phosphate, strawberry flavor, synthetic black iron oxide, synthetic red iron oxide, titanium dioxide, and white wax.
  • the pharmaceutical composition is formulated for parenteral administration, enteral administration, mucosal administration, nasal administration, oral administration, or intra-articular administration.
  • the administration comprises inhalation, intravenous injection, infusion, subcutaneous injection, or intramuscular injection.
  • the disclosure provides a method of treating COVID-19 in a subject in need thereof, comprising administering to the subject an antibody or antigen-binding fragment described herein or a pharmaceutical composition described herein.
  • the disclosure provides a method of preventing COVID-19 in a subject in need thereof, comprising administering to the subject an antibody or antigen-binding fragment described herein or a pharmaceutical composition described herein
  • the disclosure provides a method of reducing symptoms associated with COVID- 19 in a subject in need thereof, comprising administering to the subject an antibody or antigen-binding fragment described herein or a pharmaceutical composition described herein.
  • the disclosure provides a method of treating, preventing, or reducing symptoms associated with COVID-19 in a subject in need thereof, comprising detecting a SARS- CoV-2 viral genome or a SARS-CoV-2 antigen in a sample collected from the subject, wherein detection of the SARS-CoV-2 antigen comprises contacting the sample with an antibody or antigen-binding fragment described herein; and administering a treatment to the subject, wherein the treatment prevents, reduces, or eliminates a SARS-CoV-2 infection or a variant thereof; and/or prevents, reduces, or eliminates symptoms associated with COVID-19, wherein the treatment comprises an antibody or antigen-binding fragment described herein or a pharmaceutical composition described herein.
  • the methods of provided herein include administering the antibody or antigenbinding fragment described herein to a subject in need thereof.
  • the subject can be a human or an animal.
  • the subject can have or be suspected of having a SARS-CoV-2 infection or a variant thereof.
  • the subject has or is suspected of having a SARS- CoV-2 infection or a variant thereof.
  • the subject has at least one symptom associated with a SARS-CoV-2 infection or a variant thereof.
  • the subject is an animal or a human.
  • a subject can be determined to have a SARS-CoV-2 infection or a variant thereof using any appropriate method known in the art. Testing can be performed on a sample collected from the subject, which can include saliva, a nasopharyngeal swab, nasal midturbinate swab, anterior nasal swab, blood, plasma, urine, feces, bronchoalveolar lavage, or another sample in which a SARS-CoV-2 infection or a variant thereof can be measured.
  • Samples can be tested using any test type known in the art to detect a previous or active SARS-CoV-2 infection. Illustrative test types include viral tests, diagnostic tests, and/or screening tests.
  • Viral tests including nucleic acid amplification tests (NAATs) and antigen tests, are used as diagnostic tests to detect infection with SARS-CoV-2 or a variant thereof and to inform a subject’s medical care. Viral tests can also be used as screening tests to reduce the transmission of SARS-CoV-2 or a variant thereof by identifying infected subjects who need to isolate from others.
  • NAATs nucleic acid amplification tests
  • antigen tests are used as diagnostic tests to detect infection with SARS-CoV-2 or a variant thereof and to inform a subject’s medical care.
  • Viral tests can also be used as screening tests to reduce the transmission of SARS-CoV-2 or a variant thereof by identifying infected subjects who need to isolate from others.
  • NAATs are high-sensitivity, high-specificity tests for diagnosing SARS-CoV-2 infection or a variant thereof by detecting the presence of a SARS-CoV-2 viral genome by measuring, for example, a gene of the viral genome.
  • NAATs detect one or more viral ribonucleic acid (RNA) genes and indicate a current infection or a recent infection but, due to prolonged viral RNA detection, are not always direct evidence for the presence of virus capable of replicating or being transmitted to others.
  • RNA viral ribonucleic acid
  • Most NAATs need to be processed in a laboratory, and time to results can vary ( ⁇ l-3 days), but some NAATs are point-of- care tests with results available in about 15-45 minutes. MostNAATs produce qualitative results.
  • NAATs can be performed on upper respiratory specimens, such as nasopharyngeal, nasal mid-turbinate, anterior nasal, or saliva. RNA is extracted from the sample and subject to reverse transcription prior to amplification. Illustrative examples of NAATs include rt- PCR, PCR tests and rapid PCR tests, wherein PCR primers are designed to amplify S ARS- CoV-2 genes for subsequent detection. Viral loads can be calculated by in RNA copies of the target gene per mL. A positive result from NAATs imply the subject has a SARS-CoV- 2 infection.
  • Antigen tests are immunoassays that detect the presence of a specific viral antigen. Antigen tests generally have similar specificity, but are less sensitive than most NAATs. Most are less expensive than NAATs and can be processed at the point of care with results available in minutes and thus can be used in screening programs to quickly identify those who are likely to be contagious. Because of the performance characteristics of antigen tests, it may be necessary to confirm some antigen test results (a negative test in persons with symptoms or a positive test in persons without symptoms) with a laboratory-based NAAT. Use of the Antigen Testing Algorithm is recommended to determine when confirmatory testing is needed. Antigen tests can be performed on nasopharyngeal or anterior nasal specimens.
  • antigen test examples include use lateral flow devices that immunodetect the presence of SARS-CoV-2 antigen, such as Innova Rapid SARS-CoV-2 Antigen Test (Xiamen Biotime Biotechnology, Fujian, China), Spring Healthcare SARS-CoV-2 Antigen Rapid Test Cassette (Shanghai ZJ Bio-Tech, Shanghai, China), E25Bio Rapid Diagnostic Test (E25Bio, Cambridge, MA, USA), Encode SARS- CoV-2 Antigen Rapid Test Device (Zhuhai Encode Medical Engineering, Zhuhai, China), SureScreen COVID-19 Rapid Antigen Test Cassette, and SureScreen COVID-19 Rapid Fluorescence Antigen Test (both from SureScreen Diagnostics, Derby, UK).
  • a positive result from an antigen test implies the subject has a SARS-CoV-2 infection.
  • a serological test can be performed in a sample to determine is the subject has had a prior SARS-CoV-2 infection.
  • the serological tests detect the presence of antibodies associated with COVID-19 in blood.
  • the antibodies are a developed in the blood of a subject due to the immune response to COVID-19.
  • a subject suspected of having a SARS-CoV-2 infection has been determined to have exposure to a known person with an active SARS-CoV-2 infection, or can have symptoms associated with a SARS-CoV-2 or a variant thereof infection or CO VID-19.
  • the methods provided herein treat, prevent, or alleviate one or more symptoms.
  • the symptom is at least one acute symptom.
  • An acute symptom is a symptom that resolves in less than a month, or resolves within a timeframe that is expected for a given disease or disorder.
  • a subject suffering from a coronavirus infection e.g. COVID-19
  • a variant thereof may experience acute symptoms such as fever, sore throat, cough, shortness of breath, and chest pain that resolve within 2 to 4 weeks.
  • the severity of the one or more symptoms is reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or eliminated completely.
  • the duration of the one or more symptoms is reduced by at least one day, at least two days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days.
  • the symptom is at least one chronic symptom.
  • a chronic symptom is a symptom that persists in a subject suffering from a disease or disorder after the time considered normal for that disease or disorder.
  • a subject suffering from a coronavirus infection e.g. COVID-19
  • a prolonged inflammatory response can contribute to chronic symptoms.
  • chronic symptoms are at least one of fatigue, muscle aches and pains, poor sleep, cough, breathlessness, orthopnea, leg swelling, exercise intolerance due to COVID-19 induced heart failure, pulmonary embolism, pulmonary fibrosis, COVID-19-related ARDS, palpitations with mild exertion, night sweats, organ damage (e.g. cardiac or respiratory organ damage), and poor temperature control.
  • organ damage e.g. cardiac or respiratory organ damage
  • the symptom of a SARS-CoV-2 infection or COVID-19 is one or more of hyperactive immune response, fever, gastrointestinal symptoms, respiratory symptoms, anosmia (loss of smell), dysgeusia (loss of taste), cough, headache, throat ache, pain when swallowing, dyspnea, difficult breathing, shortness of breath, nausea, vomiting, reduced 02 saturation, diarrhea, rhinorrhea, abdominal pain, myalgia, fever, conjunctivitis, and loss of appetite.
  • a hyperactive immune response can include, for example, a cytokine release syndrome, a “cytokine storm”, or the like.
  • the hyperactive immune response comprises elevated levels of at least one of interleukin 6 (IL-6), C-reactive protein (CRP), D-dimer, interferon (IFN), interferon alpha (IFN-a), interferon gamma (IFN-y), interleukin 1 beta (IL- 1 (3), and/or CXCL10.
  • IL-6 interleukin 6
  • CRP C-reactive protein
  • IFN interferon
  • IFN-a interferon alpha
  • IFN-y interferon gamma
  • IL- 1 (3) interleukin 1 (3)
  • CXCL10 interleukin 6
  • the methods provided herein include administration of an antibody or antigenbinding fragment to a subject in need thereof.
  • the antibody or antigen-binding fragment or pharmaceutical composition can be administered by any suitable means.
  • administration is enteral, parenteral, mucosal, nasal, oral, by inhalation, by intravenous injection, by infusion, by subcutaneous injection, by intramuscular injection, or intra-articular.
  • Dosing can be by any suitable route, for example, by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
  • Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.
  • the antibody or antigen-binding fragment or pharmaceutical composition can be administered as a unit dose.
  • the unit dose is a therapeutically effective amount.
  • the therapeutically effective amount to be administered as a unit dose to a subject will be in the range of about 0.01 to about 100 mg/kg of patient body weight whether by one or more administrations.
  • the masked antibody used is about 0.01 to about 45 mg/kg, about 0.01 to about 40 mg/kg, about 0.01 to about 35 mg/kg, about 0.01 to about 30 mg/kg, about 0.01 to about 29 mg/kg, about 0.01 to about 28 mg/kg, about 0.01 to about 27 mg/kg, about 0.01 to about 26 mg/kg, about 0.01 to about 25 mg/kg, about 0.01 to about 24 mg/kg, about 0.01 to about 23 mg/kg, about 0.01 to about 22 mg/kg, about 0.01 to about 21 mg/kg, about 0.01 to about 20 mg/kg, about 0.01 to about 19 mg/kg, about 0.01 to about 18 mg/kg, about 0.01 to about 17 mg/kg, about 0.01 to about 16 mg/kg, about 0.01 to about 15 mg/kg, about 0.01 to about
  • a unit dose is about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1 100 mg, about 1200 mg, about 1300 mg or about 1400 mg on day 1 of a cycle.
  • a cycle can be 1 to 21 days in duration. Dosing can occur once per week, twice per week, three times per week, four times per week, five times per week, six times per week or seven times per week.
  • the dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as infusions. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs.
  • One illustrative dosage may be in the range from about 0.05 mg/kg to about 10 mg/kg.
  • one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg (or any combination thereof) may be administered to the subject.
  • Such doses may be administered intermittently, for example, every week or every three weeks (e.g., such that the patient receives from about two to about twenty, or, for example, about six doses of antibody or antigen-binding fragment described herein or the pharmaceutical composition described herein).
  • An initial higher loading dose, followed by one or more lower doses may be administered.
  • kits for detecting a SARS-CoV-2 spike protein in a sample comprising contacting a sample with the antibody or antigen-binding fragment described herein.
  • the antibody or antigen-binding fragment is labeled with a tag, detection dye or a secondary antibody.
  • the sample is an environmental sample, sewage, wastewater, urine, feces, saliva, nasal swab, mucous, blood, plasma, bronchoalveolar lavage, or one or more cells.
  • the sample is collected from a subject.
  • the subject has or is suspected of having a SARS-CoV-2 infection or COVID-19.
  • the antibody or antigen-binding fragment is labeled with a tag, detection dye or a secondary antibody.
  • the disclosure provides a method of diagnosing a SARS-CoV-2 infection or COVID-19 in subject, comprising detecting a SARS-CoV-2 spike protein in a sample, comprising contacting a sample with the antibody or antigen-binding fragment described herein.
  • any assay platform or type known in the art for detecting an antigen e.g. a SARS- CoV-2 spike protein
  • an antibody or antigen-binding fragment targeting said antigen may be used in the methods of detection and diagnosis described herein.
  • the method comprises use of an electrochemical assay, biosensor, or immunoassay.
  • the detection method for conducting an assay may be selected from a group including but not limited to absorbance, chemiluminescence, fluorescence, magnetic, and/or electrochemical.
  • detecting the SARS-CoV-2 spike protein further comprises use of an ELISA technique or a lateral flow assay.
  • CLIA Chemiluminescent Microparticle Immunoassay
  • chemiluminescent microparticle assay may be used in the methods of detection and diagnosis described herein.
  • chemiluminescent microparticle assay techniques are based on the principle that a chemiluminescent label, when treated via a trigger reagent, will emit light at a characteristic wavelength (i.e., chemiluminescence).
  • the reactants necessary for CMIA can include microparticles coated with a capture agent (e.g. an antibody or antigen-binding fragment) that binds the antigen (e.g. a SARS- CoV-2 spike protein) being measured, a chemiluminescent detection agent and a triggering agent (e.g., chemical or electrochemical).
  • a capture agent e.g. an antibody or antigen-binding fragment
  • a chemiluminescent detection agent e.g., chemical or electrochemical
  • the reaction sequence for performing CMIA can include mixing the microparticles coated with a capture agent specific for the analyte with a sample in a reaction vessel to form an immune complex; washing the captured immune complex to remove unbound material; mixing the captured immune complex with a chemiluminescent detection agent; washing the captured immune complex- chemiluminescent detection agent; and mixing the captured immune complex- chemiluminescent detection agent with a triggering agent to initiate light emission.
  • Chemiluminophores useful in CMIA include acridinium (e.g. acridinium-9- carboxamide), luminol, dioxetane, ruthenium complexes and similar chemiluminescent derivatives.
  • Microparticles useful in CMIA include diamagnetic, magnetic and paramagnetic microparticles. Examples of commercially available automated instruments with which chemiluminescent microparticle assay assays can be conducted include: Architect i-Systems and the Abbott Prism (all available from Abbott Laboratories, Abbott Park, Hl.).
  • Electrochemical detection systems may be used in the methods of detection and diagnosis described herein.
  • Basic procedures for electrochemical detection are known in the art.
  • An illustrative example includes immobilization of a primary binding molecule (e.g. an antibody or antigen-binding fragment), followed by exposure to a sequence of solutions or samples containing the antigen (e.g. a SARS-CoV-2 spike protein), followed by exposure to a secondary antibody conjugated to an enzyme label (AP-Ab, rat anti mouse IgG and alkaline phosphatase), and p-aminophenyl phosphate (PAPP).
  • AP-Ab enzyme label
  • rat anti mouse IgG and alkaline phosphatase p-aminophenyl phosphate
  • the AP converts PAPP to p-aminophenol (PAPR, the “R” is intended to distinguish the reduced form from the oxidized form, PAPO, the quinoneimine), which is electrochemically reversible at potentials that do not interfere with reduction of oxygen and water at pH 9.0, where AP exhibits optimum activity.
  • PAPR does not cause electrode fouling, unlike phenol whose precursor, phenylphosphate, is often used as the enzyme substrate.
  • Picomole detection limits for PAPR and femtogram detection limits for IgG achieved in microelectrochemical immunoassays using PAPP volumes ranging from 20 pL to 360 pL have been reported previously. In capillary immunoassays with electrochemical detection, the lowest detection limit reported thus far is 3000 molecules of mouse IgG using a volume of 70 pL and a 30 min or 25 min assay time.
  • Biosensor detection systems may be used in the methods of detection and diagnosis described herein.
  • Biosensors such as optical biosensors, electronic biosensor, piezoelectric biosensor, gravimetric biosensors, pyroelectric biosensors, thermal, and/or magnetic biosensors may be used in conjunction with an antibody or antigen-binding fragments described herein.
  • Biosensors can be detection reagents conjugated to an antibody or antigen-binding fragments described herein, such that contact with a SARS-CoV-2 antigen results in the change of a detectable signal.
  • detection reagents can include, without limitation, nanoparticles (e.g.
  • Immunoassay detection systems may be used in the methods of detection and diagnosis described herein.
  • An immunoassay generally refers to any detection method that measures an antigen (e.g. a SARS-CoV-2 spike protein), wherein the antigen is bound specifically by an immune- molecule (e.g. an antibody or antigen-binding fragment described herein).
  • an immune- molecule e.g. an antibody or antigen-binding fragment described herein.
  • Conventional immunoassays include, without limitation, an ELISA, an RIA, flow cytometry, FACS, tissue immunohistochemistry, Western blot or immunoprecipitation.
  • Assays may be performed in solution or may use a solid (insoluble) support (e.g., polystyrene, nitrocellulose, or beads), using any standard methods (e.g., as described in Current Protocols in Immunology, Coligan et al., ed.; John Wiley & Sons, New York, 1992). Immunoassays usually are classified in one of several ways. For example, immunoassays can be classified according to the mode of detection used, i.e., enzyme immunoassays, radio immunoassays, fluorescence polarization immunoassays, chemiluminescence immunoassays, turbidimetric assays, etc.
  • mode of detection i.e., enzyme immunoassays, radio immunoassays, fluorescence polarization immunoassays, chemiluminescence immunoassays, turbidimetric assays, etc.
  • Another grouping method is according to the assay procedure used, i.e., competitive assay formats, sandwich-type assay formats as well as assays based on precipitation or agglutination principles.
  • assay procedure used i.e., competitive assay formats, sandwich-type assay formats as well as assays based on precipitation or agglutination principles.
  • washing steps are included in the procedure (so- called heterogeneous assays) or whether reaction and detection are performed without a washing step (so-called homogeneous assays).
  • Lateral flow devices may be used in the methods of detection and diagnosis described herein. Lateral flow devices flow devices are known in the art for use in detection of antigens (Lateral Flow Immunoassay, edited by Raphael Wong, Harley Tse, 2009, Springer; Paper-based diagnostics: Current Status and Future applications, Kevin J. Land, Springer 2019).
  • a non-limiting illustrative workflow for a lateral flow assay may include a membrane such as a nitrocellulose membrane which comprises an antibody or antigen-binding fragment, which further comprises a detectable label. If the membrane is contacted with a sample, an antigen (e.g. a SARS-CoV-2 spike protein) to be detected will bind to the antibody or antigen-binding fragment.
  • an antigen e.g. a SARS-CoV-2 spike protein
  • the resulting complex will move driven by capillary forces on the membrane and will be immobilized on a test line on the membrane comprising a means for detecting the antibody or antigen-binding fragment or the antigen; typically a secondary antibody binding to the immunoglobulin class or classes of the antibody or the antibodies to be detected such as IgG and/or IgA and/or IgM.
  • a means for detecting the antibody or antigen-binding fragment or the antigen typically a secondary antibody binding to the immunoglobulin class or classes of the antibody or the antibodies to be detected such as IgG and/or IgA and/or IgM.
  • nanoparticles or beads are used as labels, for example gold nanoparticles or latex beads.
  • Test kits include one or more reagents useful for practicing one or more immunoassays according to the disclosure.
  • a test kit generally includes a package with one or more containers holding the reagents, as one or more separate compositions or, optionally, as admixture where the compatibility of the reagents will allow.
  • the test kit can also include other material(s) that may be desirable from a user standpoint, such as a buffer(s), a diluent(s), a standard(s), and/or any other material useful in sample processing, washing, or conducting any other step of the assay.
  • a test kit includes: (a) a labeled detection agent specific for an antigen (e.g. an antibody or antigen-binding fragment described herein that binds a SARS- CoV-2 spike protein); and (b) a labeled species-specific antibody, wherein the labeled species-specific antibody is specific for the species from which the biological sample was obtained.
  • the labeled detection agent includes a labeled antianalyte antibody.
  • the labeled anti-analyte antibody can be labeled with the same label as the labeled species-specific antibody or can be labeled with a different label.
  • the labeled anti-analyte antibody and the labeled species-specific antibody can be packaged in the same container or in different containers.
  • Test kits according to the disclosure can also include at least one indirect label. If the label employed generally requires an indicator reagent to produce a detectable signal, the test kit preferably includes one or more suitable indicator reagents.
  • Test kits according to the disclosure can additionally include a solid phase and a capture agent, such as an anti-analyte capture antibody, affixed to the solid phase.
  • the solid phase includes one or more microparticles (e.g., magnetic or paramagnetic microparticles), electrodes, and/or a microplate.
  • Test kits designed for multiplex assays conveniently contain one or more solid phases including a plurality of anti-analyte capture antibodies that are specific for a plurality of different analytes.
  • a test kit designed for multiplex electrochemical immunoassays can contain a solid phase including a plurality of electrodes, with each electrode bearing a different anti-analyte capture antibody.
  • a test kit intended for multiplex “sandwich” immunoassays can include a plurality of different labeled anti-analyte antibodies that are specific for the plurality of different analytes, wherein each different labeled anti-analyte antibody is labeled with a distinct label.
  • Test kits preferably include instructions for carrying out one or more of the methods of detection provided in the disclosure.
  • Instructions included in kits of the disclosure can be affixed to packaging material or can be included as a package insert. While the instructions are typically written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like.
  • the term “instructions” can include the address of an internet site that provides the instructions.
  • Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein.
  • the foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)).
  • the nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
  • the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen.
  • immunoglobulin (Ig) molecules i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen.
  • Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab’ and F(ab')2 fragments, and an Fab expression library.
  • bind or “immunoreacts with” is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react (i.e., bind) with other polypeptides or binds at much lower affinity (Kd > 10-6) with other polypeptides.
  • an “isolated” antibody or antigen-binding fragment as described herein refers to an antibody or antigen-binding fragment that is substantially pure.
  • An antibody or antigenbinding fragment that results following the first steps of production may exist in a crude solution that contains the antibody or antigen-binding fragment comprises and additional contaminants, such as other nucleic acids, polypeptides, cell debris, media components, or other reagents used during production.
  • An isolated antibody or antigen-binding fragment has been substantially isolated from the contaminants through any purification process known in the art.
  • the basic antibody structural unit is known to comprise a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa).
  • the amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains.
  • Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ea., 2nd ed. Raven Press, N.Y. (1989)).
  • the variable regions of each light/heavy chain pair form the antibody binding site.
  • MAb monoclonal antibody
  • CDRs complementarity determining regions
  • isotype refers to the antibody class (e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE antibody) that is encoded by the heavy chain constant domain genes.
  • antibody class e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE antibody
  • each wild type human IgG constant region (including all domains, i.e., CHI domain, hinge, CH2 domain, and CH3 domain) is cataloged in the UniProt database available on-line, e.g., as P01857 (IgGl), P01859 (IgG2), P01860 (IgG3), and P01861 (IgG4), or different allotypes thereof (SEQ ID NOs: 1, 6, 11, and 16, respectively).
  • a domain of a heavy chain constant region is of an "IgGl isotype," “IgG2 isotype,” “IgG3 isotype,” or “IgG4 isotype,” if the domain comprises the amino acid sequence of the corresponding domain of the respective isotype, or a variant thereof (that has a higher homology to the corresponding domain of the respective isotype than it does to that of the other isotypes).
  • antibody molecules obtained from humans relate to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgGl, IgG2, and others.
  • the light chain may be a kappa chain or a lambda chain.
  • epitopic determinants include any protein determinant capable of specific binding to an immunoglobulin, a scFv, or a T-cell receptor.
  • epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • An antibody is said to specifically bind an antigen when the dissociation constant is ⁇ 1 pM; preferably ⁇ 100 nM and most preferably ⁇ 10 nM.
  • immunological binding and “immunological binding properties” and “specific binding” and “binding” and “selective binding” refer to the non- covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific.
  • the strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (KD) of the interaction, wherein a smaller KD represents a greater affinity.
  • KD dissociation constant
  • One such method entails measuring the rates of antigen-binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions.
  • both the “on rate constant” (K on ) and the “off rate constant” (K O ff) can be determined by calculation of the concentrations and the actual rates of association and dissociation.
  • K on the “on rate constant”
  • K O fff K O ff
  • An antibody of the present disclosure is said to specifically bind to a SARS- CoV-2 spike protein epitope when the equilibrium binding constant (KD) is about 1 pM, preferably about 100 nM, more preferably about 10 nM, and most preferably about 100 pM to about 1 pM, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art.
  • KD equilibrium binding constant
  • amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide- containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur- containing side chains is cysteine and methionine.
  • Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine valine, glutamic- aspartic, and asparagine-glutamine.
  • amino acid sequences of antibodies or immunoglobulin molecules are contemplated as being encompassed by the present disclosure, providing that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99%.
  • conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains.
  • amino acids are generally divided into families: (1) acidic amino acids are aspartate, glutamate; (2) basic amino acids are lysine, arginine, histidine; (3) nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine.
  • the hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine.
  • the hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine.
  • Other families of amino acids include (i) serine and threonine, which are the aliphatic-hydroxy family; (ii) asparagine and glutamine, which are the amide containing family; (iii) alanine, valine, leucine and isoleucine, which are the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine, which are the aromatic family.
  • a “humanized antibody” is a genetically engineered antibody in which CDRs are grafted into human "acceptor” antibody sequences (see, e.g., Queen, US 5,530,101 and 5,585,089; Winter, US 5,225,539; Carter, US 6,407,213; Adair, US 5,859,205; and Foote, US 6,881,557).
  • the acceptor antibody sequences can be, for example, a mature human antibody sequence, a composite of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence.
  • An example acceptor sequence for the heavy chain is the germline VH exon VH1-2 (also referred to in the literature as HV1-2) (Shin et al, 1991, EMBO J.
  • a humanized antibody is an antibody having some or all CDRs entirely or substantially from any source, and variable region framework sequences and constant regions, if present, entirely or substantially from human antibody sequences.
  • a humanized heavy chain has at least one, two and usually all three CDRs entirely or substantially originating in a source separate from a heavy chain variable region framework sequence and heavy chain constant region, if present, substantially from human heavy chain variable region framework and constant region sequences.
  • a humanized light chain has at least one, two and usually all three CDRs entirely or substantially originating in a source separate from a light chain variable region framework sequence and light chain constant region, if present, substantially from human light chain variable region framework and constant region sequences.
  • a humanized antibody comprises a humanized heavy chain and a humanized light chain.
  • a CDR in a humanized antibody is substantially from a corresponding CDR in a non-human antibody when at least 60%, 85%, 90%, 95% or 100% of corresponding residues (as defined by Kabat) are identical between the respective CDRs.
  • the variable region framework sequences of an antibody chain or the constant region of an antibody chain are substantially from a human variable region framework sequence or human constant region respectively when at least 85%, 90%, 95% or 100% of corresponding residues defined by Kabat are identical.
  • operably linked refers to the arrangement of various nucleic acid molecule elements relative to each such that the elements are functionally connected and are able to interact with each other.
  • Such elements may include, without limitation, a promoter, an enhancer and/or a regulatory element, a polyadenylation sequence, one or more introns and/or exons, and a coding sequence of a polynucleotide to be expressed (e.g. a polynucleotide encoding an antibody or antigen-binding fragment described herein).
  • the nucleic acid sequence elements when properly oriented or operably linked, act together to modulate the activity of one another, and ultimately may affect the level of expression of the polynucleotide.
  • modulate is meant increasing, decreasing, or maintaining the level of activity of a particular element.
  • the position of each element relative to other elements may be expressed in terms of the 5' terminus and the 3' terminus of each element, and the distance between any particular elements may be referenced by the number of intervening nucleotides, or base pairs, between the elements.
  • the term “enhancer” refers to nucleic acid sequences that regulate, either directly or indirectly, the transcription of corresponding nucleic acid coding sequences to which they are operably linked (e.g., a transgene).
  • An enhancer may function alone to regulate transcription or may act in concert with one or more other regulatory sequences (e.g., promoters, minimal promoters, or silencers).
  • an enhancer is typically operably linked to a polynucleotide encoding an antibody or antigen-binding fragment described herein.
  • An “enhancer” as described herein may, in some embodiments, function as a promoter in the absence of a promoter sequence other than the enhancer itself
  • agent is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.
  • patient includes human and veterinary subjects.
  • the disclosure also includes Fv, Fab, Fab’ and F(ab')2 anti-SARS-CoV-2 spike protein antibody fragments, single chain anti-SARS-CoV-2 spike protein antibodies, bispecific anti-SARS-CoV-2 spike protein antibodies, heteroconjugate anti-SARS-CoV-2 spike protein antibodies, trispecific antibodies, immunoconjugates and fragments thereof.
  • Bispecific antibodies are antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for SARS-CoV-2 spike protein.
  • the second binding target is any other antigen, and advantageously is a cellsurface protein or receptor or receptor subunit.
  • the serum was aliquoted and subsequently used for measuring the level of neutralizing antibodies against prototype SARS-CoV-2 by plaque-reduction neutralization test (PRNT).
  • PRNT plaque-reduction neutralization test
  • PBMCs from the four patients with the highest PRNT activities were isolated by the FicollTM density gradient centrifugation technique.
  • B cells were isolated from PBMCs by EasySepTM human B cell isolation kit (STEMCELL Technologies) following the manufacturer’s instructions. After that, isolated B cells were transformed by Epstein- Barr virus (EBV) obtained from the B95-8 lymphoblastoid cell line supernatant.
  • EBV- transformed B cells were cultured in 384-well plates at 37°C for seven days.
  • EBV-transformed B cells with the top-rank neutralizing activities were fused with SHM-33 myeloma cells using the cell electrofusion technique to create stable human hybridoma clones.
  • individual selected EBV-transformed B cell pools were mixed with myeloma cells followed by washing with BTX cytofusion medium. The cell mixture was transferred to a cytofusion cuvette, and electrofusion was performed using BTX ECM 2001 fusion system. After fusion, hybridoma cells were cultured in 96-well plates for ten days in a ClonaCell-HY Medium E (STEMCELL Technologies) containing hypoxanthine- aminopterin-thymidine (HAT).
  • HAT hypoxanthine- aminopterin-thymidine
  • Example 2 and 3 The supernatant from each well was screened based on binding and neutralizing activities against prototype SARS-CoV-2 by ELISA, as mentioned in Example 2 and 3 below.
  • Hybridoma cells with high neutralizing activities were subcloned by a limiting dilution technique to achieve hybridoma monocl onality. After 7-10 days of culturing, hybridoma clones were screened by ELISA. Finally, selected human hybridoma clones with high neutralizing activities were expanded to produce SARS-CoV-2 neutralizing antibodies.
  • Example 2 Screening of human antibodies against SARS-CoV-2 by binding activity
  • ELISA technique was utilized to screen for high binding anti-SARS-CoV-2 antibodies.
  • Each well of the MaxiSorp 96-well ELISA plate (ThermoFisher Scientific) was coated with 10 ng of prototype SARS-CoV-2 RBD-His proteins (Sino Biological) at 4°C overnight.
  • the coated ELISA plate was washed and blocked with 0.05% Tween-20 in PBS buffer (PBST).
  • PBST PBS buffer
  • 100 pl culture supernatant samples from EBV-transformed B cells or human hybridoma cells were added to the coated wells. The plate was incubated for 1 hour at 37°C and washed three times with PBST.
  • a secondary antibody probing step was performed by adding 100 pl/well of goat anti -human IgG (Fey fragment specific)-HRP (Jackson ImmunoResearch), incubating for 1 hour at 37°C, and washing with PBST 3 times. Next, the plate was added with o-Phenylenediamine dihydrochloride (OPD) substrate solution (100 pl/well) and incubated in the dark for 20 minutes at room temperature. The reaction was stopped by adding 2N H2SO4 solution (50 pl/well). Finally, the absorbance was measured at 492 nm by Cytation 5 (BioTek)
  • Example 3 Screening of human antibodies against SARS-CoV-2 by neutralizing activity
  • ELISA technique was employed to evaluate the RBD/ACE2 neutralizing activity from EBV-transformed B cells and human hybridoma cells that produced RBD-specific antibodies (positive binding clones).
  • an ELISA plate was coated with recombinant ACE2 human Fc tag proteins (Genscript) at 4°C overnight.
  • the coated ELISA plate was washed and blocked with PBST.
  • 50 pl culture supernatant samples from the positive binding clones or a control antibody were pre-incubated with 50 pl of RBD mouse Fc tag proteins (Genscript) at 37°C for 30 minutes and individual samples were transferred to the coated plate. After incubation at 37°C for 1 hour, the plate was washed by PBST three times.
  • Goat anti-mouse Ig /-chain specific)-HRP Jackson ImmunoResearch
  • Goat anti-mouse Ig /-chain specific)-HRP Jackson ImmunoResearch
  • the plate was washed three times by PBST before adding 100 pl of OPD and then incubated in the dark at room temperature for 20 minutes.
  • the stop solution was added to each well, and the absorbance at 492 nm was determined by Cytation 5 (BioTek).
  • RNA-Seq whole transcriptome shotgun sequencing technique
  • total RNA was extracted from Human hybridoma clones producing neutralizing antibodies against SARS-CoV-2.
  • a barcoded cDNA library was generated through RT-PCR using a random hexamer.
  • Next-generation sequencing was performed on an Illumina HiSeq sequencer. Contigs were assembled, and data was mined for all viable antibody sequences (i.e., those not containing stop codons). Sequence analysis was performed separately to identify variable heavy and variable light domains. The complementarity determining regions (CDRs) were identified using the Kabat definition.
  • Example 5 Binding profile of human monoclonal antibodies against SARS- CoV-2
  • the Expi293 transient expression system (GibcoTM) was used to produce recombinant human monoclonal antibodies against SARS-CoV-2.
  • the binding profile of purified antibodies disclosed herein was assessed by ELISA, as mentioned in Example 2 with the following modification. Instead of using the culture supernatant, purified antibodies at 2 pg/ml were used as starting samples, then 11 rounds of 2-fold dilution were performed for each sample until the final concentration of 0.977 ng/mL was reached.
  • ELISA binding data of the recombinant human monoclonal antibodies against RBD of SARS-CoV-2 spike protein are illustrated in FIG. 1.
  • the EC50 values of these antibodies are reported in Table 5.1.
  • Example 6 Binding kinetics of human monoclonal antibodies against SARS- CoV-2
  • Binding kinetic values for different SARS-CoV-2 monoclonal antibodies are shown in Table 6.1. All antibodies exhibited KD values in the subnanomolar range. The 3D6 antibody showed the highest binding affinity, with a KD value of 4.01 x lO' 11 M, followed by 1A5, 2E10, 3H8, 1D1, 2B4, 3D2, and 1C10, respectively.
  • the SARS-CoV-2 surrogate virus neutralization test (ePass, GenScript) was used to assess the neutralizing activity of antibodies disclosed herein. Briefly, the individual human anti-SARS-CoV-2 monoclonal antibody was mixed with prototype RBD-HRP tag proteins and incubated at 37°C for 30 minutes. Then, the mixture was added to each well of the human ACE2-coated plate. After incubation at 37°C for 15 minutes, the plate was washed 4 times with a washing solution. In consequence, the free and non-neutralizing antibody-bound HRP-RBD were captured on the plate. Next, the plate was added with 100 pl TMB substrate solution to each well and incubated in the dark for 20 minutes. Finally, a stop solution was subsequently added to each well. The absorbance of the final solution was determined at 450 nm immediately.
  • Biolayer interferometry (BLI) technique was used to cluster recombinant human monoclonal antibodies disclosed herein based on binding epitopes on RBD of prototype SARS-CoV-2 spike protein.
  • the experiment was performed using ForteBio Octet HTX in an in-tandem format, i.e., a biosensor was immobilized with a prototype SARS-CoV-2 RBD-His protein and then presented with a saturating monoclonal antibody (1st mAb) followed by a competing monoclonal antibody (2nd mAb) in a pairwise combinatorial manner.
  • the assays were performed at 32°C with a shaking speed of 1,000 rpm.
  • a prototype SARS-CoV-2 RBD-His protein (3 pg/ml solution) was allowed to be captured onto an anti-His (AHC) biosensor tip for 300 seconds.
  • the first and second anti-SARS- CoV-2 monoclonal antibodies (10 and 5 pg/ml, respectively) were sequentially presented to the biosensor tip for 300 seconds.
  • the binding response was evaluated at each step after subtracting the self-blocking background signal.
  • the data analysis was performed with Octet Data Analysis HT 10.0 software using the Epitope Binning mode.
  • Example 9 SARS-CoV-2 pseudovirus neutralization assay of human monoclonal antibodies
  • Pseudovirus Neutralization Assay kit (Luc reporter, GenScript) was used to evaluate the potency of human anti-SARS-CoV-2 monoclonal antibodies disclosed herein for neutralizing infectivity of SARS-CoV-2 (prototype, alpha (B.l.1.7), beta (B.1.351), gamma (P. l), and delta (B.1.617.2) variants).
  • the monoclonal antibodies were diluted in 4-fold serial dilution with Opti-MEM, and 25 pl of the solution was transferred to an assay plate. Then, 25 pl of pseudovirus with luciferase reporter in Opti-MEM was added to wells and incubated at room temperature for 1 hour.
  • luciferase reagent 50 pl was added to the assay plate and subsequently incubated for 5 minutes at room temperature, and the bioluminescence signal was measured.

Abstract

The present disclosure relates to antibodies and antigen-binding fragments that bind specifically to the receptor-binding domain of SARS-CoV-2, compositions thereof, and methods of production. Also provided are methods of use, including methods of SARS-CoV-2 detection, methods for treating or preventing SARS-CoV-2 infections and associated symptoms.

Description

HUMAN MONOCLONAL ANTIBODIES AGAINST THE RECEPTOR-BINDING DOMAIN OF SARS-COV-2 SPIKE PROTEIN
TECHNICAL FIELD
[0001] The present invention relates to antibodies and antigen-binding fragments that bind specifically to the receptor-binding domain of SARS-CoV-2 and methods for treating or preventing SARS-CoV-2 infections with said antibodies and antigen-binding fragments.
BACKGROUND
[0002] Coronavirus disease 2019 (COVID-19) is an infectious disease from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has rapidly spread globally, causing a pandemic. The SARS-CoV-2 is an RNA virus with the characteristic of multiple spike glycoproteins on its envelope. The receptor-binding domain (RBD) on the spike proteins binds specifically with the cellular receptor angiotensin-converting enzyme 2 (ACE2) of its host cells, resulting in fusion cascade and virus entry. This virus can transmit very easily through respiratory droplets and aerosols with a reproduction number of up to 9. It replicates in the upper airway during the incubation period before developing symptoms. Globally, as of 30 July 2021, there have been 196,553,009 confirmed cases of COVID-19, including 4,200,412 deaths, reported to WHO.
[0003] A monoclonal antibody with neutralizing activity against SARS-CoV-2 could be used to block the virus entry and replication. Several spike protein-specific antibodies, especially against RBD, show efficiency for neutralizing SARS-CoV-2. There is need in the art for monoclonal antibodies with neutralizing activity against SARS-CoV-2.
SUMMARY
[0004] In one aspect, the disclosure provides an antibody or antigen-binding fragment that binds to a SARS-CoV-2 spike protein.
[0005] In some embodiments, the SARS-CoV-2 spike protein comprises any one of SEQ ID NOs: 129-136, or a sequence that shares at least 90%, at least 95%, or least 98% identity thereto.
[0006] In some embodiments, the antibody or antigen-binding fragment binds the SARS- CoV-2 spike protein with a KD value of about 10-12 M, about 10-11 M, about 10-10 M, about 10-9 M, or about 10-8 M. [0007] In some embodiments, the antibody or antigen-binding fragment inhibits the binding of the SARS-CoV-2 spike protein to an ACE2 receptor with an IC50 of about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL, about 65 ng/mL, about 70 ng/mL, about 75 ng/mL, or about 80 ng/mL.
[0008] In some embodiments, the antibody or antigen-binding fragment described herein comprises a heavy-chain complementarity-determining region 1 (HCDR1), a heavy-chain complementarity-determining region 2 (HCDR2), a heavy-chain complementaritydetermining region 3 (HCDR3), a light-chain complementarity determining region 1 (LCDR1), a light-chain complementarity determining region 2 (LCDR2), and a light-chain complementarity determining region 3 (LCDR3).
[0009] In some embodiments, the antibody or antigen-binding fragment described herein comprises: a) any one of the HCDR1 sequences set forth in Table 2; b) any one of the HCDR2 sequences set forth in Table 2; c) any one of the HCDR3 sequences set forth in Table 2; d) any one of the LCDR1 sequences set forth in Table 2; e) any one of the LCDR2 sequences set forth in Table 2; and f) any one of the LCDR3 sequences set forth in Table 2, wherein no more than 1, 2, or 3, amino acids in the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and/or LCDR3 sequences are substituted, deleted or inserted.
[0010] In some embodiments, the HCDR1 comprises SEQ ID NO: 6; the HCDR2 comprises SEQ ID NO: 7; and the HCDR3 comprises SEQ ID NO: 8.
[0011] In some embodiments, the HCDR1 comprises SEQ ID NO: 14; the HCDR2 comprises SEQ ID NO: 15; and the HCDR3 comprises SEQ ID NO: 16.
[0012] In some embodiments, the HCDR1 comprises SEQ ID NO: 22; the HCDR2 comprises SEQ ID NO: 23; and the HCDR3 comprises SEQ ID NO: 24.
[0013] In some embodiments, the HCDR1 comprises SEQ ID NO: 30; the HCDR2 comprises SEQ ID NO: 31; and the HCDR3 comprises SEQ ID NO: 32.
[0014] In some embodiments, the HCDR1 comprises SEQ ID NO: 38; the HCDR2 comprises SEQ ID NO: 39; and the HCDR3 comprises SEQ ID NO: 40.
[0015] In some embodiments, the HCDR1 comprises SEQ ID NO: 46; the HCDR2 comprises SEQ ID NO: 47; and the HCDR3 comprises SEQ ID NO: 48.
[0016] In some embodiments, the HCDR1 comprises SEQ ID NO: 54; the HCDR2 comprises SEQ ID NO: 55; and the HCDR3 comprises SEQ ID NO: 56. [0017] In some embodiments, the HCDR1 comprises SEQ ID NO: 62; the HCDR2 comprises SEQ ID NO: 63; and the HCDR3 comprises SEQ ID NO: 64
[0018] In some embodiments, the LCDR1 comprises SEQ ID NO: 2; the LCDR2 comprises SEQ ID NO: 3; and the LCDR3 comprises SEQ ID NO: 4.
[0019] In some embodiments, the LCDR1 comprises SEQ ID NO: 10; the LCDR2 comprises SEQ ID NO: 11; and the LCDR3 comprises SEQ ID NO: 12.
[0020] In some embodiments, the LCDR1 comprises SEQ ID NO: 18; the LCDR2 comprises SEQ ID NO: 19; and the LCDR3 comprises SEQ ID NO: 20.
[0021] In some embodiments, the LCDR1 comprises SEQ ID NO: 26; the LCDR2 comprises SEQ ID NO: 27; and the LCDR3 comprises SEQ ID NO: 28.
[0022] In some embodiments, the LCDR1 comprises SEQ ID NO: 34; the LCDR2 comprises SEQ ID NO: 35; and the LCDR3 comprises SEQ ID NO: 36.
[0023] In some embodiments, the LCDR1 comprises SEQ ID NO: 42; the LCDR2 comprises SEQ ID NO: 43; and the LCDR3 comprises SEQ ID NO: 44.
[0024] In some embodiments, the LCDR1 comprises SEQ ID NO: 50; the LCDR2 comprises SEQ ID NO: 51; and the LCDR3 comprises SEQ ID NO: 52.
[0025] In some embodiments, the LCDR1 comprises SEQ ID NO: 58; the LCDR2 comprises SEQ ID NO: 59; and the LCDR3 comprises SEQ ID NO: 60.
[0026] In some embodiments, the antibody or antigen-binding fragment described herein comprises: a) a heavy-chain variable region (VH) comprising a sequence set forth in Table 2, or a sequence at least that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto; and b) a light-chain variable region (VL) comprising a sequence set forth in Table 2, or a sequence at least that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto.
[0027] In some embodiments, the antibody or antigen-binding fragment described herein comprises: a) the VH comprises SEQ ID NO: 5, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 1, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0028] In some embodiments, the antibody or antigen-binding fragment described herein comprises: a) the VH comprises SEQ ID NO: 13, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 9, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0029] In some embodiments, the antibody or antigen-binding fragment described herein comprises: a) the VH comprises SEQ ID NO: 21, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 17, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0030] In some embodiments, the antibody or antigen-binding fragment described herein comprises: a) the VH comprises SEQ ID NO: 29, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 25, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0031] In some embodiments, the antibody or antigen-binding fragment described herein comprises: a) the VH comprises SEQ ID NO: 37, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 33, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0032] In some embodiments, the antibody or antigen-binding fragment described herein comprises: a) the VH comprises SEQ ID NO: 45, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 41, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0033] In some embodiments, the antibody or antigen-binding fragment described herein comprises: a) the VH comprises SEQ ID NO: 53, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 49, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0034] In some embodiments, the antibody or antigen-binding fragment described herein comprises: a) the VH comprises SEQ ID NO: 61, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 57, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto. [0035] In some embodiments, the antibody or antigen-binding fragment described herein further comprises a constant region. In some embodiments, the constant region is selected from an IgA, IgM, IgGl, IgG2, IgG3, or IgG4 constant region.
[0036] In some embodiments, the antibody or antigen-binding fragment described herein is humanized.
[0037] In some embodiments, the antibody or antigen-binding fragment described herein is recombinant.
[0038] In some embodiments, the antibody or antigen-binding fragment described herein is multispecific.
[0039] In some embodiments, the antibody or antigen-binding fragment described herein neutralizes a SARS-CoV-2 virus or a variant thereof.
[0040] In one aspect, the disclosure provides a pharmaceutical composition, comprising an antibody or antigen-binding fragment described herein and a pharmaceutically acceptable carrier, excipient, and/or diluent. The disclosure further provides a pharmaceutical composition comprising the antibody or antigen-binding fragment described herein, for use in the treatment of COVID-19.
[0041] In some embodiments, the pharmaceutical composition further comprises one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents is selected from an antibody, an antiviral, a steroid, an antiinflammatory, an immune booster, vitamin C, vitamin D, vitamin E, or any combination thereof.
[0042] In some embodiments, the pharmaceutical composition is formulated for parenteral administration, enteral administration, mucosal administration, nasal administration, oral administration, or intra-articular administration. In some embodiments, the administration comprises inhalation, intravenous injection, infusion, subcutaneous injection, or intramuscular injection.
[0043] In one aspect, the disclosure provides a kit comprising an antibody or antigenbinding fragment described herein or a pharmaceutical composition described herein and instructions for use.
[0044] In one aspect, the disclosure provides a nucleic acid comprising a polynucleotide sequence encoding the antibody or antigen-binding fragment described herein.
[0045] In one aspect, the disclosure provides a nucleic acid comprising: a) any one of the sequences encoding a heavy-chain complementarity-determining region 1 (HCDR1) set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; b) any one of the sequences encoding a heavy-chain complementarity-determining region 2 (HCDR2) set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; c) any one of the sequences encoding a heavy-chain complementarity-determining region 3 (HCDR3) set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; d) any one of the sequences encoding a light-chain complementarity determining region 1 (LCDR1) set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; e) any one of the sequences encoding a light-chain complementarity determining region 2 (LCDR2) set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and f) any one of the sequences encoding a light-chain complementarity determining region 3 (LCDR3) set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0046] In some embodiments, the nucleic acid described herein comprises: a) the sequence encoding the HCDR1 comprises SEQ ID NO: 70; b) the sequence encoding the HCDR2 comprises SEQ ID NO: 71; and c) the sequence encoding the HCDR3 comprises SEQ ID NO: 72.
[0047] In some embodiments, the nucleic acid described herein comprises: a) the sequence encoding the HCDR1 comprises SEQ ID NO: 78; b) the sequence encoding the HCDR2 comprises SEQ ID NO: 79; and c) the sequence encoding the HCDR3 comprises SEQ ID NO: 80.
[0048] In some embodiments, the nucleic acid described herein comprises: a) the sequence encoding the HCDR1 comprises SEQ ID NO: 86; b) the sequence encoding the HCDR2 comprises SEQ ID NO: 87; and c) the sequence encoding the HCDR3 comprises SEQ ID NO: 88.
[0049] In some embodiments, the nucleic acid described herein comprises: a) the sequence encoding the HCDR1 comprises SEQ ID NO: 94; b) the sequence encoding the HCDR2 comprises SEQ ID NO: 95; and c) the sequence encoding the HCDR3 comprises SEQ ID NO: 96.
[0050] In some embodiments, the nucleic acid described herein comprises: a) the sequence encoding the HCDR1 comprises SEQ ID NO: 102; b) the sequence encoding the HCDR2 comprises SEQ ID NO: 103; and c) the sequence encoding the HCDR3 comprises SEQ ID NO: 104.
[0051] In some embodiments, the nucleic acid described herein comprises: a) the sequence encoding the HCDR1 comprises SEQ ID NO: 110; b) the sequence encoding the HCDR2 comprises SEQ ID NO: 111; and c) the sequence encoding the HCDR3 comprises SEQ ID NO: 112.
[0052] In some embodiments, the nucleic acid described herein comprises: a) the sequence encoding the HCDR1 comprises SEQ ID NO: 118; b) the sequence encoding the HCDR2 comprises SEQ ID NO: 119; and c) the sequence encoding the HCDR3 comprises SEQ ID NO: 120.
[0053] In some embodiments, the nucleic acid described herein comprises: a) the sequence encoding the HCDR1 comprises SEQ ID NO: 126; b) the sequence encoding the HCDR2 comprises SEQ ID NO: 127; and c) the sequence encoding the HCDR3 comprises SEQ ID NO: 128.
[0054] In some embodiments, the nucleic acid described herein comprises: a) the sequence encoding the LCDR1 comprises SEQ ID NO: 66; b) the sequence encoding the LCDR2 comprises SEQ ID NO: 67; and c) the sequence encoding the LCDR3 comprises SEQ ID NO: 68.
[0055] In some embodiments, the nucleic acid described herein comprises: a) the sequence encoding the LCDR1 comprises SEQ ID NO: 74; b) the sequence encoding the LCDR2 comprises SEQ ID NO: 75; and c) the sequence encoding the LCDR3 comprises SEQ ID NO: 76.
[0056] In some embodiments, the nucleic acid described herein comprises: a) the sequence encoding the LCDR1 comprises SEQ ID NO: 82; b) the sequence encoding the LCDR2 comprises SEQ ID NO: 83; and c) the sequence encoding the LCDR3 comprises SEQ ID NO: 84.
[0057] In some embodiments, the nucleic acid described herein comprises: a) the sequence encoding the LCDR1 comprises SEQ ID NO: 90; b) the sequence encoding the LCDR2 comprises SEQ ID NO: 91; and c) the sequence encoding the LCDR3 comprises SEQ ID NO: 92.
[0058] In some embodiments, the nucleic acid described herein comprises: a) the sequence encoding the LCDR1 comprises SEQ ID NO: 98; b) the sequence encoding the LCDR2 comprises SEQ ID NO: 99; and c) the sequence encoding the LCDR3 comprises SEQ ID NO: 100.
[0059] In some embodiments, the nucleic acid described herein comprises: a) the sequence encoding the LCDR1 comprises SEQ ID NO: 106; b) the sequence encoding the LCDR2 comprises SEQ ID NO: 107; and c) the sequence encoding the LCDR3 comprises SEQ ID NO: 108.
[0060] In some embodiments, the nucleic acid described herein comprises: a) the sequence encoding the LCDR1 comprises SEQ ID NO: 114; b) the sequence encoding the LCDR2 comprises SEQ ID NO: 115; and c) the sequence encoding the LCDR3 comprises SEQ ID NO: 116.
[0061] In some embodiments, the nucleic acid described herein comprises: a) the sequence encoding the LCDR1 comprises SEQ ID NO: 122; b) the sequence encoding the LCDR2 comprises SEQ ID NO: 123; and c) the sequence encoding the LCDR3 comprises SEQ ID NO: 124.
[0062] In some embodiments, the nucleic acid described herein comprises: a) a sequence encoding a heavy-chain variable region (VH) comprising a sequence set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto; and b) a sequence encoding a light-chain variable region (VL) comprising a sequence set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto.
[0063] In some embodiments, the nucleic acid described herein comprises: a) the VH comprises SEQ ID NO: 69, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 65, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0064] In some embodiments, the nucleic acid described herein comprises: a) the VH comprises SEQ ID NO: 77, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 73, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0065] In some embodiments, the nucleic acid described herein comprises: a) the VH comprises SEQ ID NO: 85, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 81, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0066] In some embodiments, the nucleic acid described herein comprises: a) the VH comprises SEQ ID NO: 93, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 89, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0067] In some embodiments, the nucleic acid described herein comprises: a) the VH comprises SEQ ID NO: 101, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 97, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0068] In some embodiments, the nucleic acid described herein comprises: a) the VH comprises SEQ ID NO: 109, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 105, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0069] In some embodiments, the nucleic acid described herein comprises: a) the VH comprises SEQ ID NO: 117, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 113, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0070] In some embodiments, the nucleic acid described herein comprises: a) the VH comprises SEQ ID NO: 125, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b) the VL comprises SEQ ID NO: 121, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0071] In some embodiments, the nucleic acid provided herein is codon optimized.
[0072] In one aspect, the disclosure provides a vector comprising a nucleic acid described herein. In some embodiments, the nucleic acid is operably linked to a promoter.
[0073] In one aspect, the disclosure provides a cell line for producing an antibody or antigen-binding fragment described herein, a nucleic acid described herein, or a vector described herein. [0074] In one aspect, the disclosure provides a method of producing an antibody or antigen-binding fragment described herein, comprising a) culturing a cell line described herein under conditions sufficient for expression of the antibody or antigen-binding fragment; and b) purifying the antibody or antigen-binding fragment.
[0075] In one aspect, the disclosure provides a method of producing an antibody or antigen-binding fragment that binds to a SARS-CoV-2 spike protein comprising: a) contacting a cell with a nucleic acid described herein or a vector described herein; b) culturing the cell under conditions sufficient to express the antibody or antigen-binding fragment; and c) purifying the antibody or antigen-binding fragment.
[0076] In one aspect, the disclosure provides a method of treating COVID-19 in a subject in need thereof, comprising administering to the subject an antibody or antigen-binding fragment described herein or a pharmaceutical composition described herein.
[0077] In one aspect, the disclosure provides a method of preventing COVID-19 in a subject in need thereof, comprising administering to the subject an antibody or antigenbinding fragment described herein or a pharmaceutical composition described herein.
[0078] In one aspect, the disclosure provides a method of reducing one or more symptoms associated with COVID-19 in a subject in need thereof, comprising administering to the subject an antibody or antigen-binding fragment described herein or a pharmaceutical composition described herein.
[0079] In some embodiments, the severity of the one or more symptoms is reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or eliminated completely. In some embodiments, the duration of the one or more symptoms is reduced by at least one day, at least two days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days.
[0080] In one aspect, the disclosure provides a method of treating, preventing, or reducing symptoms associated with COVID-19 in a subject in need thereof, comprising: a) detecting a SARS-CoV-2 viral genome or a SARS-CoV-2 antigen in a sample collected from the subject; and b) administering a treatment to the subject, wherein the treatment i) prevents, reduces, or eliminates a SARS-CoV-2 infection or a variant thereof; and/or ii) prevents, reduces, or eliminates symptoms associated with COVID-19, wherein the treatment comprises an antibody or antigen-binding fragment described herein or a pharmaceutical composition described herein. [0081] In some embodiments, the subject has or is suspected of having a SARS-CoV-2 infection or a variant thereof. In some embodiments, the subject has at least one symptom associated with a SARS-CoV-2 infection or a variant thereof. In some embodiments, the subject is a human.
[0082] In some embodiments, administration is enteral, parenteral, mucosal, nasal, oral, by inhalation, by intravenous injection, by infusion, by subcutaneous injection, by intramuscular injection, or intra-articular.
[0083] In one aspect, the disclosure provides a method for detecting a SARS-CoV-2 spike protein in a sample, comprising contacting a sample with the antibody or antigen-binding fragment described herein.
[0084] In some embodiments, the sample is an environmental sample, sewage, wastewater, urine, feces, saliva, nasal swab, mucous, blood, plasma, bronchoalveolar lavage, or one or more cells. In some embodiments, the sample is collected from a subject. In some embodiments, the subject has or is suspected of having a SARS-CoV-2 infection or COVID-19. In some embodiments, the antibody or antigen-binding fragment is labeled with a tag, detection dye or a secondary antibody.
[0085] In some embodiments, the method further comprises use of an electrochemical assay, biosensor, or immunoassay.
[0086] In one aspect, the disclosure provides a method of diagnosing an individual as having COVID-19, comprising detecting a SARS-CoV-2 spike protein in a sample, comprising contacting a sample with the antibody or antigen-binding fragment described herein. In some embodiments, the antibody or antigen-binding fragment is labeled with a detection dye or a secondary antibody.
[0087] In some embodiments, the method further comprises use of an ELISA technique or a lateral flow assay.
[0088] In some embodiments, the sample is saliva, nasal swab, mucous, blood, plasma, bronchoalveolar lavage, or one or more cells. In some embodiments, the sample is collected from a subject. In some embodiments, the subject has or is suspected of having a SARS-CoV-2 infection or COVID-19.
BRIEF DESCRIPTION OF THE DRAWINGS
[0089] FIG. 1 is a plot showing binding of human monoclonal antibodies disclosed herein against the receptor binding domain (RBD) of SARS-CoV-2 spike protein. [0090] FIG. 2 is a plot showing neutralization of SARS-CoV-2 by the antibodies described herein using a surrogate virus neutralization test (sVNT) (GenScript ePass™ SARS-CoV- 2 Neutralization Antibody Detection Kit).
[0091] FIG. 3 is a plot showing neutralization of SARS-CoV-2 by the antibodies described herein using a pseudovirus neutralization assay.
[0092] FIG. 4 is a plot showing neutralization of SARS-CoV-2 (alpha (B. l.1.7) variant) by the antibodies described herein using a pseudovirus neutralization assay.
[0093] FIG. 5 is a plot showing neutralization of SARS-CoV-2 (beta ( B.1.351) variant) by the antibodies described herein using a pseudovirus neutralization assay.
[0094] FIG. 6 is a plot showing neutralization of SARS-CoV-2 (gamma (P. l) variant) by the antibodies described herein using a pseudovirus neutralization assay.
[0095] FIG. 7 is a plot showing neutralization of SARS-CoV-2 (delta (B.1.617.2) variant) by the antibodies described herein using a pseudovirus neutralization assay.
DETAILED DESCRIPTION
[0096] This disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to the SARS-CoV2 spike protein. The antibodies and antigen-binding fragments may be isolated. The antibodies and antigen-binding fragments can be used in pharmaceutical compositions and kits.
[0097] This disclosure provides nucleic acids and vectors encoding the antibodies and antigen-binding fragments described herein.
[0098] The disclosure provides cell lines used in the production of the antibodies and antigen-binding fragments described herein.
[0099] The disclosure provides methods for producing the antibodies and antigen-binding fragments described herein.
[0100] The disclosure provides methods of using the antibodies and binding fragments described herein. The methods can be methods of treatment and prevention of a SARS- CoV-2 infection or COVID-19. The antibodies and binding fragments thereof can be used in a method of preventing, reducing, or eliminating symptoms associated with a SARS- CoV-2 infection or COVID-19. The antibodies and binding fragments thereof can be used in a method of detecting SARS-CoV-2 in a sample. SARS-CoV-2 Spike Protein Antigens
[0101] The coronavirus (i.e. SARS-CoV-1, MERS-CoV, and SARS-CoV-2) viral genome encodes spike (S), envelope (E), membrane (M), and nucleocapsid (N) structural proteins, among which the surface glycoprotein i.e. spike protein) is responsible for binding the host receptor via the receptor-binding domain (RBD) in its SI subunit, as well as the subsequent membrane fusion and viral entry driven by its S2 subunit. The RBD contains a core subdomain and a receptor-binding motif (RBM). While the core subdomains are highly similar between the three coronaviruses, their RBMs are markedly different, leading to different receptor specificity: SARS-CoV and SARS-CoV-2 recognize the angiotensin-converting enzyme 2 (ACE or ACE2), whereas MERS-CoV binds the dipeptidyl peptidase 4 (DPP4). Binding of the spike protein RBM to the ACE2 facilitates viral entry; a possible membrane fusion process has been proposed.
[0102] The spike protein, the RBD, and the RBM provide a set of unique protein structures to act as antigens with unique epitopes for antibody based targeting and binding of the coronavirus (e.g. SARS-CoV-2), for detection of the coronavirus, treatment of coronavirus infection, treatment disease phenotypes (e.g. COVID-19), and treatment of symptoms associated with disease phenotypes. The SARS-CoV-2 spike protein has formed the basis for vaccines to immunize populations against SARS-CoV-2 infection and COVID-19 by acting as the antigen (i.e. immunogen) of the vaccine to which the host receiving the vaccine develops antibodies against.
[0103] In one aspect, the disclosure provides antibodies and binding fragments thereof that specifically bind a SARS-CoV-2 spike protein antigen. Any region or epitope within a SARS-CoV-2 spike protein antigen is envisaged as a putative binding site for the antibodies and binding fragments thereof provided herein. For example, putative binding sites can be, without limitation, on the following SARS-CoV-2 spike protein regions: the SI subunit, S2 subunit, the SD-1 subdomain, the S1/S2 cleavage region, the S2 subunit, the receptor-binding domain, and the receptor binding motif.
[0104] Illustrative sequences of SARS-CoV-2 spike protein antigens are shown in Table 1. In some embodiments, the SARS-CoV-2 spike protein antigen comprises a sequence, or a portion thereof, set forth in Table 1. In some embodiments, the SARS-CoV-2 spike protein antigen comprises SEQ ID NO: 129, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto. In some embodiments, the SARS- CoV-2 spike protein antigen comprises SEQ ID NO: 130, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto. In some embodiments, the SARS-CoV-2 spike protein antigen comprises SEQ ID NO: 131, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto. In some embodiments, the SARS-CoV-2 spike protein antigen comprises SEQ ID NO: 132, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto. In some embodiments, the SARS-CoV-2 spike protein antigen comprises SEQ ID NO: 133, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto. In some embodiments, the SARS-CoV-2 spike protein antigen comprises SEQ ID NO: 134, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto. In some embodiments, the SARS-
CoV-2 spike protein antigen comprises SEQ ID NO: 135, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto.
[0105] It is to be understood that the antibody or antigen-binding fragment provided herein binds to a portion, or an epitope, of the SARS-CoV-2 spike protein. Put another way, the antibody or antigen-binding fragment provided herein can bind to any one or more amino acid subsequence(s) of the SARS-CoV-2 spike protein.
[0106] Table 1. Illustrative SARS-CoV-2 Spike Protein Antigen Sequences
Figure imgf000015_0001
Figure imgf000016_0001
Figure imgf000017_0001
Figure imgf000018_0001
SARS-CoV-2 antigen-binding Antibodies and Fragments
[0107] In one aspect, the disclosure provides antibodies and antigen-binding fragments that bind to a SARS-CoV-2 spike protein. In some embodiments, the antibody or antigenbinding fragment binds a sequence, or a portion thereof, set forth in Table 1. In some embodiments, the antibody or antigen-binding fragment binds a SARS-CoV-2 spike protein antigen comprises SEQ ID NO: 129, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto. In some embodiments, the antibody or antigen-binding fragment binds a SARS-CoV-2 spike protein antigen comprises SEQ ID NO: 130, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto. In some embodiments, the antibody or antigenbinding fragment binds a SARS-CoV-2 spike protein antigen comprises SEQ ID NO: 131, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto. In some embodiments, the antibody or antigen-binding fragment binds a SARS-CoV-2 spike protein antigen comprises SEQ ID NO: 132, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto. In some embodiments, the antibody or antigen-binding fragment binds a SARS-CoV-2 spike protein antigen comprises SEQ ID NO: 133, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto. In some embodiments, the antibody or antigen-binding fragment binds a SARS-CoV-2 spike protein antigen comprises SEQ ID NO: 134, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto. In some embodiments, the antibody or antigenbinding fragment binds a SARS-CoV-2 spike protein antigen comprises SEQ ID NO: 135, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto. In some embodiments, the antibody or antigen-binding fragment binds a SARS-CoV-2 spike protein antigen comprises SEQ ID NO: 136, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto.
[0108] The antibodies and binding fragments provided herein comprise a variable heavy chain (i.e. VH or HC) region and a variable light chain (i.e. VL or LC) region. In some embodiments, the antibody or binding fragment comprises two identical VH regions. In some embodiments, the antibody or binding fragment comprises two identical VL regions. In some embodiments, the antibody or fragment comprises two different VL regions. In some embodiments, the antibody or fragment comprises two different VH regions. In some embodiments, the two VH regions are covalently bound. VH regions can be covalently be bound by, for example, disulfide bonds. In some embodiments, the VL regions are covalently bound to the VH regions.
[0109] VL and VH regions each have one variable region and three constant regions. Within the variable regions of light and heavy chains are hypervariable sequences called complementarity-determining regions (CDRs) flanked by framework regions. The binding specificity of an antibody is conferred by its combination of CDRs. There are three CDRs on the light chain (LCDR1, LCDR2, and LCDR3) and three on the heavy chain (HCDR1, HCDR2, and HCDR3) of an antibody molecule. Together, these form the 3-dimensional cavity that will bind (hold) an epitope on an antigen. Although CDRs are hypervariable, a particular CDR on one antibody may also be found on antibodies with different specificities, as it is the total combination of CDRs regions that determine binding specificity. Identification of the CDRs is useful for changing the “speciation” of an antibody, for example changing a mouse antibody to a humanized form suitable for human use, because one would want to preserve the CDRs so as not to eliminate the binding specificity. Using the numbering system of Kabat et al, (NIH Publication No. 91-3242, 1991) in which the signal sequences of the heavy and light chains are indicated with negative numbers, the CDRs of the light chain are between amino acids 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (95 a-f, LCDR3). The complementarity-determining regions of the heavy chain are between amino acids 31-35 (35 a-b, HCDR1), 50-65 (52 a-c, HCDR2), and 95-102 (100 a-k, HCDR3). Insertions of extra amino acids into the complementaritydetermining regions can be observed and their locations are represented above in parentheses, e.g. 95 a-f. Deletions are also observed, for example in CDR3 of some types of heavy chains. Illustrative CDRs of the disclosure are provided in Table 2.
[0110] In some embodiments, the antibody is isolated. In some embodiments, the antibody is humanized. In some embodiments, the antibody is recombinant. In some embodiments, the antibody is multispecific
Figure imgf000020_0001
[oni] The antibodies and antigen-binding fragments provided herein comprises a heavychain complementarity-determining region 1 (HCDR1), a heavy-chain complementaritydetermining region 2 (HCDR2), a heavy-chain complementarity-determining region 3 (HCDR3), a light-chain complementarity determining region 1 (LCDR1), a light-chain complementarity determining region 2 (LCDR2), and a light-chain complementarity determining region 3 (LCDR3).
[0112] In some embodiments, the antibody or antigen-binding fragment comprises any one of the HCDR1 sequences set forth in Table 2; any one of the HCDR2 sequences set forth in Table 2; any one of the HCDR3 sequences set forth in Table 2; any one of the LCDR1 sequences set forth in Table 2; any one of the LCDR2 sequences set forth in Table 2; and any one of the LCDR3 sequences set forth in Table 2. In some embodiments, no more than 1, 2, or 3, amino acids in the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and/or LCDR3 sequences are substituted, deleted or inserted.
[0113] In some embodiments, the antibody or antigen-binding fragment comprises a HCDR1 comprising SEQ ID NO: 6; a HCDR2 comprising SEQ ID NO: 7; and a HCDR3 comprising SEQ ID NO: 8.
[0114] In some embodiments, the antibody or antigen-binding fragment comprises a HCDR1 comprising SEQ ID NO: 14; a HCDR2 comprising SEQ ID NO: 15; and a HCDR3 comprising SEQ ID NO: 16.
[0115] In some embodiments, the antibody or antigen-binding fragment comprises a HCDR1 comprising SEQ ID NO: 22; a HCDR2 comprising SEQ ID NO: 23; and a HCDR3 comprising SEQ ID NO: 24.
[0116] In some embodiments, the antibody or antigen-binding fragment comprises a HCDR1 comprising SEQ ID NO: 30; a HCDR2 comprising SEQ ID NO: 31; and a HCDR3 comprising SEQ ID NO: 32.
[0117] In some embodiments, the antibody or antigen-binding fragment comprises a HCDR1 comprising SEQ ID NO: 38; a HCDR2 comprising SEQ ID NO: 39; and a HCDR3 comprising SEQ ID NO: 40.
[0118] In some embodiments, the antibody or antigen-binding fragment comprises a HCDR1 comprising SEQ ID NO: 46; a HCDR2 comprising SEQ ID NO: 47; and a HCDR3 comprising SEQ ID NO: 48.
[0119] In some embodiments, the antibody or antigen-binding fragment comprises a HCDR1 comprising SEQ ID NO: 54; a HCDR2 comprising SEQ ID NO: 55; and a HCDR3 comprising SEQ ID NO: 56.
[0120] In some embodiments, the antibody or antigen-binding fragment comprises a HCDR1 comprising SEQ ID NO: 62; a HCDR2 comprising SEQ ID NO: 63; and a HCDR3 comprising SEQ ID NO: 64.
[0121] In some embodiments, the antibody or antigen-binding fragment comprises a LCDR1 comprising SEQ ID NO: 2; a LCDR2 comprising SEQ ID NO: 3; and a LCDR3 comprising SEQ ID NO: 4.
[0122] In some embodiments, the antibody or antigen-binding fragment comprises a LCDR1 comprising SEQ ID NO: 10; a LCDR2 comprising SEQ ID NO: 11; and aLCDR3 comprising SEQ ID NO: 12. [0123] In some embodiments, the antibody or antigen-binding fragment comprises a LCDR1 comprising SEQ ID NO: 18; a LCDR2 comprising SEQ ID NO: 19; and aLCDR3 comprising SEQ ID NO: 20.
[0124] In some embodiments, the antibody or antigen-binding fragment comprises a LCDR1 comprising SEQ ID NO: 26; a LCDR2 comprising SEQ ID NO: 27; and a LCDR3 comprising SEQ ID NO: 28.
[0125] In some embodiments, the antibody or antigen-binding fragment comprises a LCDR1 comprising SEQ ID NO: 34; a LCDR2 comprising SEQ ID NO: 35; and aLCDR3 comprising SEQ ID NO: 36.
[0126] In some embodiments, the antibody or antigen-binding fragment comprises a LCDR1 comprising SEQ ID NO: 42; a LCDR2 comprising SEQ ID NO: 43; and a LCDR3 comprising SEQ ID NO: 44.
[0127] In some embodiments, the antibody or antigen-binding fragment comprises a LCDR1 comprising SEQ ID NO: 50; a LCDR2 comprising SEQ ID NO: 51; and aLCDR3 comprising SEQ ID NO: 52.
[0128] In some embodiments, the antibody or antigen-binding fragment comprises a LCDR1 comprising SEQ ID NO: 58; a LCDR2 comprises SEQ ID NO: 59; and a LCDR3 comprising SEQ ID NO: 60.
Heavy and Light Chain Variable Regions
[0129] The antibody or antigen-binding fragment comprises a heavy-chain variable region (VH) comprising a sequence set forth in Table 2, or a sequence at least that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto; and a light-chain variable region (VL) comprising a sequence set forth in Table 2, or a sequence at least that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto.
[0130] In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising SEQ ID NO: 5, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and a VL comprising SEQ ID NO: 1, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0131] In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising SEQ ID NO: 13, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and a VL comprising SEQ ID NO: 9, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0132] In some embodiments, the antibody or antigen-binding fragment comprise a VH comprising SEQ ID NO: 21, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and a VL comprising SEQ ID NO: 17, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0133] In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising SEQ ID NO: 29, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and a VL comprising SEQ ID NO: 25, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0134] In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising SEQ ID NO: 37, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and a VL comprising SEQ ID NO: 33, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0135] In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising SEQ ID NO: 45, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and a VL comprising SEQ ID NO: 41, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0136] In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising SEQ ID NO: 53, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and a VL comprising SEQ ID NO: 49, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0137] In some embodiments, the antibody or antigen-binding fragment comprises a VH comprising SEQ ID NO: 61, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and a VL comprising SEQ ID NO: 57, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto
[0138] Table 2. Illustrative CDRs and VH/VL Regions
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Constant Region
[0139] The antibody or antigen-binding fragment provided herein may further comprise a constant region. A constant region can be an Fc region. An "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C- terminal region of the heavy chain of an antibody that mediates the binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (Clq) of the classical complement system. Thus, an Fc region of an antibody of isotype IgG comprises the heavy chain constant region of the antibody excluding the first constant region immunoglobulin domain (CHI). In IgG, IgA and IgD antibody isotypes, the Fc region comprises CH2 and CH3 constant domains in each of the antibody’s two heavy chains; IgM and IgE Fc regions comprise three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. For IgG, the Fc region comprises immunoglobulin domains consisting of the hinge, CH2 and CH3. [0140] “Hinge”, “hinge domain”, or “hinge region” refers to the domain of a heavy chain constant region that joins the CHI domain to the CH2 domain and comprises upper, middle, and lower portions. (Roux et al. (1998) J. Immunol.161 :4083). Depending on its amino acid sequence, the hinge provides varying levels of flexibility between the antigenbinding domain and effector region of an antibody and also provides sites for intermolecular disulfide bonding between the two heavy chain constant regions. In some embodiments, the antibody or antigen-binding fragment comprises a hinge region.
[0141] For purposes herein, the Fc region is defined as starting at amino acid 216 and ending at amino acid 447, wherein the numbering is according to the EU index as in Kabat. Kabat et al. (1991) Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD, and according to FIGs.3c-3f of U.S. Pat. App. Pub. No. 2008/0248028. The Fc may be a native (or naturally-occurring or wildtype) Fc, including any allotypic variant, or a variant Fc (e.g., a non- naturally occurring Fc), comprising, e.g., 1, 2, 3, 4, 5, 1-5, 1-10 or 5-10 or more amino acid mutations, e.g., substitutions, additions or deletions. For example, a variant Fc may comprise an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a wildtype Fc. Modified or mutated Fes may have enhanced or reduced effector function and/or half-life. The CH2 and CH3 regions are the primary site of effector functions and FcRn binding. Fc may refer to this region in isolation or in the context of an Fc-comprising protein polypeptide such as a “binding protein comprising an Fc region,” also referred to as an “Fc fusion protein” (e.g., an antibody or immunoadhesin).
[0142] The constant regions of the antibodies provided herein may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The constant region mediates an effector function, which refers to the interaction of an antibody Fc region with an Fc receptor or ligand, or a biochemical event that results therefrom. Exemplary "effector functions" include Clq binding, complement dependent cytotoxicity (CDC), Fc receptor binding, FcyR- mediated effector functions such as ADCC and antibody dependent cell-mediated phagocytosis (ADCP), and downregulation of a cell surface receptor (e.g., the B cell receptor; BCR). Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain).
[0143] An Fc receptor (FcR) is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind to an IgG antibody comprise receptors of the FcyR family, including allelic variants and alternatively spliced forms of these receptors. The FcyR family consists of three activating (FcyRI, FcyRIII, and FcyRIV in mice; FcyRIA, FcyRIIA, and FcyRIIIA in humans) and one inhibitory (FcyRIIB) receptor. The majority of innate effector cell types coexpress one or more activating FcyR and the inhibitory FcyRIIB, whereas natural killer (NK) cells selectively express one activating Fc receptor (FcyRIII in mice and FcyRIIIA in humans) but not the inhibitory FcyRIIB in mice and humans. Human IgGl binds to most human Fc receptors and is considered equivalent to murine IgG2a with respect to the types of activating Fc receptors that it binds to.
[0144] A constant region can be a constant region within the light chain. The light chain constant region can be a lambda (t) light chain region or a kappa (K) light chain region. In some embodiments, the light chain constant region comprises a lambda light chain region. In some embodiments, the light chain constant region comprises a kappa light chain region. [0145] The constant region may be from any of the commonly known immunoglobulin isotypes, including but not limited to IgA, secretory IgA, IgG and IgM. The IgG isotype is divided in subclasses in certain species: IgGl, IgG2, IgG3 and IgG4 in humans, and IgGl, IgG2a, IgG2b and IgG3 in mice. Immunoglobulins, e.g., human IgGl, exist in several allotypes, which differ from each other in at most a few amino acids. In some embodiments, the constant region is selected from IgA, IgG, and IgM. In some embodiments, the constant region is selected from IgGl, IgG2, IgG3, and IgG4 constant regions.
[0146] In some embodiments, the SARS-CoV-2 binding antibody or antigen-binding fragment comprises a constant region selected from a sequence in Table 2.1, or a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto. In some embodiments, the antibody or antigen-binding fragment comprises a constant region comprises any one of SEQ ID NOs: 137-147, or a sequence sharing at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or at least 99% identity thereto.
[0147] Table 2.1 Illustrative Constant Region Sequences
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Binding and Biological Activity
[0148] The antibody and antigen-binding fragments provided herein binds a SARS-CoV- 2 spike protein. Binding and binding affinities can be characterized using any method known in the art. Binding and binding affinities can be expressed as a dissociation constant (KD) value. Dissociation constant values can be determined using, for example and without limitation, surface plasmon resonance, surface plasmon resonance imaging, interferometry, ELISA, titration curves, radioligand binding assay, affinity chromatography, fluorescence energy resonance transfer, and/or isothermal titration calorimetry. It can be useful to determine the KD value using more than one method to validate the binding properties reported by a given characterization method.
[0149] In some embodiments, the antibody or antigen-binding fragment binds the SARS- CoV-2 spike protein with a KD value of about 10'12M, about 1O'UM, about 10'10M, about 10'9M, or about 10'8 M.
[0150] Biological activity refers to an effect the antibody has on a system of biological molecules. For example, a biological activity can be blocking a SARS-CoV-2 spike protein from binding a target receptor (e.g. an ACE2 receptor). In this case, an antibody or antigenbinding fragment in the presence of both a SARS-CoV-2 spike protein and an ACE2 receptor will bind the SARS-CoV-2 spike protein and form a complex such that the SARS- CoV-2 spike protein does not bind the ACE2 receptor, or has reduced or otherwise altered binding.
[0151] In another example, a biological activity is the prevention, reduction, or elimination of the ability of a SARS-CoV-2 virus to infect a cell. An antibody or antigen-binding fragment of the disclosure is said to neutralize a SARS-CoV-2 virus if, when in the presence of a cell expressing an ACE2 receptor and a SARS-CoV-2 virus, the ability of the virus to infect the cell is prevented, reduced, or eliminated; said antibody or antigenbinding fragment can also be referred to as neutralizing, have neutralizing activity, or can neutralize SARS-CoV-2. Neutralizing activity can arise from the antibody or antigenbinding fragment binding a SARS-CoV-2 spike protein on a SARS-CoV-2 virus and prevent, reduce or otherwise alter binding of the spike protein to an ACE2 receptor on a cell. Binding of the antibody or antigen-binding fragment to ACE2 receptor can thus be used as a surrogate measurement for classifying the antibody or antigen-binding fragment as having SARS-CoV-2 neutralizing activity, as described herein. In some embodiments, the antibody or antigen-binding fragment neutralizes a SARS-CoV-2 virus or a variant thereof.
[0152] Biological activity of the antibody or antigen-binding fragment can be determined using any method known in the art. Biological activity can be expressed in terms of IC50 or EC50 values. IC50 and EC50 values quantify the biological activity as the concentration of antibody or antigen-binding fragment required to achieve half of the maximum biological activity. For example, IC50 can be used to express the concentration of antibody or antigen-binding fragment required for reducing the binding of the spike protein to an ACE2 receptor by 50% of the binding observed in the absence of the antibody or antigenbinding fragment. In another example, EC50 can be used to express the concentration of antibody or antigen-binding fragment that is required to reduce infection of ACE2 expressing cells by SARS-CoV-2 by 50% of the infection observed in the absence of the antibody or antigen-binding fragment.
[0153] In some embodiments, the antibody or antigen-binding fragment inhibits the binding of the SARS-CoV-2 spike protein to an ACE2 receptor with an IC50 of about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL, about 65 ng/mL, about 70 ng/mL, about 75 ng/mL, or about 80 ng/mL.
Polynucleotides and Vectors
[0154] The disclosure provides a nucleic acid comprising a polynucleotide sequence encoding the antibody antigen-binding fragment described herein. In some embodiments, the sequence is codon optimized.
[0155] Methods for obtaining, producing, handling, manipulating, purifying, detecting, visualizing, and the like (e.g. nuclease mediated digestion, ligation, cloning, electrophoresis, staining, etc.) of nucleic acids and polynucleotides described herein are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).
[0156] The disclosure provides a nucleic acid comprising: a.) any one of the sequences encoding a heavy-chain complementarity-determining region 1 (HCDR1) set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; b.) any one of the sequences encoding a heavy-chain complementarity-determining region 2 (HCDR2) set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; c.) any one of the sequences encoding a heavy-chain complementarity-determining region 3 (HCDR3) set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; d.) any one of the sequences encoding a light-chain complementarity determining region 1 (LCDR1) set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; e.) any one of the sequences encoding a light-chain complementarity determining region 2 (LCDR2) set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and f.) any one of the sequences encoding a light-chain complementarity determining region 3 (LCDR3) set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0157] In some embodiments, the nucleic acid comprises a.) a sequence encoding the HCDR1 comprising SEQ ID NO: 70; b.) a sequence encoding the HCDR2 comprising SEQ ID NO: 71; and c.) a sequence encoding the HCDR3 comprising SEQ ID NO: 72.
[0158] In some embodiments, the nucleic acid comprises a.) a sequence encoding the HCDR1 comprising SEQ ID NO: 78; b.) a sequence encoding the HCDR2 comprising SEQ ID NO: 79; and c.) a sequence encoding the HCDR3 comprising SEQ ID NO: 80.
[0159] In some embodiments, the nucleic acid comprises a.) a sequence encoding the HCDR1 comprising SEQ ID NO: 86; b.) a sequence encoding the HCDR2 comprising SEQ ID NO: 87; and c.) a sequence encoding the HCDR3 comprising SEQ ID NO: 88.
[0160] In some embodiments, the nucleic acid comprises a.) a sequence encoding the HCDR1 comprising SEQ ID NO: 94; b.) a sequence encoding the HCDR2 comprising SEQ ID NO: 95; and c.) a sequence encoding the HCDR3 comprising SEQ ID NO: 96.
[0161] In some embodiments, the nucleic acid comprises a.) a sequence encoding the HCDR1 comprising SEQ ID NO: 102; b.) a sequence encoding the HCDR2 comprising SEQ ID NO: 103; and c.) a sequence encoding the HCDR3 comprising SEQ ID NO: 104. [0162] In some embodiments, the nucleic acid comprises a.) a sequence encoding the HCDR1 comprises SEQ ID NO: 110; b.) a sequence encoding the HCDR2 comprising SEQ ID NO: 111; and c.) a sequence encoding the HCDR3 comprising SEQ ID NO: 112. [0163] In some embodiments, the nucleic acid comprises a.) a sequence encoding the HCDR1 comprising SEQ ID NO: 118; b.) a sequence encoding the HCDR2 comprising SEQ ID NO: 119; and c.) a sequence encoding the HCDR3 comprising SEQ ID NO: 120. [0164] In some embodiments, the nucleic acid comprises a.) a sequence encoding the HCDR1 comprising SEQ ID NO: 126; a sequence encoding the HCDR2 comprising SEQ ID NO: 127; and c.) a sequence encoding the HCDR3 comprising SEQ ID NO: 128.
[0165] In some embodiments, the nucleic acid comprises a.) a sequence encoding the LCDR1 comprising SEQ ID NO: 66; b.) a sequence encoding the LCDR2 comprising SEQ ID NO: 67; and c.) a sequence encoding the LCDR3 comprising SEQ ID NO: 68.
[0166] In some embodiments, the nucleic acid comprises a.) a sequence encoding the LCDR1 comprising SEQ ID NO: 74; b.) a sequence encoding the LCDR2 comprising SEQ ID NO: 75; and c.) a sequence encoding the LCDR3 comprising SEQ ID NO: 76.
[0167] In some embodiments, the nucleic acid comprises a.) a sequence encoding the LCDR1 comprising SEQ ID NO: 82; b.) a sequence encoding the LCDR2 comprising SEQ ID NO: 83; and c.) a sequence encoding the LCDR3 comprising SEQ ID NO: 84.
[0168] In some embodiments, the nucleic acid comprises a.) a sequence encoding the LCDR1 comprising SEQ ID NO: 90; b.) a sequence encoding the LCDR2 comprising SEQ ID NO: 91; and c.) a sequence encoding the LCDR3 comprising SEQ ID NO: 92.
[0169] In some embodiments, the nucleic acid comprises a.) a sequence encoding the LCDR1 comprising SEQ ID NO: 98; b.) a sequence encoding the LCDR2 comprising SEQ ID NO: 99; and c.) a sequence encoding the LCDR3 comprising SEQ ID NO: 100.
[0170] In some embodiments, the nucleic acid comprises a.) a sequence encoding the LCDR1 comprising SEQ ID NO: 106; b.) a sequence encoding the LCDR2 comprising SEQ ID NO: 107; and c.) a sequence encoding the LCDR3 comprising SEQ ID NO: 108. [0171] In some embodiments, the nucleic acid comprises a.) a sequence encoding the LCDR1 comprises SEQ ID NO: 114; b.) a sequence encoding the LCDR2 comprising SEQ ID NO: 115; and c.) a sequence encoding the LCDR3 comprising SEQ ID NO: 116.
[0172] In some embodiments, the nucleic acid comprises a.) a sequence encoding the LCDR1 comprising SEQ ID NO: 122; b.) a sequence encoding the LCDR2 comprising SEQ ID NO: 123; and c.) a sequence encoding the LCDR3 comprising SEQ ID NO: 124. [0173] The disclosure provides a nucleic acid comprising a sequence encoding a heavychain variable region (VH) comprising a sequence set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto; and a sequence encoding a light-chain variable region (VL) comprising a sequence set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto.
[0174] In some embodiments, the sequence encoding VH comprises SEQ ID NO: 69, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and the sequence encoding VL comprises SEQ ID NO: 65, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0175] In some embodiments, the sequence encoding VH comprises SEQ ID NO: 77, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and the sequence encoding VL comprises SEQ ID NO: 73, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0176] In some embodiments, the sequence encoding VH comprises SEQ ID NO: 85, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and the sequence encoding VL comprises SEQ ID NO: 81, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0177] In some embodiments, the sequence encoding VH comprises SEQ ID NO: 93, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and the sequence encoding VL comprises SEQ ID NO: 89, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0178] In some embodiments, the sequence encoding VH comprises SEQ ID NO: 101, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and the sequence encoding VL comprises SEQ ID NO: 97, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0179] In some embodiments, the sequence encoding VH comprises SEQ ID NO: 109, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and the sequence encoding VL comprises SEQ ID NO: 105, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto. [0180] In some embodiments, the sequence encoding VH comprises SEQ ID NO: 117, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and the sequence encoding VL comprises SEQ ID NO: 113, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0181] In some embodiments, the sequence encoding VH comprises SEQ ID NO: 125, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and the sequence encoding VL comprises SEQ ID NO: 121, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
[0182] Table 3. Illustrative Polynucleotide Sequences
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
[0183] Vectors
[0184] The term “vector” as used in the application refers to nucleic acid molecules, usually double-stranded DNA, which may have inserted into it another nucleic acid molecule (the insert nucleic acid molecule) such as, but not limited to, a polynucleotide encoding an antibody or antigen-binding fragment described herein. The vector is used to transport the insert nucleic acid molecule into a suitable host cell. A vector may contain the necessary elements that permit transcribing the insert nucleic acid molecule, and, optionally, translating the transcript into a polypeptide. The insert nucleic acid molecule may be derived from the host cell, or may be derived from a different cell or organism. Once in the host cell, the vector can replicate independently of, or coincidental with, the host chromosomal DNA, and several copies of the vector and its inserted nucleic acid molecule may be generated.
[0185] The term “vector” may thus also be defined as a gene delivery vehicle that facilitates gene transfer into a target cell. This definition includes both non-viral and viral vectors. Non-viral vectors include but are not limited to cationic lipids, liposomes, nanoparticles, polyethylene glycol (PEG), polyethylenimine (PEI), etc. Viral vectors are derived from viruses and include but are not limited to retroviral, lentiviral, adeno- associated viral, adenoviral, herpesviral, hepatitis viral vectors or the like. Typically, but not necessarily, viral vectors are replication-deficient as they have lost the ability to propagate in a given cell since viral genes essential for replication have been eliminated from the viral vector. However, some viral vectors can also be adapted to replicate specifically in a given cell, such as, e.g., a cancer cell, and are typically used to trigger the (cancer) cell-specific (onco)lysis
[0186] The disclosure provides vectors comprising the polynucleotides described herein. In some embodiments, the vector comprises a polynucleotide described herein. In some embodiments, the vector comprises a promoter operably linked to the polynucleotide described herein. A promoter is said to be operably linked to a sequence (e.g. a polynucleotide described herein) if the promoter can initiate transcription of the sequence. For example, a promoter operably linked to a polynucleotide encoding an antibody or antigen-binding fragment described herein will recruit transcriptional initiating and promoting proteins (e.g. RNA polymerase and transcription factors) and initiate transcription of the polynucleotide to form an mRNA transcript encoding the antibody or antigen-binding fragment. The mRNA transcript may then be translated to produce a polypeptide comprising the antibody or antigen-binding fragment.
[0187] The vector can be any vector known in the art suitable for expressing the antibody or antigen-binding fragments described herein. In some embodiments, the vector is an expression vector. In some embodiments, the expression vector comprises a promoter. Any suitable promoter known in the art can be used. The promoter will be selected based on the target host cell. For example, a vector will promote expression of the polynucleotides encoding the antibody or antigen-binding fragments described herein in a mammalian cell if the promoter is mammalian cell specific. A suitable promoter can promote strong expression of the polynucleotide encoding an antibody or antigen-binding fragment. In some embodiments, the promoter is inducible. In some embodiments, the promoter is constitutive. Illustrative examples of suitable promoters for expression in a cell include, without limitation, a cytomegalovirus (CMV) promoter or a elongation factor alpha (EFla) promoter.
[0188] In some embodiments, the vector comprises an enhancer sequence. An enhancer sequence is a nucleic acid sequences that regulates, either directly or indirectly, the transcription of corresponding nucleic acid coding sequences to which they are operably linked (e.g., a sequence encoding an antibody or antigen-binding fragment described herein). An enhancer may function alone to regulate transcription or may act in concert with one or more other regulatory sequences (e.g., promoters). Any suitable enhancer sequence known in the art may be used.
[0189] In some embodiments, the vector comprises a sequence encoding a selectable marker. A selectable marker can be a polypeptide that serves as a visualization marker of positive transduction or transfection of the vector described herein. Illustrative examples of a polypeptide that serves as a visualization marker include, without limitation, a fluorescent protein (e.g. green fluorescent protein or red fluorescent protein) or a protein that catalyzes a visualization reagent (e.g. beta-galactosidase or luciferase). For example, a cell that has been transfected or transduced with a vector encoding a selectable marker comprising GFP will show a GFP -mediated fluorescent signal when visualized in a fluorescence microscope, whereas a cell that is not transfected or transduced with a vector encoding a selectable marker comprising GFP will not show a GFP-mediated fluorescence signal when visualized in a fluorescence microscope. A selectable marker can also be a polypeptide that allows selective killing of cells using that do not contain the vector. Illustrative examples of a polypeptide that serves as a polypeptide that allows selective killing of cells that do not contain the vector include, without limitation, puromycin acetyltransferase, hygromycin phosphotransferase, and glutamine synthase. For example, a cell that has been transfected or transduced with a vector encoding a selectable marker comprising puromycin acetyltransferase will not be killed when contacted with puromycin, whereas a cell that is not transfected or transduced with a vector encoding a selectable marker comprising puromycin acetyltransferase will be killed when contacted with puromycin. Any suitable selective markers known in the art may be used in the vectors described herein.
[0190] In some embodiments, the vector comprises a sequence encoding an intron sequence. An intron sequence can be included in a 5' untranslated region of the polynucleotide encoding an antibody or antigen-binding fragment following the promoter/enhancer to increase export of transcribed mRNA to the cytoplasm from the nucleus. Any suitable intron sequence known in the art may be used in the vectors described herein.
[0191] In some embodiments, the vector comprises a sequence encoding a polyadenylation (poly(A)) signal. The poly(A) signal is a region is a polynucleotide sequence on the 3’ end of the polynucleotide sequence encoding a polypeptide (e.g. an antibody or antigen-binding fragment). The poly(A) signal can regulate mRNA transport, stability, translation, and can promote expression of the polypeptide. Any suitable poly(A) signal sequence known in the art may be used in the vectors described herein.
[0192] In some embodiments, the vector comprises a sequence encoding a Kozak sequence. In some embodiments, the vector comprises a sequence encoding a ribosomal binding site. In some embodiments, the vector comprises a sequence encoding an internal ribosome entry site. The Kozak sequence, ribosomal binding site, and internal ribosome entry sites are nucleic acids that function as a translation initiation and/or ribosomal recruitment sites to ensure that a polypeptide is correctly translated from an mRNA transcript. Any suitable Kozak sequence known in the art may be used in the vectors described herein.
[0193] In some embodiments, the vector comprises a sequence encoding a signal peptide. The sequence encoding a signal peptide can be a polynucleotide sequence located on the 5’ end of the polynucleotide sequence encoding a polypeptide (e.g. an antibody or antigenbinding fragment). The signal peptide facilitates export of the polypeptide from the cell in which they are expressed, and is cleaved from the polypeptide prior to export from the cell. Any suitable sequence encoding a signal peptide may be used in the vectors described herein.
Methods of Production
[0194] The disclosure methods of producing the antibody or antigen-binding fragment described herein. The disclosure provides a method of producing the antibody of antigenbinding fragment that binds to a SARS-CoV-2 spike protein comprising: a.) contacting a cell with the nucleic acid described herein or a vector described herein; b.) culturing the cell under conditions sufficient to express the antibody or antigen-binding fragment; and purifying the antigen-binding fragment.
[0195] Cell Line Generation
[0196] The disclosure provides a cell line for producing the antibody or antigen-binding fragment.
[0197] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.
[0198] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). One method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.
[0199] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.
[0200] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).
[0201] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present disclosure, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the disclosure.
[0202] Host cells for use in cell line generation can be any cell known in the art for expressing an antibody or antigen-binding fragment. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell. In some embodiments, the host cell is a human embryonic kidney cell 293 (HEK293) cell. In some embodiments, the host cell is a baby hamster kidney (BKH) cell. In some embodiments, the host cell is a mouse myeloma cell. In some embodiments, the host cell is a human embryonic retinal cell. In some embodiments, the host cell is a bacterial cell. In some embodiments, the host cell is a yeast cell.
[0203] Host cells may be allowed to grow for 1-2 days in an enriched media following transduction or transfection with the nucleic acids or vectors described herein, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to generate cell lines which express the antibody molecule. Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1.
[0204] Established cell lines for producing the antibody or antigen-binding fragments described herein can be expanded and banked indefinitely under cryogenic conditions. Banked cells can be thawed and further expanded under suitable conditions for production of the antibody or antigen-binding fragments at a desired scale.
[0205] Conditions for expressing an antibody or anti en-binding fragment in a cell
[0206] The disclosure provides a method of producing the antibody or antigen-binding fragment described herein, comprising culturing a cell line comprising a nucleic acid described herein or a vector described herein under conditions sufficient for expression of the antibody or antigen-binding fragment, and purifying the antibody or antigen-binding fragment.
[0207] A cell comprising the nucleic acids and/or vectors described herein can be cultured under suitable conditions to express the antibody or antigen-binding fragment. Expression of an antibody or antigen-binding fragment described herein in the cell is referred to as “recombinant” expression, and the produced antibody or antigen-binding fragment can be referred to as “recombinant.” In some embodiments, the antibody or antigen-binding fragment is recombinant. [0208] For long-term, high-yield production of recombinant polypeptide (e.g. an antibody or antigen-binding fragment described herein) can be achieved using stable expression. Stable expression of the recombinant polypeptide can be achieved with use of a constitutive promoter.
[0209] Any suitable cell culture medium for the culture of cell lines described herein, such as mammalian cells expressing an antibody or antigen-binding fragment described herein, may be used. The culture can be an in vitro cell culture. Cell culture media formulations are well known in the art. Typically, cell culture media are comprised of buffers, salts, carbohydrates, amino acids, vitamins and trace essential elements. The cell culture medium may or may not contain serum, peptone, and/or proteins. Various tissue culture media, including serum-free and defined culture media, are commercially available. Illustrative examples include, without limitation, one or a combination of the following cell culture medias: RPMI-1640 Medium, RPMI-1641 Medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium Eagle, F-12K Medium, Ham's F12 Medium, Iscove's Modified Dulbecco's Medium, McCoy's 5 A Medium, Leibovitz's L- 15 Medium, and serum-free media such as EX-CELL™ 300 Series (JRH Biosciences, Lenexa, Kansas), among others. Cell culture media may be supplemented with additional or increased concentrations of components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements and the like, depending on the requirements of the cells to be cultured and/or the desired cell culture parameters. For example, cell culture media may be supplemented with polyamines such as putrescine, spermidine and spermine, to improve cell growth, cell viability, and/or recombinant protein production in association with a particular host cell. Cell culture media may be serum-free, protein-free, and/or peptone-free media. "Serum- free" applies to a cell culture medium that does not contain animal sera, such as fetal bovine serum. "Protein-free" applies to cell culture media free from exogenously added protein, such as transferring, protein growth factors IGF-I, or insulin. Protein-free media may or may not contain peptones. "Peptone-free" applies to cell culture media which contains no exogenous protein hydrolysates such as animal and/or plant protein hydrolysates. Eliminating serum and/or hydrolysates from cell culture media has the advantage of reducing lot to lot variability and enhancing processing steps, such as filtration. However, when serum and/or peptone are removed from the cell culture media, cell growth, viability and/or protein expression may be diminished or less than optimal. As such, serum-free and/or peptone-free cell culture medium may be highly enriched for amino acids, trace elements and the like. See, for example, US Patent Nos. 5,122,469 and 5,633,162. Although there are many media formulations, there is a need to develop defined media formulations that perform as well or preferably better than those containing animal sera and/or peptones.
[0210] By cell culture or "culture" is meant the growth and propagation of cells outside of a multicellular organism or tissue. Suitable culture conditions for mammalian cells are known in the art. See e.g. Animal cell culture: A Practical Approach, D. Rickwood, ed., Oxford University Press, New York (1992). Mammalian cells may be cultured in suspension or while attached to a solid substrate. Fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, shake flasks, or stirred tank bioreactors, with or without microcarriers, and operated in a batch, fed batch, continuous, semi-continuous, or perfusion mode are available for mammalian cell culture. Cell culture media and/or concentrated feed media may be added to the culture continuously or at intervals during the culture. For example, a culture may be fed once per day, every other day, every three days, or may be fed when the concentration of a specific medium component, which is being monitored, falls outside a desired range.
[0211] Cells described herein may be cultured in small scale cultures, such as, for example, in 100 ml containers having about 30 ml of media, 250 ml containers having about 80 to about 90 ml of media, 250 ml containers having about 150 to about 200 ml of media. Alternatively, the cultures can be large scale such as for example 1000 ml containers having about 300 to about 1000 ml of media, 3000 ml containers having about 500 ml to about 3000 ml of media, 8000 ml containers having about 2000 ml to about 8000 ml of media, and 15000 ml containers having about 4000 ml to about 15000 ml of media. Large scale cell cultures, such as for clinical manufacturing of protein therapeutics, are typically maintained for days, or even weeks, while the cells produce the desired protein(s). During this time the culture can be supplemented with a concentrated feed medium containing components, such as nutrients and amino acids, which are consumed during the course of the culture. Concentrated feed medium may be based on just about any cell culture media formulation. Such a concentrated feed medium can contain most of the components of the cell culture medium at, for example, about 5x, 6x, 7x, 8x, 9x, lOx, 12x, 14x, 16x, 20x, 3 Ox, 50x, lOOx, 200x, 400x, 600x, 800x, or about lOOOx of their normal amount. Concentrated feed media are often used in fed batch culture processes. [0212] methods according to the present invention may be used to improve the production of recombinant proteins in both single phase and multiple phase culture processes. In a single phase process, cells are inoculated into a culture environment and the disclosed methods are employed during the single production phase. In a multiple stage process, cells are cultured in two or more distinct phases. For example cells may be cultured first in one or more growth phases, under environmental conditions that maximize cell proliferation and viability, then transferred to a production phase, under conditions that maximize protein production. In a commercial process for production of a protein by mammalian cells, there are commonly multiple, for example, at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 growth phases that occur in different culture vessels preceding a final production phase. The growth and production phases may be preceded by, or separated by, one or more transition phases. In multiple phase processes, the methods according to the present invention can be employed at least during the production phase, although they may also be employed in a preceding growth phase. A production phase can be conducted at large scale. A large scale process can be conducted in a volume of at least about 100, about 500, about 1000, about 2000, about 3000, about 5,000, about 7,000, about 8,000, about 10,000, about 15,000, or about 20,000 liters. A growth phase may occur at a higher temperature than a production phase. For example, a growth phase may occur at a first temperature from about 35°C to about 38°C, and a production phase may occur at a second temperature from about 29°C to about 37°C, optionally from about 30°C to about 36°C or from about 30°C to about 34°C. In addition, chemical inducers of protein production, such as, for example, caffeine, butyrate, and hexamethylene bisacetamide (HMBA), may be added at the same time as, before, and/or after a temperature shift. If inducers are added after a temperature shift, they can be added from one hour to five days after the temperature shift, optionally from one to two days after the temperature shift.
[0213] Collecting and purifying an antibody or anti en-binding fragment
[0214] The antibody and antigen-binding fragments expressed by cells described herein can be purified or isolated after expression. Antibodies may be isolated or purified in a variety of ways known to those skilled in the art. Standard purification methods include chromatographic techniques, including ion exchange, hydrophobic interaction, affinity, sizing or gel filtration, and reversed-phase, carried out at atmospheric pressure or at high pressure using systems such as FPLC and HPLC. Purification methods also include electrophoretic, immunological, precipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful, variety of natural proteins bind antibodies, and these proteins can be used for purification of antibody and antigen-binding fragments described herein. For example, the bacterial proteins A and G bind to the Fc region, and the bacterial protein L binds to the Fab region. Purification can often be enabled by fusions or tags. For example, antibody and antigen-binding fragments may be purified using glutathione resin if a GST fusion is employed, Ni+2 affinity chromatography if a His tag is employed, or immobilized antiflag antibody if a flag tag is used. For general guidance in suitable purification techniques, see Protein Purification: Principles and Practice, 3rd Ed., Scopes, Springer- Verlag, NY, 1994, hereby expressly incorporated by reference.
[0215] The degree of purification necessary will vary depending on the use of the antibody or antigen-binding fragment. In some instances no purification is necessary. For example characterization of an antibody or antigen-binding fragment may take place directly from the media. In some instances, a high degree of purification is necessary. For example, high purity preparations of the antibody or antigen-binding fragment are required for use in a medicament or pharmaceutical compositions described herein, methods of detection described herein, and methods of treatment described herein.
Pharmaceutical Compositions and Kits
[0216] The disclosure provides a pharmaceutical compositions comprising the antibody or antigen-binding fragment described herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, excipient, and/or diluent.
[0217] The disclosure provides a kit comprising the antibody or antigen-binding fragment described herein. In some embodiments, the kit comprises a pharmaceutical composition described herein. In some embodiments, the kit comprises instructions for use.
[0218] The disclosure provides a pharmaceutical composition comprising the antibody or antigen-binding fragment described herein, for use in the treatment of COVID-19.
[0219] The pharmaceutical composition can be formulated for various types of administration or storage conditions. The formulation will be optimized, often in a non- obvious way, according to the desired administration or storage conditions using a subset of carriers, excipients, and/or diluents, each having at specific concentration or range of acceptable concentrations. [0220] The formulation can include, for example, one or more salts (a buffering salt), one or more polyols and one or more excipients. The formulations of the present disclosure may also contain buffering agents, or preservatives. The pH range of the formulation can be buffered in a solution at a pH in the range of about 4 to 8; in the range of about 4 to 7; in the range of about 4 to 6; in the range of about 5 to 6; or in the range of about 5.5 to 6.5. [0221] Examples of salts include those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, boric, formic, malonic, succinic, and the like. Such salts can also be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts. Examples of buffering agents include phosphate, citrate, acetate, and 2-(N-morpholino)ethanesulfonic acid (MES).
[0222] The formulations of the present disclosure may include a buffer system. As used in this application, the terms "buffer" or "buffer system" is meant a compound that, usually in combination with at least one other compound, provides a buffering system in solution that exhibits buffering capacity, that is, the capacity to neutralize, within limits, either acids or bases (alkali) with relatively little or no change in the original pH.
[0223] Buffers include borate buffers, phosphate buffers, calcium buffers, and combinations and mixtures thereof. Borate buffers include, for example, boric acid and its salts, for example, sodium borate or potassium borate. Borate buffers also include compounds such as potassium tetraborate or potassium metaborate that produce borate acid or its salt in solutions.
[0224] A phosphate buffer system includes one or more monobasic phosphates, dibasic phosphates and the like. Particularly useful phosphate buffers are those selected from phosphate salts of alkali and/or alkaline earth metals. Examples of suitable phosphate buffers include one or more of sodium dibasic phosphate (ISfel PCU), sodium monobasic phosphate (Na^PCE) and potassium monobasic phosphate (KH2PO4). The phosphate buffer components frequently are used in amounts from 0.01% or to 0.5% (w/v), calculated as phosphate ion.
[0225] Other known buffer compounds can optionally be added to the formulations, for example, citrates, sodium bicarbonate, TRIS, and the like. Other ingredients in the solution, while having other functions, may also affect the buffer capacity. For example, EDTA, often used as a complexing agent, can have a noticeable effect on the buffer capacity of a solution. [0226] Illustrative salts for use in the formulation of the disclosure include sodium chloride, sodium acetate, sodium acetate trihydrate and sodium citrate.
[0227] The concentration of salt in the formulations according to the disclosure is between about 10 mM and 500mM, between about 25m and 250 mM, between about 25nM and 150mM.
[0228] The sodium acetate trihydrate is at a concentration in the range of about 10 mM to 100 mM. For example, the sodium acetate trihydrate is at about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mM. Preferably, the sodium acetate trihydrate is at 25mM.
[0229] The sodium chloride at a concentration in the range of about 50 mM to 500 mM. For example, the sodium chloride is at about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 or 500 mM.
[0230] The sodium citrate is at a concentration in the range of about 10 mM to 100 mM For example the sodium citrate is at about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mM.
[0231] In some embodiments, the salt is sodium acetate trihydrate at a concentration in the range of about 25 mm to 100 mm and sodium chloride at a concentration in the range of about 150 mm to 500 mm.
[0232] The formulation can include one or more polyols as a bulking agent and/or stabilizing excipients. Polyols include for example, trehalose, mannitol, maltose, lactose, sucrose, sorbitol, or glycerol. The polyols is at a concentration in the range of about 0.1% to 50% or 5% to 25%. For example, the polyol is at about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50%.
[0233] In some embodiments, the polyol is trehalose at a concentration in the range of about 1% to 50% or 5% to 25%. For example, the trehalose is at about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50%. The trehalose can be at a concentration of about 10% or about 20%. The trehalose can be at a concentration of about 20%.
[0234] In some embodiments, the polyol is sorbitol at a concentration in the range of about 1% to about 10%. In some embodiments, the polyol is glycerol at a concentration in the range of about 1% to about 10%.
[0235] In some embodiments, the polyol is mannitol at a concentration in the range of about 0.1% to about 10%. In some embodiments, the polyol is maltose at a concentration in the range of about 1% to about 10%. [0236] The formulation includes one or more excipients and/ or surfactants to suppress or otherwise reduce antibody aggregation. Suitable excipients to reduce antibody aggregation include, by way of non-limiting example, a surfactant such as, by way of non-limiting example, Polysorbate 20 or Polysorbate 80. In some embodiments, the Polysorbate 20 or Polysorbate 80 is present at a concentration in the range of about 0.01 to 1 % or about 0.0 Ito 0.05%. For example the Polysorbate 20 or Polysorbate 80 is at a concentration of about 0.01. 0.02, 0.03, 0.04, 0.05, 0.06, 0.07. 0.08, 0.09, 0.1, 0.2, 0.3. 0.4, 0.5, 0.6, 0.7, 0.8. 0.9, or 1.0%.
[0237] The surfactant can be Polysorbate 80 at a concentration in the range of about 0.01 to 0.05%. The Polysorbate 80 concentration can be 0.02%.
[0238] The formulation includes one or more excipients to reduce antibody oxidation. Suitable excipients to reduce antibody oxidation include, by way of non-limiting example, antioxidants. Antioxidants include for example, methionine, D-arginine, BHT or ascorbic acid. The antioxidant is present at a concentration in the range of about 0.01 % to 1% ; 0.1% to 1%; or 0.1% to 0.5%. In some embodiments, the antioxidant is methionine. In some embodiments, the methionine is present at a concentration in the range of about 0.01 % to 1% ; 0.1% to 1%; or 0.1% to 0.5%. For example, the methionine is present at a concentration of about 0.01. 0.02, 0.03, 0.04, 0.05, 0.06, 0.07. 0.08, 0.09, 0.1, 0.2, 0.3. 0.4, 0.5, 0.6, 0.7, 0.8. 0.9, or 1.0%. The methionine concentration can be about 0.1%.
[0239] The formulation includes one or more chelating agents, such as, for example, ethylenediaminetetraacetic acid (EDTA). The chelating agent is at a concentration in the range of 0.01% to 1% ; 0.1% to 1%; or 0.1% to 0.5%. For example, the chelating agent is present at a concentration of about 0.01%. 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%. The chelating agent can be EDTA at a concentration of about 0.1%.
[0240] In some embodiments, the formulation includes one or more excipients to increase stability. In some embodiments, the excipient to increase stability is human serum albumin. In some embodiments, the human serum albumin is present in the range of about 1 mg to about 5 mg.
[0241] In some embodiments, the formulation includes magnesium stearate (Mg stearate), an amino acid, or both mg-stearate and an amino acid. Suitable amino acids include for example, leucine, arginine, histidine, or combinations thereof. [0242] In some embodiments, the formulation includes low moisture microcrystalline cellulose, such as Avicel, polyethylene glycols (PEG), or a starch.
[0243] Further examples of pharmaceutically acceptable carriers and excipients useful for the formulations of the present disclosure include, but are not limited to binders, fillers, disintegrants, lubricants, anti-microbial agents, antioxidant, and coating agents.
[0244] Binders can be corn starch, potato starch, other starches, gelatin, natural and synthetic gums such as acacia, xanthan, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone (e.g., povidone, crospovidone, copovidone, etc), methyl cellulose, Methocel, pre-gelatinized starch (e.g., STARCH 1500® and STARCH 1500 LM®, sold by Colorcon, Ltd.), hydroxypropyl methyl cellulose, microcrystalline cellulose (FMC Corporation, Marcus Hook, PA, USA), Emdex, Plasdone, or mixtures thereof.
[0245] Fillers can be talc, calcium carbonate (e.g., granules or powder), dibasic calcium phosphate, tribasic calcium phosphate, calcium sulfate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, dextrose, fructose, honey, lactose anhydrate, lactose monohydrate, lactose and aspartame, lactose and cellulose, lactose and microcrystalline cellulose, maltodextrin, maltose, mannitol, microcrystalline cellulose & guar gum, molasses, sucrose, or mixtures thereof.
[0246] Disintegrants can be agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, (such as Explotab), potato or tapioca starch, other starches, pre-gelatinized starch, clays, other algins, other celluloses, gums (like gellan), low-substituted hydroxypropyl cellulose, ployplasdone, or mixtures thereof.
[0247] Lubricants can be calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, compritol, stearic acid, sodium lauryl sulfate, sodium stearyl fumarate, (such as Pruv), vegetable based fatty acids lubricant, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar, syloid silica gel (AEROSIL 200, W.R. Grace Co., Baltimore, MD USA), a coagulated aerosol of synthetic silica (Deaussa Co., Piano, TX USA), a pyrogenic silicon dioxide (CAB-O-SIL, Cabot Co., Boston, MA USA), or mixtures thereof. [0248] Anti-caking agents can be calcium silicate, magnesium silicate, silicon dioxide, colloidal silicon dioxide, talc, or mixtures thereof.
[0249] Antimicrobial agents can be benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, butyl paraben, cetylpyridinium chloride, cresol, chlorobutanol, dehydroacetic acid, ethylparaben, methylparaben, phenol, phenylethyl alcohol, phenoxyethanol, phenylmercuric acetate, phenylmercuric nitrate, potassium sorbate, propylparaben, sodium benzoate, sodium dehydroacetate, sodium propionate, sorbic acid, thimersol, thymo, or mixtures thereof.
[0250] Antioxidants can be ascorbic acid, BHA, BHT, EDTA, or mixture thereof, and COATING AGENTS: sodium carboxymethyl cellulose, cellulose acetate phthalate, ethylcellulose, gelatin, pharmaceutical glaze, hydroxypropyl cellulose, hydroxypropyl methylcellulose (hypromellose), hydroxypropyl methyl cellulose phthalate, methylcellulose, polyethylene glycol, polyvinyl acetate phthalate, shellac, sucrose, titanium dioxide, carnauba wax, microcrystalline wax, gellan gum, maltodextrin, methacrylates, microcrystalline cellulose and carrageenan or mixtures thereof.
[0251] The formulation can also include other excipients and categories thereof including but not limited to Pluronic®, Pol oxamers (such as Lutrol® and Pol oxamer 188), ascorbic acid, glutathione, protease inhibitors (e.g. soybean trypsin inhibitor, organic acids), pH lowering agents, creams and lotions (like maltodextrin and carrageenans); materials for chewable tablets (like dextrose, fructose, lactose monohydrate, lactose and aspartame, lactose and cellulose, maltodextrin, maltose, mannitol, microcrystalline cellulose and guar gum, sorbitol crystalline); parenterals (like mannitol and povidone); plasticizers (like dibutyl sebacate, plasticizers for coatings, polyvinylacetate phthalate); powder lubricants (like glyceryl behenate); soft gelatin capsules (like sorbitol special solution); spheres for coating (like sugar spheres); spheronization agents (like glyceryl behenate and microcrystalline cellulose); suspending/gelling agents (like carrageenan, gellan gum, mannitol, microcrystalline cellulose, povidone, sodium starch glycolate, xanthan gum); sweeteners (like aspartame, aspartame and lactose, dextrose, fructose, honey, maltodextrin, maltose, mannitol, molasses, sorbitol crystalline, sorbitol special solution, sucrose); wet granulation agents (like calcium carbonate, lactose anhydrous, lactose monohydrate, maltodextrin, mannitol, microcrystalline cellulose, povidone, starch), caramel, carboxymethyl cellulose sodium, cherry cream flavor and cherry flavor, citric acid anhydrous, citric acid, confectioner's sugar, D&C Red No. 33, D&C Yellow #10 Aluminum Lake, disodium edetate, ethyl alcohol 15%, FD&C Yellow No. 6 aluminum lake, FD&C Blue # 1 Aluminum Lake, FD&C Blue No. 1, FD&C blue no. 2 aluminum lake, FD&C Green No.3, FD&C Red No. 40, FD&C Yellow No. 6 Aluminum Lake, FD&C Yellow No. 6, FD&C Yellow No.10, glycerol palmitostearate, glyceryl monostearate, indigo carmine, lecithin, manitol, methyl and propyl parabens, mono ammonium glycyrrhizinate, natural and artificial orange flavor, pharmaceutical glaze, pol oxamer 188, Polydextrose, polysorbate 20, polysorbate 80, polyvidone, pregelatinized com starch, pregelatinized starch, red iron oxide, saccharin sodium, sodium carboxymethyl ether, sodium chloride, sodium citrate, sodium phosphate, strawberry flavor, synthetic black iron oxide, synthetic red iron oxide, titanium dioxide, and white wax.
[0252] In some embodiments, the pharmaceutical composition is formulated for parenteral administration, enteral administration, mucosal administration, nasal administration, oral administration, or intra-articular administration. In some embodiments, the administration comprises inhalation, intravenous injection, infusion, subcutaneous injection, or intramuscular injection.
Methods of Treatment and Prevention
[0253] The disclosure provides a method of treating COVID-19 in a subject in need thereof, comprising administering to the subject an antibody or antigen-binding fragment described herein or a pharmaceutical composition described herein.
[0254] The disclosure provides a method of preventing COVID-19 in a subject in need thereof, comprising administering to the subject an antibody or antigen-binding fragment described herein or a pharmaceutical composition described herein
[0255] The disclosure provides a method of reducing symptoms associated with COVID- 19 in a subject in need thereof, comprising administering to the subject an antibody or antigen-binding fragment described herein or a pharmaceutical composition described herein.
[0256] The disclosure provides a method of treating, preventing, or reducing symptoms associated with COVID-19 in a subject in need thereof, comprising detecting a SARS- CoV-2 viral genome or a SARS-CoV-2 antigen in a sample collected from the subject, wherein detection of the SARS-CoV-2 antigen comprises contacting the sample with an antibody or antigen-binding fragment described herein; and administering a treatment to the subject, wherein the treatment prevents, reduces, or eliminates a SARS-CoV-2 infection or a variant thereof; and/or prevents, reduces, or eliminates symptoms associated with COVID-19, wherein the treatment comprises an antibody or antigen-binding fragment described herein or a pharmaceutical composition described herein.
[0257] The methods of provided herein include administering the antibody or antigenbinding fragment described herein to a subject in need thereof. The subject can be a human or an animal. The subject can have or be suspected of having a SARS-CoV-2 infection or a variant thereof. In some embodiments, the subject has or is suspected of having a SARS- CoV-2 infection or a variant thereof. In some embodiments, the subject has at least one symptom associated with a SARS-CoV-2 infection or a variant thereof. In some embodiments, the subject is an animal or a human.
[0258] A subject can be determined to have a SARS-CoV-2 infection or a variant thereof using any appropriate method known in the art. Testing can be performed on a sample collected from the subject, which can include saliva, a nasopharyngeal swab, nasal midturbinate swab, anterior nasal swab, blood, plasma, urine, feces, bronchoalveolar lavage, or another sample in which a SARS-CoV-2 infection or a variant thereof can be measured. [0259] Samples can be tested using any test type known in the art to detect a previous or active SARS-CoV-2 infection. Illustrative test types include viral tests, diagnostic tests, and/or screening tests.
[0260] Viral tests, including nucleic acid amplification tests (NAATs) and antigen tests, are used as diagnostic tests to detect infection with SARS-CoV-2 or a variant thereof and to inform a subject’s medical care. Viral tests can also be used as screening tests to reduce the transmission of SARS-CoV-2 or a variant thereof by identifying infected subjects who need to isolate from others.
[0261] NAATs are high-sensitivity, high-specificity tests for diagnosing SARS-CoV-2 infection or a variant thereof by detecting the presence of a SARS-CoV-2 viral genome by measuring, for example, a gene of the viral genome. NAATs detect one or more viral ribonucleic acid (RNA) genes and indicate a current infection or a recent infection but, due to prolonged viral RNA detection, are not always direct evidence for the presence of virus capable of replicating or being transmitted to others. Most NAATs need to be processed in a laboratory, and time to results can vary (~l-3 days), but some NAATs are point-of- care tests with results available in about 15-45 minutes. MostNAATs produce qualitative results. NAATs can be performed on upper respiratory specimens, such as nasopharyngeal, nasal mid-turbinate, anterior nasal, or saliva. RNA is extracted from the sample and subject to reverse transcription prior to amplification. Illustrative examples of NAATs include rt- PCR, PCR tests and rapid PCR tests, wherein PCR primers are designed to amplify S ARS- CoV-2 genes for subsequent detection. Viral loads can be calculated by in RNA copies of the target gene per mL. A positive result from NAATs imply the subject has a SARS-CoV- 2 infection.
[0262] Antigen tests are immunoassays that detect the presence of a specific viral antigen. Antigen tests generally have similar specificity, but are less sensitive than most NAATs. Most are less expensive than NAATs and can be processed at the point of care with results available in minutes and thus can be used in screening programs to quickly identify those who are likely to be contagious. Because of the performance characteristics of antigen tests, it may be necessary to confirm some antigen test results (a negative test in persons with symptoms or a positive test in persons without symptoms) with a laboratory-based NAAT. Use of the Antigen Testing Algorithm is recommended to determine when confirmatory testing is needed. Antigen tests can be performed on nasopharyngeal or anterior nasal specimens. Illustrative examples of antigen test include use lateral flow devices that immunodetect the presence of SARS-CoV-2 antigen, such as Innova Rapid SARS-CoV-2 Antigen Test (Xiamen Biotime Biotechnology, Fujian, China), Spring Healthcare SARS-CoV-2 Antigen Rapid Test Cassette (Shanghai ZJ Bio-Tech, Shanghai, China), E25Bio Rapid Diagnostic Test (E25Bio, Cambridge, MA, USA), Encode SARS- CoV-2 Antigen Rapid Test Device (Zhuhai Encode Medical Engineering, Zhuhai, China), SureScreen COVID-19 Rapid Antigen Test Cassette, and SureScreen COVID-19 Rapid Fluorescence Antigen Test (both from SureScreen Diagnostics, Derby, UK). A positive result from an antigen test implies the subject has a SARS-CoV-2 infection.
[0263] A serological test can be performed in a sample to determine is the subject has had a prior SARS-CoV-2 infection. The serological tests detect the presence of antibodies associated with COVID-19 in blood. The antibodies are a developed in the blood of a subject due to the immune response to COVID-19.
[0264] A subject suspected of having a SARS-CoV-2 infection has been determined to have exposure to a known person with an active SARS-CoV-2 infection, or can have symptoms associated with a SARS-CoV-2 or a variant thereof infection or CO VID-19. [0265] In some embodiments, the methods provided herein treat, prevent, or alleviate one or more symptoms. In some embodiments, the symptom is at least one acute symptom. An acute symptom is a symptom that resolves in less than a month, or resolves within a timeframe that is expected for a given disease or disorder. For example, a subject suffering from a coronavirus infection (e.g. COVID-19), or a variant thereof, may experience acute symptoms such as fever, sore throat, cough, shortness of breath, and chest pain that resolve within 2 to 4 weeks.
[0266] In some embodiments, the severity of the one or more symptoms is reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or eliminated completely.
[0267] In some embodiments, the duration of the one or more symptoms is reduced by at least one day, at least two days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days.
[0268] In some embodiments, the symptom is at least one chronic symptom. A chronic symptom is a symptom that persists in a subject suffering from a disease or disorder after the time considered normal for that disease or disorder. For example, a subject suffering from a coronavirus infection (e.g. COVID-19), or a variant thereof, may experience chronic symptoms that may persist beyond 1 month, beyond 2 months, beyond 3 months, beyond 4 months, beyond 5 months, or beyond 6 months following infection. A prolonged inflammatory response can contribute to chronic symptoms. In some embodiments, chronic symptoms are at least one of fatigue, muscle aches and pains, poor sleep, cough, breathlessness, orthopnea, leg swelling, exercise intolerance due to COVID-19 induced heart failure, pulmonary embolism, pulmonary fibrosis, COVID-19-related ARDS, palpitations with mild exertion, night sweats, organ damage (e.g. cardiac or respiratory organ damage), and poor temperature control.
[0269] In some embodiments, the symptom of a SARS-CoV-2 infection or COVID-19 is one or more of hyperactive immune response, fever, gastrointestinal symptoms, respiratory symptoms, anosmia (loss of smell), dysgeusia (loss of taste), cough, headache, throat ache, pain when swallowing, dyspnea, difficult breathing, shortness of breath, nausea, vomiting, reduced 02 saturation, diarrhea, rhinorrhea, abdominal pain, myalgia, fever, conjunctivitis, and loss of appetite. [0270] A hyperactive immune response can include, for example, a cytokine release syndrome, a “cytokine storm”, or the like. In some embodiments, the hyperactive immune response comprises elevated levels of at least one of interleukin 6 (IL-6), C-reactive protein (CRP), D-dimer, interferon (IFN), interferon alpha (IFN-a), interferon gamma (IFN-y), interleukin 1 beta (IL- 1 (3), and/or CXCL10.
Administration and Dosage
[0271] The methods provided herein include administration of an antibody or antigenbinding fragment to a subject in need thereof. The antibody or antigen-binding fragment or pharmaceutical composition (and/or any additional therapeutic agent) can be administered by any suitable means. In some embodiments, administration is enteral, parenteral, mucosal, nasal, oral, by inhalation, by intravenous injection, by infusion, by subcutaneous injection, by intramuscular injection, or intra-articular.
[0272] Dosing can be by any suitable route, for example, by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.
[0273] The antibody or antigen-binding fragment or pharmaceutical composition (and/or any additional therapeutic agent) can be administered as a unit dose. In some embodiments, the unit dose is a therapeutically effective amount.
[0274] The therapeutically effective amount to be administered as a unit dose to a subject will be in the range of about 0.01 to about 100 mg/kg of patient body weight whether by one or more administrations. In some embodiments, the masked antibody used is about 0.01 to about 45 mg/kg, about 0.01 to about 40 mg/kg, about 0.01 to about 35 mg/kg, about 0.01 to about 30 mg/kg, about 0.01 to about 29 mg/kg, about 0.01 to about 28 mg/kg, about 0.01 to about 27 mg/kg, about 0.01 to about 26 mg/kg, about 0.01 to about 25 mg/kg, about 0.01 to about 24 mg/kg, about 0.01 to about 23 mg/kg, about 0.01 to about 22 mg/kg, about 0.01 to about 21 mg/kg, about 0.01 to about 20 mg/kg, about 0.01 to about 19 mg/kg, about 0.01 to about 18 mg/kg, about 0.01 to about 17 mg/kg, about 0.01 to about 16 mg/kg, about 0.01 to about 15 mg/kg, about 0.01 to about 14 mg/kg, about 0.01 to about 13 mg/kg, about 0.01 to about 12 mg/kg, about 0.01 to about 1 1 mg/kg, about 0.01 to about 10 mg/kg, about 0.01 to about 9 mg/kg, about 0.01 to about 8 mg/kg, about 0.01 to about 7 mg/kg, about 0.01 to about 6 mg/kg, about 0.01 to about 5 mg/kg, about 0.01 to about 4 mg/kg, about 0.01 to about 3 mg/kg, about 0.01 to about 2 mg/kg, or about 0.01 to about 1 mg/kg administered daily, for example. In some embodiments, a unit dose is about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1 100 mg, about 1200 mg, about 1300 mg or about 1400 mg on day 1 of a cycle. A cycle can be 1 to 21 days in duration. Dosing can occur once per week, twice per week, three times per week, four times per week, five times per week, six times per week or seven times per week. The dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as infusions. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One illustrative dosage may be in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg (or any combination thereof) may be administered to the subject. Such doses may be administered intermittently, for example, every week or every three weeks (e.g., such that the patient receives from about two to about twenty, or, for example, about six doses of antibody or antigen-binding fragment described herein or the pharmaceutical composition described herein). An initial higher loading dose, followed by one or more lower doses may be administered.
Methods of Detecting a SARS-CoV-2 Spike Protein
[0275] Provided herein are methods for detecting a SARS-CoV-2 spike protein in a sample, comprising contacting a sample with the antibody or antigen-binding fragment described herein. In some embodiments, the antibody or antigen-binding fragment is labeled with a tag, detection dye or a secondary antibody.
[0276] In some embodiments, the sample is an environmental sample, sewage, wastewater, urine, feces, saliva, nasal swab, mucous, blood, plasma, bronchoalveolar lavage, or one or more cells. In some embodiments, the sample is collected from a subject. In some embodiments, the subject has or is suspected of having a SARS-CoV-2 infection or COVID-19. In some embodiments, the antibody or antigen-binding fragment is labeled with a tag, detection dye or a secondary antibody.
[0277] In one aspect, the disclosure provides a method of diagnosing a SARS-CoV-2 infection or COVID-19 in subject, comprising detecting a SARS-CoV-2 spike protein in a sample, comprising contacting a sample with the antibody or antigen-binding fragment described herein.
[0278] Any assay platform or type known in the art for detecting an antigen (e.g. a SARS- CoV-2 spike protein) and is enabled by an antibody or antigen-binding fragment targeting said antigen may be used in the methods of detection and diagnosis described herein. In some embodiments, the method comprises use of an electrochemical assay, biosensor, or immunoassay.
[0279] In some embodiments, the detection method for conducting an assay may be selected from a group including but not limited to absorbance, chemiluminescence, fluorescence, magnetic, and/or electrochemical. In some embodiments, detecting the SARS-CoV-2 spike protein further comprises use of an ELISA technique or a lateral flow assay.
Illustrative Assay Platforms
[0280] Chemiluminescent Microparticle Immunoassay (CMIA)
[0281] A chemiluminescent microparticle assay (CMIA) may be used in the methods of detection and diagnosis described herein. Generally, chemiluminescent microparticle assay techniques are based on the principle that a chemiluminescent label, when treated via a trigger reagent, will emit light at a characteristic wavelength (i.e., chemiluminescence).
[0282] The reactants necessary for CMIA can include microparticles coated with a capture agent (e.g. an antibody or antigen-binding fragment) that binds the antigen (e.g. a SARS- CoV-2 spike protein) being measured, a chemiluminescent detection agent and a triggering agent (e.g., chemical or electrochemical). The reaction sequence for performing CMIA can include mixing the microparticles coated with a capture agent specific for the analyte with a sample in a reaction vessel to form an immune complex; washing the captured immune complex to remove unbound material; mixing the captured immune complex with a chemiluminescent detection agent; washing the captured immune complex- chemiluminescent detection agent; and mixing the captured immune complex- chemiluminescent detection agent with a triggering agent to initiate light emission.
[0283] Chemiluminophores useful in CMIA include acridinium (e.g. acridinium-9- carboxamide), luminol, dioxetane, ruthenium complexes and similar chemiluminescent derivatives. Microparticles useful in CMIA include diamagnetic, magnetic and paramagnetic microparticles. Examples of commercially available automated instruments with which chemiluminescent microparticle assay assays can be conducted include: Architect i-Systems and the Abbott Prism (all available from Abbott Laboratories, Abbott Park, Hl.).
[0284] Electrochemical Detection Systems
[0285] Electrochemical detection systems may be used in the methods of detection and diagnosis described herein. Basic procedures for electrochemical detection are known in the art. An illustrative example includes immobilization of a primary binding molecule (e.g. an antibody or antigen-binding fragment), followed by exposure to a sequence of solutions or samples containing the antigen (e.g. a SARS-CoV-2 spike protein), followed by exposure to a secondary antibody conjugated to an enzyme label (AP-Ab, rat anti mouse IgG and alkaline phosphatase), and p-aminophenyl phosphate (PAPP). The AP converts PAPP to p-aminophenol (PAPR, the “R” is intended to distinguish the reduced form from the oxidized form, PAPO, the quinoneimine), which is electrochemically reversible at potentials that do not interfere with reduction of oxygen and water at pH 9.0, where AP exhibits optimum activity. PAPR does not cause electrode fouling, unlike phenol whose precursor, phenylphosphate, is often used as the enzyme substrate. Picomole detection limits for PAPR and femtogram detection limits for IgG achieved in microelectrochemical immunoassays using PAPP volumes ranging from 20 pL to 360 pL have been reported previously. In capillary immunoassays with electrochemical detection, the lowest detection limit reported thus far is 3000 molecules of mouse IgG using a volume of 70 pL and a 30 min or 25 min assay time.
[0286] Biosensors
[0287] Biosensor detection systems may be used in the methods of detection and diagnosis described herein. Biosensors such as optical biosensors, electronic biosensor, piezoelectric biosensor, gravimetric biosensors, pyroelectric biosensors, thermal, and/or magnetic biosensors may be used in conjunction with an antibody or antigen-binding fragments described herein. Biosensors can be detection reagents conjugated to an antibody or antigen-binding fragments described herein, such that contact with a SARS-CoV-2 antigen results in the change of a detectable signal. Such detection reagents can include, without limitation, nanoparticles (e.g. dielectic, gold, silver, iron, polymeric, or semiconductor nanoparticles), nanostructures, nanowires, films, hydrogels, solid-phase surfaces, films, quantum dots, coatings (e.g. SiCh, TiCh, AI2O3, SisNf), fluorophores, and/or lanthanides. [0288] Immunoassays
[0289] Immunoassay detection systems may be used in the methods of detection and diagnosis described herein. A multitude of immunoassay platforms are known in the art. An immunoassay generally refers to any detection method that measures an antigen (e.g. a SARS-CoV-2 spike protein), wherein the antigen is bound specifically by an immune- molecule (e.g. an antibody or antigen-binding fragment described herein). Conventional immunoassays include, without limitation, an ELISA, an RIA, flow cytometry, FACS, tissue immunohistochemistry, Western blot or immunoprecipitation. Assays may be performed in solution or may use a solid (insoluble) support (e.g., polystyrene, nitrocellulose, or beads), using any standard methods (e.g., as described in Current Protocols in Immunology, Coligan et al., ed.; John Wiley & Sons, New York, 1992). Immunoassays usually are classified in one of several ways. For example, immunoassays can be classified according to the mode of detection used, i.e., enzyme immunoassays, radio immunoassays, fluorescence polarization immunoassays, chemiluminescence immunoassays, turbidimetric assays, etc. Another grouping method is according to the assay procedure used, i.e., competitive assay formats, sandwich-type assay formats as well as assays based on precipitation or agglutination principles. In certain instances, a further distinction is made depending on whether washing steps are included in the procedure (so- called heterogeneous assays) or whether reaction and detection are performed without a washing step (so-called homogeneous assays).
[0290] Lateral Flow Devices
[0291] Lateral flow devices may be used in the methods of detection and diagnosis described herein. Lateral flow devices flow devices are known in the art for use in detection of antigens (Lateral Flow Immunoassay, edited by Raphael Wong, Harley Tse, 2009, Springer; Paper-based diagnostics: Current Status and Future applications, Kevin J. Land, Springer 2019). A non-limiting illustrative workflow for a lateral flow assay may include a membrane such as a nitrocellulose membrane which comprises an antibody or antigen-binding fragment, which further comprises a detectable label. If the membrane is contacted with a sample, an antigen (e.g. a SARS-CoV-2 spike protein) to be detected will bind to the antibody or antigen-binding fragment. The resulting complex will move driven by capillary forces on the membrane and will be immobilized on a test line on the membrane comprising a means for detecting the antibody or antigen-binding fragment or the antigen; typically a secondary antibody binding to the immunoglobulin class or classes of the antibody or the antibodies to be detected such as IgG and/or IgA and/or IgM. Preferably nanoparticles or beads are used as labels, for example gold nanoparticles or latex beads.
[0292] Test Kits
[0293] The disclosure also provides test kits. Test kits according to the disclosure include one or more reagents useful for practicing one or more immunoassays according to the disclosure. A test kit generally includes a package with one or more containers holding the reagents, as one or more separate compositions or, optionally, as admixture where the compatibility of the reagents will allow. The test kit can also include other material(s) that may be desirable from a user standpoint, such as a buffer(s), a diluent(s), a standard(s), and/or any other material useful in sample processing, washing, or conducting any other step of the assay.
[0294] In one embodiment, a test kit includes: (a) a labeled detection agent specific for an antigen (e.g. an antibody or antigen-binding fragment described herein that binds a SARS- CoV-2 spike protein); and (b) a labeled species-specific antibody, wherein the labeled species-specific antibody is specific for the species from which the biological sample was obtained. In particular embodiments, the labeled detection agent includes a labeled antianalyte antibody. The labeled anti-analyte antibody can be labeled with the same label as the labeled species-specific antibody or can be labeled with a different label. The labeled anti-analyte antibody and the labeled species-specific antibody can be packaged in the same container or in different containers.
[0295] Test kits according to the disclosure can also include at least one indirect label. If the label employed generally requires an indicator reagent to produce a detectable signal, the test kit preferably includes one or more suitable indicator reagents.
[0296] Test kits according to the disclosure can additionally include a solid phase and a capture agent, such as an anti-analyte capture antibody, affixed to the solid phase. In exemplary embodiments, the solid phase includes one or more microparticles (e.g., magnetic or paramagnetic microparticles), electrodes, and/or a microplate. Test kits designed for multiplex assays conveniently contain one or more solid phases including a plurality of anti-analyte capture antibodies that are specific for a plurality of different analytes. Thus, for example, a test kit designed for multiplex electrochemical immunoassays can contain a solid phase including a plurality of electrodes, with each electrode bearing a different anti-analyte capture antibody. Alternatively, a test kit intended for multiplex “sandwich” immunoassays can include a plurality of different labeled anti-analyte antibodies that are specific for the plurality of different analytes, wherein each different labeled anti-analyte antibody is labeled with a distinct label.
[0297] Test kits according to the disclosure preferably include instructions for carrying out one or more of the methods of detection provided in the disclosure. Instructions included in kits of the disclosure can be affixed to packaging material or can be included as a package insert. While the instructions are typically written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term “instructions” can include the address of an internet site that provides the instructions.
Definitions
[0298] Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
[0299] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:
[0300] As used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural references unless the content clearly dictates otherwise.
[0301] As used in this specification, the term “and/or” is used in this disclosure to mean either “and” or “or” unless indicated otherwise.
[0302] Throughout this specification, unless the context requires otherwise, the words “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
[0303] As used in this application, the terms “about” and “approximately” are used as equivalents. Any numerals used in this application with or without about/approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
[0304] As used herein, the term “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab’ and F(ab')2 fragments, and an Fab expression library. By “specifically bind” or “immunoreacts with” is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react (i.e., bind) with other polypeptides or binds at much lower affinity (Kd > 10-6) with other polypeptides.
[0305] An “isolated” antibody or antigen-binding fragment as described herein refers to an antibody or antigen-binding fragment that is substantially pure. An antibody or antigenbinding fragment that results following the first steps of production may exist in a crude solution that contains the antibody or antigen-binding fragment comprises and additional contaminants, such as other nucleic acids, polypeptides, cell debris, media components, or other reagents used during production. An isolated antibody or antigen-binding fragment has been substantially isolated from the contaminants through any purification process known in the art.
[0306] The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ea., 2nd ed. Raven Press, N.Y. (1989)). The variable regions of each light/heavy chain pair form the antibody binding site.
[0307] The term “monoclonal antibody” (MAb) or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs contain an antigen-binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.
[0308] As used herein, "isotype" refers to the antibody class (e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE antibody) that is encoded by the heavy chain constant domain genes. The full-length amino acid sequence of each wild type human IgG constant region (including all domains, i.e., CHI domain, hinge, CH2 domain, and CH3 domain) is cataloged in the UniProt database available on-line, e.g., as P01857 (IgGl), P01859 (IgG2), P01860 (IgG3), and P01861 (IgG4), or different allotypes thereof (SEQ ID NOs: 1, 6, 11, and 16, respectively). As used herein, a domain of a heavy chain constant region, e.g., the hinge, is of an "IgGl isotype," "IgG2 isotype," "IgG3 isotype," or "IgG4 isotype," if the domain comprises the amino acid sequence of the corresponding domain of the respective isotype, or a variant thereof (that has a higher homology to the corresponding domain of the respective isotype than it does to that of the other isotypes). In general, antibody molecules obtained from humans relate to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgGl, IgG2, and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.
[0309] As used herein, the term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin, a scFv, or a T-cell receptor. The term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin or T- cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. An antibody is said to specifically bind an antigen when the dissociation constant is < 1 pM; preferably < 100 nM and most preferably < 10 nM.
[0310] As used herein, the terms “immunological binding” and “immunological binding properties” and “specific binding” and “binding” and “selective binding” refer to the non- covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (KD) of the interaction, wherein a smaller KD represents a greater affinity. Immunological binding properties of selected polypeptides are quantified using methods well known in the art. One such method entails measuring the rates of antigen-binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions. Thus, both the “on rate constant” (Kon) and the “off rate constant” (KOff) can be determined by calculation of the concentrations and the actual rates of association and dissociation. (See Nature 361 : 186-87 (1993)). The ratio of KOff /Kon enables the cancellation of all parameters not related to affinity, and is equal to the dissociation constant KD. (See, generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). An antibody of the present disclosure is said to specifically bind to a SARS- CoV-2 spike protein epitope when the equilibrium binding constant (KD) is about 1 pM, preferably about 100 nM, more preferably about 10 nM, and most preferably about 100 pM to about 1 pM, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art. [0311] Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide- containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur- containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine valine, glutamic- aspartic, and asparagine-glutamine.
[0312] As discussed herein, minor variations in the amino acid sequences of antibodies or immunoglobulin molecules are contemplated as being encompassed by the present disclosure, providing that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99%. In particular, conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into families: (1) acidic amino acids are aspartate, glutamate; (2) basic amino acids are lysine, arginine, histidine; (3) nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. The hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine. The hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine. Other families of amino acids include (i) serine and threonine, which are the aliphatic-hydroxy family; (ii) asparagine and glutamine, which are the amide containing family; (iii) alanine, valine, leucine and isoleucine, which are the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine, which are the aromatic family.
[0313] A “humanized antibody” is a genetically engineered antibody in which CDRs are grafted into human "acceptor" antibody sequences (see, e.g., Queen, US 5,530,101 and 5,585,089; Winter, US 5,225,539; Carter, US 6,407,213; Adair, US 5,859,205; and Foote, US 6,881,557). The acceptor antibody sequences can be, for example, a mature human antibody sequence, a composite of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence. An example acceptor sequence for the heavy chain is the germline VH exon VH1-2 (also referred to in the literature as HV1-2) (Shin et al, 1991, EMBO J. 10:3641-3645) and for the hinge region (JH), exon JH-6 (Mattila et al, 1995, Eur. J. Immunol. 25:2578-2582). For the light chain, an example acceptor sequence is exon VK2-30 (also referred to in the literature as KV2-30) and for the hinge region exon JK-4 (Hieter et al, 1982, J. Biol. Chem. 257: 1516-1522). Thus, a humanized antibody is an antibody having some or all CDRs entirely or substantially from any source, and variable region framework sequences and constant regions, if present, entirely or substantially from human antibody sequences. Similarly a humanized heavy chain has at least one, two and usually all three CDRs entirely or substantially originating in a source separate from a heavy chain variable region framework sequence and heavy chain constant region, if present, substantially from human heavy chain variable region framework and constant region sequences. Similarly a humanized light chain has at least one, two and usually all three CDRs entirely or substantially originating in a source separate from a light chain variable region framework sequence and light chain constant region, if present, substantially from human light chain variable region framework and constant region sequences. Other than nanobodies and dAbs, a humanized antibody comprises a humanized heavy chain and a humanized light chain. A CDR in a humanized antibody is substantially from a corresponding CDR in a non-human antibody when at least 60%, 85%, 90%, 95% or 100% of corresponding residues (as defined by Kabat) are identical between the respective CDRs. The variable region framework sequences of an antibody chain or the constant region of an antibody chain are substantially from a human variable region framework sequence or human constant region respectively when at least 85%, 90%, 95% or 100% of corresponding residues defined by Kabat are identical.
[0314] The term “operably linked” as used herein refers to the arrangement of various nucleic acid molecule elements relative to each such that the elements are functionally connected and are able to interact with each other. Such elements may include, without limitation, a promoter, an enhancer and/or a regulatory element, a polyadenylation sequence, one or more introns and/or exons, and a coding sequence of a polynucleotide to be expressed (e.g. a polynucleotide encoding an antibody or antigen-binding fragment described herein). The nucleic acid sequence elements, when properly oriented or operably linked, act together to modulate the activity of one another, and ultimately may affect the level of expression of the polynucleotide. By modulate is meant increasing, decreasing, or maintaining the level of activity of a particular element. The position of each element relative to other elements may be expressed in terms of the 5' terminus and the 3' terminus of each element, and the distance between any particular elements may be referenced by the number of intervening nucleotides, or base pairs, between the elements.
[0315] As used in the application, the term “enhancer” refers to nucleic acid sequences that regulate, either directly or indirectly, the transcription of corresponding nucleic acid coding sequences to which they are operably linked (e.g., a transgene). An enhancer may function alone to regulate transcription or may act in concert with one or more other regulatory sequences (e.g., promoters, minimal promoters, or silencers). In the context of the application, an enhancer is typically operably linked to a polynucleotide encoding an antibody or antigen-binding fragment described herein. An “enhancer” as described herein may, in some embodiments, function as a promoter in the absence of a promoter sequence other than the enhancer itself
[0316] The term “agent” is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.
[0317] The term patient includes human and veterinary subjects.
[0318] The disclosure also includes Fv, Fab, Fab’ and F(ab')2 anti-SARS-CoV-2 spike protein antibody fragments, single chain anti-SARS-CoV-2 spike protein antibodies, bispecific anti-SARS-CoV-2 spike protein antibodies, heteroconjugate anti-SARS-CoV-2 spike protein antibodies, trispecific antibodies, immunoconjugates and fragments thereof. [0319] Bispecific antibodies are antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for SARS-CoV-2 spike protein. The second binding target is any other antigen, and advantageously is a cellsurface protein or receptor or receptor subunit.
[0320] All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an admission that any is pertinent prior art, nor does it constitute any admission as to the contents or date of the same. The disclosure having now been described by way of written description, those of skill in the art will recognize that the disclosure can be practiced in a variety of embodiments and that the foregoing description and examples below are for purposes of illustration and not limitation of the claims that follow.
EXAMPLES
Example 1: Generation of human antibodies against SARS-CoV-2
[0321] Blood was collected from 10 hospitalized and recovered Covid- 19 patients with high IgG titers against the SARS-CoV-2 spike protein from June to July 2020 with approval by an Institutional Review Board. The blood samples of approximately 5 ml were allowed to clot at room temperature for 15 minutes, then centrifuged at 2000g for 10 minutes to isolate the serum.
[0322] The serum was aliquoted and subsequently used for measuring the level of neutralizing antibodies against prototype SARS-CoV-2 by plaque-reduction neutralization test (PRNT). PBMCs from the four patients with the highest PRNT activities were isolated by the Ficoll™ density gradient centrifugation technique. B cells were isolated from PBMCs by EasySep™ human B cell isolation kit (STEMCELL Technologies) following the manufacturer’s instructions. After that, isolated B cells were transformed by Epstein- Barr virus (EBV) obtained from the B95-8 lymphoblastoid cell line supernatant. The EBV- transformed B cells were cultured in 384-well plates at 37°C for seven days. Later on, cells in each well were transferred to 96-well plates containing irradiated heterologous human PBMC feeder cells. After incubation for three days, the supernatant from each well was screened based on binding and neutralizing activities against prototype SARS-CoV-2 by ELISA, as mentioned in Example 2 and 3 below.
[0323] Next, EBV-transformed B cells with the top-rank neutralizing activities were fused with SHM-33 myeloma cells using the cell electrofusion technique to create stable human hybridoma clones. Briefly, individual selected EBV-transformed B cell pools were mixed with myeloma cells followed by washing with BTX cytofusion medium. The cell mixture was transferred to a cytofusion cuvette, and electrofusion was performed using BTX ECM 2001 fusion system. After fusion, hybridoma cells were cultured in 96-well plates for ten days in a ClonaCell-HY Medium E (STEMCELL Technologies) containing hypoxanthine- aminopterin-thymidine (HAT). The supernatant from each well was screened based on binding and neutralizing activities against prototype SARS-CoV-2 by ELISA, as mentioned in Example 2 and 3 below. [0324] Hybridoma cells with high neutralizing activities were subcloned by a limiting dilution technique to achieve hybridoma monocl onality. After 7-10 days of culturing, hybridoma clones were screened by ELISA. Finally, selected human hybridoma clones with high neutralizing activities were expanded to produce SARS-CoV-2 neutralizing antibodies.
[0325] Example 2: Screening of human antibodies against SARS-CoV-2 by binding activity
[0326] ELISA technique was utilized to screen for high binding anti-SARS-CoV-2 antibodies. Each well of the MaxiSorp 96-well ELISA plate (ThermoFisher Scientific) was coated with 10 ng of prototype SARS-CoV-2 RBD-His proteins (Sino Biological) at 4°C overnight. The coated ELISA plate was washed and blocked with 0.05% Tween-20 in PBS buffer (PBST). Next, 100 pl culture supernatant samples from EBV-transformed B cells or human hybridoma cells were added to the coated wells. The plate was incubated for 1 hour at 37°C and washed three times with PBST. A secondary antibody probing step was performed by adding 100 pl/well of goat anti -human IgG (Fey fragment specific)-HRP (Jackson ImmunoResearch), incubating for 1 hour at 37°C, and washing with PBST 3 times. Next, the plate was added with o-Phenylenediamine dihydrochloride (OPD) substrate solution (100 pl/well) and incubated in the dark for 20 minutes at room temperature. The reaction was stopped by adding 2N H2SO4 solution (50 pl/well). Finally, the absorbance was measured at 492 nm by Cytation 5 (BioTek)
[0327] Example 3: Screening of human antibodies against SARS-CoV-2 by neutralizing activity
[0328] ELISA technique was employed to evaluate the RBD/ACE2 neutralizing activity from EBV-transformed B cells and human hybridoma cells that produced RBD-specific antibodies (positive binding clones). Firstly, an ELISA plate was coated with recombinant ACE2 human Fc tag proteins (Genscript) at 4°C overnight. The coated ELISA plate was washed and blocked with PBST. Next, 50 pl culture supernatant samples from the positive binding clones or a control antibody were pre-incubated with 50 pl of RBD mouse Fc tag proteins (Genscript) at 37°C for 30 minutes and individual samples were transferred to the coated plate. After incubation at 37°C for 1 hour, the plate was washed by PBST three times. Goat anti-mouse Ig /-chain specific)-HRP (Jackson ImmunoResearch) was added to the reaction, then incubated at 37°C for 1 hour to detect RBD mouse Fc tag proteins. The plate was washed three times by PBST before adding 100 pl of OPD and then incubated in the dark at room temperature for 20 minutes. The stop solution was added to each well, and the absorbance at 492 nm was determined by Cytation 5 (BioTek).
[0329] Example 4: Sequencing of human antibody variable regions
[0330] Sequencing was performed by whole transcriptome shotgun sequencing technique (RNA-Seq). In brief, total RNA was extracted from Human hybridoma clones producing neutralizing antibodies against SARS-CoV-2. A barcoded cDNA library was generated through RT-PCR using a random hexamer. Next-generation sequencing was performed on an Illumina HiSeq sequencer. Contigs were assembled, and data was mined for all viable antibody sequences (i.e., those not containing stop codons). Sequence analysis was performed separately to identify variable heavy and variable light domains. The complementarity determining regions (CDRs) were identified using the Kabat definition.
[0331] Example 5: Binding profile of human monoclonal antibodies against SARS- CoV-2
[0332] The Expi293 transient expression system (Gibco™) was used to produce recombinant human monoclonal antibodies against SARS-CoV-2. The binding profile of purified antibodies disclosed herein was assessed by ELISA, as mentioned in Example 2 with the following modification. Instead of using the culture supernatant, purified antibodies at 2 pg/ml were used as starting samples, then 11 rounds of 2-fold dilution were performed for each sample until the final concentration of 0.977 ng/mL was reached.
[0333] ELISA binding data of the recombinant human monoclonal antibodies against RBD of SARS-CoV-2 spike protein are illustrated in FIG. 1. The EC50 values of these antibodies are reported in Table 5.1.
[0334] Table 5.1. Half-maximal effective concentration (EC50) values of human monoclonal antibodies against SARS-CoV-2
Figure imgf000077_0001
Figure imgf000078_0001
[0335] Example 6: Binding kinetics of human monoclonal antibodies against SARS- CoV-2
[0336] To determine the binding kinetics of human monoclonal antibodies disclosed herein and RBD of prototype SARS-CoV-2 spike protein, the surface plasmon resonance (SPR) technique was performed by Biacore T200 equipped with a protein G sensor chip (GE Healthcare). For the antibody-capturing step, each purified human anti-SARS-CoV- 2 monoclonal antibody (at 1 pg/ml prepared in the HBS-EP buffer) was injected into an individual flow cell in the sensor chip. Single-cycle kinetics at 25°C was determined by sequentially injecting recombinant RBD-His tag proteins at different concentrations. An HBS-EP buffer blank was also included as a negative control for baseline subtraction. The antibody binding affinity (KD) value was calculated using the Biacore T200 Evaluation Software v3.1.
[0337] Binding kinetic values for different SARS-CoV-2 monoclonal antibodies are shown in Table 6.1. All antibodies exhibited KD values in the subnanomolar range. The 3D6 antibody showed the highest binding affinity, with a KD value of 4.01 x lO'11 M, followed by 1A5, 2E10, 3H8, 1D1, 2B4, 3D2, and 1C10, respectively.
[0338] Table 6.1. Binding kinetics of human monoclonal antibodies disclosed herein and RBD of prototype SARS-CoV-2 spike protein at 25°C.
Figure imgf000078_0002
[0339] Example 7: SARS-CoV-2 surrogate virus neutralization test of human monoclonal antibodies
[0340] The SARS-CoV-2 surrogate virus neutralization test (ePass, GenScript) was used to assess the neutralizing activity of antibodies disclosed herein. Briefly, the individual human anti-SARS-CoV-2 monoclonal antibody was mixed with prototype RBD-HRP tag proteins and incubated at 37°C for 30 minutes. Then, the mixture was added to each well of the human ACE2-coated plate. After incubation at 37°C for 15 minutes, the plate was washed 4 times with a washing solution. In consequence, the free and non-neutralizing antibody-bound HRP-RBD were captured on the plate. Next, the plate was added with 100 pl TMB substrate solution to each well and incubated in the dark for 20 minutes. Finally, a stop solution was subsequently added to each well. The absorbance of the final solution was determined at 450 nm immediately.
[0341] The ePass results and the corresponding IC50 values are presented in FIG. 2 and Table 7.1. The 2E10 antibody exhibited the highest neutralizing profile (IC50 at 16.86 ng/ml).
[0342] Table 7.1. Half-maximal inhibitory concentration (IC50) values of human monoclonal antibodies against prototype SARS-CoV-2 using ePass neutralization antibody detection kit
Figure imgf000079_0001
[0343] Example 8: Epitope binning assay of human monoclonal antibodies against SARS-CoV-2
[0344] Biolayer interferometry (BLI) technique was used to cluster recombinant human monoclonal antibodies disclosed herein based on binding epitopes on RBD of prototype SARS-CoV-2 spike protein. The experiment was performed using ForteBio Octet HTX in an in-tandem format, i.e., a biosensor was immobilized with a prototype SARS-CoV-2 RBD-His protein and then presented with a saturating monoclonal antibody (1st mAb) followed by a competing monoclonal antibody (2nd mAb) in a pairwise combinatorial manner. The assays were performed at 32°C with a shaking speed of 1,000 rpm. Briefly, a prototype SARS-CoV-2 RBD-His protein (3 pg/ml solution) was allowed to be captured onto an anti-His (AHC) biosensor tip for 300 seconds. The first and second anti-SARS- CoV-2 monoclonal antibodies (10 and 5 pg/ml, respectively) were sequentially presented to the biosensor tip for 300 seconds. The binding response was evaluated at each step after subtracting the self-blocking background signal. The data analysis was performed with Octet Data Analysis HT 10.0 software using the Epitope Binning mode.
[0345] Epitope binning data of human monoclonal antibodies disclosed herein are presented in Table 8.1. The results demonstrated that several pairs of non-competing monoclonal antibodies could potentially be combined to form antibody cocktails, e.g., 3D2 and IDE
[0346] Table 8.1. Epitope binning data of human monoclonal antibodies disclosed herein against RBD of prototype SARS-CoV-2 spike protein
Figure imgf000080_0001
Figure imgf000081_0001
[0347] Grey = bi-directional competition
[0348] Light grey = no competition
[0349] White = self-competition
[0350] Example 9: SARS-CoV-2 pseudovirus neutralization assay of human monoclonal antibodies
[0351] Pseudovirus Neutralization Assay kit (Luc reporter, GenScript) was used to evaluate the potency of human anti-SARS-CoV-2 monoclonal antibodies disclosed herein for neutralizing infectivity of SARS-CoV-2 (prototype, alpha (B.l.1.7), beta (B.1.351), gamma (P. l), and delta (B.1.617.2) variants). In brief, the monoclonal antibodies were diluted in 4-fold serial dilution with Opti-MEM, and 25 pl of the solution was transferred to an assay plate. Then, 25 pl of pseudovirus with luciferase reporter in Opti-MEM was added to wells and incubated at room temperature for 1 hour. Next, 50 pl of Opti- HEK293/ACE2 single-cell suspension at 600,000 cells/ml was added into each well. After 24 hours of incubation at 37°C, 50 pl of fresh media was added to the assay plate and incubated at 37°C for 24 hours. The culture supernatant was then carefully discarded using a pipette. Finally, luciferase reagent (50 pl) was added to the assay plate and subsequently incubated for 5 minutes at room temperature, and the bioluminescence signal was measured.
[0352] The neutralization profiles of each human anti-SARS-CoV-2 monoclonal antibody are depicted in Figures 3-7 (prototype, alpha (B. l.1.7), beta (B.1.351), gamma (P.l), and delta (B.1.617.2) variants, respectively). The neutralization potency is represented as IC50 in Table 9.1. The 1D1 + 3D2 cocktail as well as 3D2 and 1D1 antibodies demonstrated IC50 of <150 ng/mL for all SARS-CoV-2 variants.
[0353] Table 9.1. SARS-CoV-2 pseudovirus neutralization test
Figure imgf000081_0002
Figure imgf000082_0001
[0354] The details of one or more embodiments of the disclosure are set forth in the accompanying description above. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, other features, objects, and advantages of the disclosure will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, 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 disclosure belongs. All patents and publications cited in this specification are incorporated by reference. [0355] The foregoing description has been presented only for the purposes of illustration and is not intended to limit the disclosure to the precise form disclosed, but by the claims appended hereto.

Claims

82
We Claim:
1. An antibody or antigen-binding fragment that binds to a SARS-CoV-2 spike protein.
2. The antibody or antigen-binding fragment of claim 1, wherein the SARS-CoV- 2 spike protein comprises any one of SEQ ID NOs: 129-136, or a sequence that shares at least 90%, at least 95%, or least 98% identity thereto.
3. The antibody or antigen-binding fragment of claim 1 or claim 2, wherein the antibody or antigen-binding fragment binds the SARS-CoV-2 spike protein with a KD value of about 10'12M, about 10'u M, about 10'10M, about 10'9 M, or about 10'8M.
4. The antibody or antigen-binding fragment of any one of claims 1-3, wherein the antibody or antigen-binding fragment inhibits the binding of the SARS-CoV-2 spike protein to an ACE2 receptor with an IC50 of about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 45 ng/mL, about 50 ng/mL, about 55 ng/mL, about 60 ng/mL, about 65 ng/mL, about 70 ng/mL, about 75 ng/mL, or about 80 ng/mL.
5. The antibody or antigen-binding fragment of any one of claims 1-4, comprising a heavy-chain complementarity-determining region 1 (HCDR1), a heavy-chain complementarity-determining region 2 (HCDR2), a heavy-chain complementaritydetermining region 3 (HCDR3), a light-chain complementarity determining region 1 (LCDR1), a light-chain complementarity determining region 2 (LCDR2), and a lightchain complementarity determining region 3 (LCDR3).
6. The antibody or antigen-binding fragment of claim 5, comprising: a. any one of the HCDR1 sequences set forth in Table 2; b. any one of the HCDR2 sequences set forth in Table 2; c. any one of the HCDR3 sequences set forth in Table 2; d. any one of the LCDR1 sequences set forth in Table 2; e. any one of the LCDR2 sequences set forth in Table 2; and f. any one of the LCDR3 sequences set forth in Table 2 wherein no more than 1, 2, or 3, amino acids in the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and/or LCDR3 sequences are substituted, deleted or inserted.
7. The antibody or antigen-binding fragment of claim 6, wherein a. the HCDR1 comprises SEQ ID NO: 6; 83 b. the HCDR2 comprises SEQ ID NO: 7; and c. the HCDR3 comprises SEQ ID NO: 8. antibody or anti gen -binding fragment of claim 6, wherein a. the HCDR1 comprises SEQ ID NO: 14; b. the HCDR2 comprises SEQ ID NO: 15; and c. the HCDR3 comprises SEQ ID NO: 16. antibody or antigen-binding fragment of claim 6, wherein a. the HCDR1 comprises SEQ ID NO: 22; b. the HCDR2 comprises SEQ ID NO: 23; and c. the HCDR3 comprises SEQ ID NO: 24. antibody or antigen-binding fragment of claim 6, wherein a. the HCDR1 comprises SEQ ID NO: 30; b. the HCDR2 comprises SEQ ID NO: 31; and c. the HCDR3 comprises SEQ ID NO: 32. antibody or antigen-binding fragment of claim 6, wherein a. the HCDR1 comprises SEQ ID NO: 38; b. the HCDR2 comprises SEQ ID NO: 39; and c. the HCDR3 comprises SEQ ID NO: 40. antibody or antigen-binding fragment of claim 6, wherein a. the HCDR1 comprises SEQ ID NO: 46; b. the HCDR2 comprises SEQ ID NO: 47; and c. the HCDR3 comprises SEQ ID NO: 48. antibody or anti gen -binding fragment of claim 6, wherein a. HCDR1 comprises SEQ ID NO: 54; b. HCDR2 comprises SEQ ID NO: 55; and c. HCDR3 comprises SEQ ID NO: 56. antibody or antigen-binding fragment of claim 6, wherein a. HCDR1 comprises SEQ ID NO: 62; b. HCDR2 comprises SEQ ID NO: 63; and c. HCDR3 comprises SEQ ID NO: 64 antibody or anti gen -binding fragment of any one of claims 1-14, wherein a. the LCDR1 comprises SEQ ID NO: 2; b. the LCDR2 comprises SEQ ID NO: 3; and 84 c. the LCDR3 comprises SEQ ID NO: 4.
16. The antibody or antigen-binding fragment of any one of claims 1-14, wherein a. the LCDR1 comprises SEQ ID NO: 10; b. the LCDR2 comprises SEQ ID NO: 11; and c. the LCDR3 comprises SEQ ID NO: 12.
17. The antibody or antigen-binding fragment of any one of claims 1-14, wherein a. the LCDR1 comprises SEQ ID NO: 18; b. the LCDR2 comprises SEQ ID NO: 19; and c. the LCDR3 comprises SEQ ID NO: 20.
18. The antibody or anti gen -binding fragment of any one of claims 1-14, wherein a. the LCDR1 comprises SEQ ID NO: 26; b. the LCDR2 comprises SEQ ID NO: 27; and c. the LCDR3 comprises SEQ ID NO: 28.
19. The antibody or antigen-binding fragment of any one of claims 1-14, wherein a. the LCDR1 comprises SEQ ID NO: 34; b. the LCDR2 comprises SEQ ID NO: 35; and c. the LCDR3 comprises SEQ ID NO: 36.
20. The antibody or antigen-binding fragment of any one of claims 1-14, wherein a. the LCDR1 comprises SEQ ID NO: 42; b. the LCDR2 comprises SEQ ID NO: 43; and c. the LCDR3 comprises SEQ ID NO: 44.
21. The antibody or antigen-binding fragment of any one of claims 1-14, wherein a. LCDR1 comprises SEQ ID NO: 50; b. LCDR2 comprises SEQ ID NO: 51; and c. LCDR3 comprises SEQ ID NO: 52.
22. The antibody or antigen-binding fragment of any one of claims 1-14, wherein a. LCDR1 comprises SEQ ID NO: 58; b. LCDR2 comprises SEQ ID NO: 59; and c. LCDR3 comprises SEQ ID NO: 60.
23. The antibody or antigen-binding fragment of any one of claims 1-4, comprising a. a heavy-chain variable region (VH) comprising a sequence set forth in Table 2, or a sequence at least that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto; and 85 b. a light-chain variable region (VL) comprising a sequence set forth in Table 2, or a sequence at least that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto.
24. The antibody or antigen-binding fragment of claim 23, wherein a. the VH comprises SEQ ID NO: 5, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b. the VL comprises SEQ ID NO: 1, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
25. The antibody or anti gen -binding fragment of claim 23, wherein a. the VH comprises SEQ ID NO: 13, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b. the VL comprises SEQ ID NO: 9, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
26. The antibody or antigen-binding fragment of claim 23, wherein a. the VH comprises SEQ ID NO: 21, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b. the VL comprises SEQ ID NO: 17, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
27. The antibody or antigen-binding fragment of claim 23, wherein a. the VH comprises SEQ ID NO: 29, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b. the VL comprises SEQ ID NO: 25, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
28. The antibody or anti gen -binding fragment of claim 23, wherein a. the VH comprises SEQ ID NO: 37, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b. the VL comprises SEQ ID NO: 33, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto. 86
29. The antibody or antigen-binding fragment of claim 23, wherein a. the VH comprises SEQ ID NO: 45, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b. the VL comprises SEQ ID NO: 41, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
30. The antibody or antigen-binding fragment of claim 23, wherein a. the VH comprises SEQ ID NO: 53, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b. the VL comprises SEQ ID NO: 49, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
31. The antibody or anti gen -binding fragment of claim 23, wherein a. the VH comprises SEQ ID NO: 61, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b. the VL comprises SEQ ID NO: 57, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
32. The antibody or antigen-binding fragment of any one of the preceding claims, further comprising a constant region.
33. The antibody or antigen-binding fragment of claim 32, wherein the constant region is selected from an IgA, IgM, IgGl, IgG2, IgG3, or IgG4 constant region.
34. The antibody or antigen-binding fragment of any one of the preceding claims, wherein the antibody is humanized.
35. The antibody or anti gen -binding fragment of any one of the preceding claims, wherein the antibody or antigen-binding fragment is recombinant.
36. The antibody or antigen-binding fragment of any one of the preceding claims, wherein the antibody or antigen-binding fragment is multispecific.
37. The antibody or antigen-binding fragment of any one of the preceding claims, wherein the antibody or antigen-binding fragment neutralizes a SARS-CoV-2 virus or a variant thereof. 87
38. A pharmaceutical composition, comprising the antibody or antigen-binding fragment of any one of the preceding claims and a pharmaceutically acceptable carrier, excipient, and/or diluent.
39. The pharmaceutical composition of claim 38, further comprising one or more additional therapeutic agents.
40. The pharmaceutical composition of claim 39, wherein the one or more additional therapeutic agents is selected from an antibody, an antiviral, a steroid, an antiinflammatory, an immune booster, vitamin C, vitamin D, vitamin E, or any combination thereof.
41. The pharmaceutical composition of any one of claims 38-40, wherein the pharmaceutical composition is formulated for parenteral administration, enteral administration, mucosal administration, nasal administration, oral administration, or intra-articular administration.
42. The pharmaceutical composition of claim 41, wherein the administration comprises inhalation, intravenous injection, infusion, subcutaneous injection, or intramuscular injection.
43. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1-37, for use in the treatment of COVID-19.
44. A kit comprising the antibody or antigen-binding fragment of any one of claims 1-37 or the pharmaceutical composition of any one of claims 38-43 and instructions for use.
45. A nucleic acid comprising a polynucleotide sequence encoding the antibody or antigen-binding fragment of any one of claims 1-37.
46. A nucleic acid comprising: a. any one of the sequences encoding a heavy-chain complementaritydetermining region 1 (HCDR1) set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; b. any one of the sequences encoding a heavy-chain complementaritydetermining region 2 (HCDR2) set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; 88 c. any one of the sequences encoding a heavy-chain complementaritydetermining region 3 (HCDR3) set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; d. any one of the sequences encoding a light-chain complementarity determining region 1 (LCDR1) set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; e. any one of the sequences encoding a light-chain complementarity determining region 2 (LCDR2) set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and f. any one of the sequences encoding a light-chain complementarity determining region 3 (LCDR3) set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto. nucleic acid of claim 46, wherein a. the sequence encoding the HCDR1 comprises SEQ ID NO: 70; b. the sequence encoding the HCDR2 comprises SEQ ID NO: 71; and c. the sequence encoding the HCDR3 comprises SEQ ID NO: 72. nucleic acid of claim 46, wherein a. the sequence encoding the HCDR1 comprises SEQ ID NO: 78; b. the sequence encoding the HCDR2 comprises SEQ ID NO: 79; and c. the sequence encoding the HCDR3 comprises SEQ ID NO: 80. nucleic acid of claim 46, wherein a. the sequence encoding the HCDR1 comprises SEQ ID NO: 86; b. the sequence encoding the HCDR2 comprises SEQ ID NO: 87; and c. the sequence encoding the HCDR3 comprises SEQ ID NO: 88. nucleic acid of claim 46, wherein a. the sequence encoding the HCDR1 comprises SEQ ID NO: 94; b. the sequence encoding the HCDR2 comprises SEQ ID NO: 95; and c. the sequence encoding the HCDR3 comprises SEQ ID NO: 96. nucleic acid of claim 46, wherein a. the sequence encoding the HCDR1 comprises SEQ ID NO: 102; b. the sequence encoding the HCDR2 comprises SEQ ID NO: 103; and c. the sequence encoding the HCDR3 comprises SEQ ID NO: 104.
52. The nucleic acid of claim 46, wherein a. the sequence encoding the HCDR1 comprises SEQ ID NO: 110; b. the sequence encoding the HCDR2 comprises SEQ ID NO: 111; and c. the sequence encoding the HCDR3 comprises SEQ ID NO: 112.
53. The nucleic acid of claim 46, wherein a. the sequence encoding the HCDR1 comprises SEQ ID NO: 118; b. the sequence encoding the HCDR2 comprises SEQ ID NO: 119; and c. the sequence encoding the HCDR3 comprises SEQ ID NO: 120.
54. The nucleic acid of claim 46, wherein a. the sequence encoding the HCDR1 comprises SEQ ID NO: 126; b. the sequence encoding the HCDR2 comprises SEQ ID NO: 127; and c. the sequence encoding the HCDR3 comprises SEQ ID NO: 128.
55. The nucleic acid of any one of claims 46-54, wherein a. the sequence encoding the LCDR1 comprises SEQ ID NO: 66; b. the sequence encoding the LCDR2 comprises SEQ ID NO: 67; and c. the sequence encoding the LCDR3 comprises SEQ ID NO: 68.
56. The nucleic acid of any one of claims 46-54, wherein a. the sequence encoding the LCDR1 comprises SEQ ID NO: 74; b. the sequence encoding the LCDR2 comprises SEQ ID NO: 75; and c. the sequence encoding the LCDR3 comprises SEQ ID NO: 76.
57. The nucleic acid of any one of claims 46-54, wherein a. the sequence encoding the LCDR1 comprises SEQ ID NO: 82; b. the sequence encoding the LCDR2 comprises SEQ ID NO: 83; and c. the sequence encoding the LCDR3 comprises SEQ ID NO: 84.
58. The nucleic acid of any one of claims 46-54, wherein a. the sequence encoding the LCDR1 comprises SEQ ID NO: 90; b. the sequence encoding the LCDR2 comprises SEQ ID NO: 91; and c. the sequence encoding the LCDR3 comprises SEQ ID NO: 92.
59. The nucleic acid of any one of claims 46-54, wherein a. the sequence encoding the LCDR1 comprises SEQ ID NO: 98; b. the sequence encoding the LCDR2 comprises SEQ ID NO: 99; and c. the sequence encoding the LCDR3 comprises SEQ ID NO: 100. nucleic acid of any one of claims 46-54, wherein a. the sequence encoding the LCDR1 comprises SEQ ID NO: 106; b. the sequence encoding the LCDR2 comprises SEQ ID NO: 107; and c. the sequence encoding the LCDR3 comprises SEQ ID NO: 108. nucleic acid of any one of claims 46-54, wherein a. the sequence encoding the LCDR1 comprises SEQ ID NO: 114; b. the sequence encoding the LCDR2 comprises SEQ ID NO: 115; and c. the sequence encoding the LCDR3 comprises SEQ ID NO: 116. nucleic acid of any one of claims 46-54, wherein a. the sequence encoding the LCDR1 comprises SEQ ID NO: 122; b. the sequence encoding the LCDR2 comprises SEQ ID NO: 123; and c. the sequence encoding the LCDR3 comprises SEQ ID NO: 124. nucleic acid of claim 46 comprising, a. a sequence encoding a heavy-chain variable region (VH) comprising a sequence set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto; and b. a sequence encoding a light-chain variable region (VL) comprising a sequence set forth in Table 3, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% thereto. nucleic acid of claim 63, wherein a. the VH comprises SEQ ID NO: 69, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b. the VL comprises SEQ ID NO: 65, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto. nucleic acid of claim 63, wherein a. the VH comprises SEQ ID NO: 77, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b. the VL comprises SEQ ID NO: 73, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto. nucleic acid of claim 63, wherein a. the VH comprises SEQ ID NO: 85, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b. the VL comprises SEQ ID NO: 81, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto. nucleic acid of claim 63, wherein a. the VH comprises SEQ ID NO: 93, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b. the VL comprises SEQ ID NO: 89, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto. nucleic acid of claim 63, wherein a. the VH comprises SEQ ID NO: 101, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b. the VL comprises SEQ ID NO: 97, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto. nucleic acid of claim 63, wherein a. the VH comprises SEQ ID NO: 109, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b. the VL comprises SEQ ID NO: 105, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto. nucleic acid of claim 63, wherein a. the VH comprises SEQ ID NO: 117, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b. the VL comprises SEQ ID NO: 113, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto. nucleic acid of claim 63, wherein 92 a. the VH comprises SEQ ID NO: 125, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto; and b. the VL comprises SEQ ID NO: 121, or a sequence that shares at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity thereto.
72. The nucleic acid of any one of claims 46-71, wherein the sequence is codon optimized.
73. A vector comprising the nucleic acid of any one of claims 46-72.
74. The vector of claim 73, wherein the polynucleotide sequence is operably linked to a promoter.
75. A cell line for producing the antibody or antigen-binding fragment of any one of claims 1-37, the nucleic acid of any one of claims 46-72, or the vector of claim 73 or claim 74.
76. A method of producing the antibody or antigen-binding fragment of any one of claims 1-37, comprising a. culturing the cell line of claim 75 under conditions sufficient for expression of the antibody or antigen-binding fragment; and b. purifying the antibody or antigen-binding fragment.
77. A method of producing an antibody or antigen-binding fragment that binds to a SARS-CoV-2 spike protein comprising: a. contacting a cell with the nucleic acid of any one of claims 46-72 or the vector of claim 73 or claim 74; b. culturing the cell under conditions sufficient to express the antibody or antigen-binding fragment; and c. purifying the antibody or antigen-binding fragment.
78. A method of treating COVID-19 in a subject in need thereof, comprising administering to the subject the antibody or antigen-binding fragment of any one of claims 1-38 or the pharmaceutical composition of any one of claims 38-43.
79. A method of preventing COVID-19 in a subject in need thereof, comprising administering to the subject the antibody or antigen-binding fragment of any one of claims 1-37 or the pharmaceutical composition of claim 38-43. 93
80. A method of reducing one or more symptoms associated with COVID-19 in a subject in need thereof, comprising administering to the subject the antibody or anti gen -binding fragment of any one of claims 1-37 or the pharmaceutical composition of claim 39-44.
81. The method of claim 80, wherein the severity of the one or more symptoms is reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or eliminated completely.
82. The method of claim 80 or 81, wherein the duration of the one or more symptoms is reduced by at least one day, at least two days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, or at least 10 days.
83. A method of treating, preventing, or reducing symptoms associated with COVID- 19 in a subject in need thereof, comprising a. detecting a SARS-CoV-2 viral genome or a SARS-CoV-2 antigen in a sample collected from the subject, wherein detection of the SARS-CoV-2 antigen comprises contacting the sample with the antibody or antigenbinding fragment of any one of claim 1-37; and b. administering a treatment to the subject, wherein the treatment i. prevents, reduces, or eliminates a SARS-CoV-2 infection or a variant thereof; and/or ii. prevents, reduces, or eliminates symptoms associated with COVID-19, wherein the treatment comprises the antibody or antigen-binding fragment of any one of claims 1-37 or the pharmaceutical composition of claim 38-43.
84. The method of any one of claims 78-83, wherein the subject has or is suspected of having a SARS-CoV-2 infection or a variant thereof.
85. The method of any one of claims 78-84, wherein the subject has at least one symptom associated with a SARS-CoV-2 infection or a variant thereof.
86. The method of any one of claims 78-85, wherein the subject is a human.
87. The method of any one of claims 78-86, wherein administration is enteral, parenteral, mucosal, nasal, oral, by inhalation, by intravenous injection, by infusion, by subcutaneous injection, by intramuscular injection, or intra-articular. 94
88. A method for detecting a SARS-CoV-2 spike protein in a sample, comprising contacting a sample with the antibody or antigen-binding fragment of any one of claims 1-37.
89. The method of claim 88, wherein the sample is an environmental sample, sewage, wastewater, urine, feces, saliva, nasal swab, mucous, blood, plasma, bronchoalveolar lavage, or one or more cells.
90. The method of claim 88 or claim 89, wherein the sample is collected from a subject.
91. The method of claim 90, wherein the subject has or is suspected of having a SARS-CoV-2 infection or COVID-19.
92. The method of any one of claims 88-91, wherein the antibody or antigenbinding fragment is labeled with a detection dye or a secondary antibody.
93. The method of any one of claims 88-92, wherein detecting a SARS-CoV-2 spike protein further comprises use of an electrochemical, biosensor, or immunoassay.
94. A method of diagnosing an individual as having COVID-19, comprising detecting a SARS-CoV-2 spike protein in a sample, comprising contacting a sample with the antibody or antigen-binding fragment of any one of claims 1-37.
95. The method of claim 94, wherein the sample is saliva, nasal swab, mucous, blood, plasma, bronchoalveolar lavage, or one or more cells.
96. The method of claim 94 or claim 95, wherein the sample is collected from a subject.
97. The method of claim 96, wherein the subject has or is suspected of having a SARS-CoV-2 infection or COVID-19.
98. The method of any one of claims 94-97, wherein the antibody or antigenbinding fragment is labeled with a detection dye or a secondary antibody.
99. The method of any one of claims 94-98, wherein detecting a SARS-CoV-2 spike protein further comprises use of an ELISA technique or a lateral flow assay.
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